Overview of SPE TechnologyMethod Development amp New Trends in Sample Preparation

An Trinh atrinhsialcomSupelco Bellefonte PA

T407048

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Agenda

bull The Importance of Sample Prepbull Overview of SPE Technologybull Understanding Retention Mechanismsbull A Systematic Approach to SPE MDbull New Developments in Sample Prep

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Analytical Chromatography Heaven

bull Short run timesbull Baseline resolutionbull Symmetric peak shapebull Good SN ratiobull No misleading peaksbull High precisionaccuracy

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The Importance of Sample Preparation

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Real World amp Real Samples

0 2 4 6Time (min)

Urine Sample without SPE

0 2 4 6Time (min)

Urine Sample with SPE

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Why is sample preparation required

Current Sample = Unsuitable for further analysishellip Whybull Too dilute- analyte(s) not concentrated enough for quantitative detectionbull Too dirty- contains other sample matrix components that interfere with

the analysisbull Present sample matrix not compatible with or harmful to the

chromatographic columnsystem

Collected Sample GC HPLC or LC-MSMS Analysis

X

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Sources of Chromatographic Errors

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Time Spend on Analytical Process

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Why is sample prep especially important in bioanalysis

bull Due to many types of sample matrices encountered- Plasma- proteins lipids and other endogenous macromolecules- Urine- contains uric acid and many nitrogenous base products- Serum- Bile- Tissue Homogenates- Perfusates- Saliva- Seminal Fluid- Caco-2 buffer- Others

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Many ToolsTechnology for Sample Prep

bull Dilute and Shootbull Filtration]bull Protein Precipitationbull Equilibrium dialysis

ultrafiltrationbull Liquid Liquid Extractionbull Solid Phase Microextraction

(SPME)bull Solid Phase Extraction (off-

line and on-line)bull Turbulent Flow

Chromatographybull Monolithic Chromatographybull Immunoaffinity

Simpler Generic Methodology

More Complicated Requires Method

Dev

Less SelectiveMinimal Sample Cleanup amp

Concentration

Greater SelectivityOptimal Sample Cleanup amp

Concentration

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How to choose the right sample prep technology

bull Should depend on three specific criteria- Requirements of the assay- Time allowed to run sample prep method- Possible investment towards method development time

bull Example - Late DiscoveryEarly Development

bull Requires rapid sample turn aroundbull Higher limits of quantitationbull Very little method development time (1-2 days)bull Protein Precipitation may be ideal choice

- Development (pre-clinical and clinical)bull Drugs more potent and dosed at lower levelsbull Requires ultra-sensitivity great selectivity and rugged method developmentbull Greater method development time (3-5 days)bull SPE is more ideal choice

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Separatory FunnelsLLE = Old Technology

bull Large solvent consumptionbull Vigorous shakingmixingbull Waiting for layers to separatebull Phase emulsions

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More Common Sample Prep ToolsTechnology

Protein Precipitationbull Advantages

- Requires little to no method development (universal)- Amenable to automation- Very simple (2-3 steps) and relatively inexpensive

bull Disadvantages- Sample dilution effect =gt requires concentration which is time

consuming due to aqueous portion of sample- Poor removal of matrix interferences =gt stress on analytical

system and increased ion-suppression resulting in poor reproducibility accuracy and sensitivity

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SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

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Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

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Overview of Solid Phase Extraction (SPE)

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Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

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Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

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Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

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SPE Vacuum Manifold (cont)

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Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

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Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

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Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

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Basic SPE Steps amp Approaches

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General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

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Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

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Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

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Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

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Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

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Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

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Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

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Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

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Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

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Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

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Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

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bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

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Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

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Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

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Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

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Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

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SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

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0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

sigma-aldrichcom

Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

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Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Agenda

bull The Importance of Sample Prepbull Overview of SPE Technologybull Understanding Retention Mechanismsbull A Systematic Approach to SPE MDbull New Developments in Sample Prep

sigma-aldrichcom

Analytical Chromatography Heaven

bull Short run timesbull Baseline resolutionbull Symmetric peak shapebull Good SN ratiobull No misleading peaksbull High precisionaccuracy

sigma-aldrichcom

The Importance of Sample Preparation

sigma-aldrichcom

Real World amp Real Samples

0 2 4 6Time (min)

Urine Sample without SPE

0 2 4 6Time (min)

Urine Sample with SPE

sigma-aldrichcom

Why is sample preparation required

Current Sample = Unsuitable for further analysishellip Whybull Too dilute- analyte(s) not concentrated enough for quantitative detectionbull Too dirty- contains other sample matrix components that interfere with

the analysisbull Present sample matrix not compatible with or harmful to the

chromatographic columnsystem

Collected Sample GC HPLC or LC-MSMS Analysis

X

sigma-aldrichcom

Sources of Chromatographic Errors

sigma-aldrichcom

Time Spend on Analytical Process

sigma-aldrichcom

Why is sample prep especially important in bioanalysis

bull Due to many types of sample matrices encountered- Plasma- proteins lipids and other endogenous macromolecules- Urine- contains uric acid and many nitrogenous base products- Serum- Bile- Tissue Homogenates- Perfusates- Saliva- Seminal Fluid- Caco-2 buffer- Others

sigma-aldrichcom

Many ToolsTechnology for Sample Prep

bull Dilute and Shootbull Filtration]bull Protein Precipitationbull Equilibrium dialysis

ultrafiltrationbull Liquid Liquid Extractionbull Solid Phase Microextraction

(SPME)bull Solid Phase Extraction (off-

line and on-line)bull Turbulent Flow

Chromatographybull Monolithic Chromatographybull Immunoaffinity

Simpler Generic Methodology

More Complicated Requires Method

Dev

Less SelectiveMinimal Sample Cleanup amp

Concentration

Greater SelectivityOptimal Sample Cleanup amp

Concentration

sigma-aldrichcom

How to choose the right sample prep technology

bull Should depend on three specific criteria- Requirements of the assay- Time allowed to run sample prep method- Possible investment towards method development time

bull Example - Late DiscoveryEarly Development

bull Requires rapid sample turn aroundbull Higher limits of quantitationbull Very little method development time (1-2 days)bull Protein Precipitation may be ideal choice

- Development (pre-clinical and clinical)bull Drugs more potent and dosed at lower levelsbull Requires ultra-sensitivity great selectivity and rugged method developmentbull Greater method development time (3-5 days)bull SPE is more ideal choice

sigma-aldrichcom

Separatory FunnelsLLE = Old Technology

bull Large solvent consumptionbull Vigorous shakingmixingbull Waiting for layers to separatebull Phase emulsions

sigma-aldrichcom

More Common Sample Prep ToolsTechnology

Protein Precipitationbull Advantages

- Requires little to no method development (universal)- Amenable to automation- Very simple (2-3 steps) and relatively inexpensive

bull Disadvantages- Sample dilution effect =gt requires concentration which is time

consuming due to aqueous portion of sample- Poor removal of matrix interferences =gt stress on analytical

system and increased ion-suppression resulting in poor reproducibility accuracy and sensitivity

sigma-aldrichcom

SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

sigma-aldrichcom

Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

sigma-aldrichcom

Overview of Solid Phase Extraction (SPE)

sigma-aldrichcom

Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

sigma-aldrichcom

Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

sigma-aldrichcom

SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

sigma-aldrichcom

Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

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NEW Developments in Sample Prep

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SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

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SupelMIP Binding Site

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Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

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Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Analytical Chromatography Heaven

bull Short run timesbull Baseline resolutionbull Symmetric peak shapebull Good SN ratiobull No misleading peaksbull High precisionaccuracy

sigma-aldrichcom

The Importance of Sample Preparation

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Real World amp Real Samples

0 2 4 6Time (min)

Urine Sample without SPE

0 2 4 6Time (min)

Urine Sample with SPE

sigma-aldrichcom

Why is sample preparation required

Current Sample = Unsuitable for further analysishellip Whybull Too dilute- analyte(s) not concentrated enough for quantitative detectionbull Too dirty- contains other sample matrix components that interfere with

the analysisbull Present sample matrix not compatible with or harmful to the

chromatographic columnsystem

Collected Sample GC HPLC or LC-MSMS Analysis

X

sigma-aldrichcom

Sources of Chromatographic Errors

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Time Spend on Analytical Process

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Why is sample prep especially important in bioanalysis

bull Due to many types of sample matrices encountered- Plasma- proteins lipids and other endogenous macromolecules- Urine- contains uric acid and many nitrogenous base products- Serum- Bile- Tissue Homogenates- Perfusates- Saliva- Seminal Fluid- Caco-2 buffer- Others

sigma-aldrichcom

Many ToolsTechnology for Sample Prep

bull Dilute and Shootbull Filtration]bull Protein Precipitationbull Equilibrium dialysis

ultrafiltrationbull Liquid Liquid Extractionbull Solid Phase Microextraction

(SPME)bull Solid Phase Extraction (off-

line and on-line)bull Turbulent Flow

Chromatographybull Monolithic Chromatographybull Immunoaffinity

Simpler Generic Methodology

More Complicated Requires Method

Dev

Less SelectiveMinimal Sample Cleanup amp

Concentration

Greater SelectivityOptimal Sample Cleanup amp

Concentration

sigma-aldrichcom

How to choose the right sample prep technology

bull Should depend on three specific criteria- Requirements of the assay- Time allowed to run sample prep method- Possible investment towards method development time

bull Example - Late DiscoveryEarly Development

bull Requires rapid sample turn aroundbull Higher limits of quantitationbull Very little method development time (1-2 days)bull Protein Precipitation may be ideal choice

- Development (pre-clinical and clinical)bull Drugs more potent and dosed at lower levelsbull Requires ultra-sensitivity great selectivity and rugged method developmentbull Greater method development time (3-5 days)bull SPE is more ideal choice

sigma-aldrichcom

Separatory FunnelsLLE = Old Technology

bull Large solvent consumptionbull Vigorous shakingmixingbull Waiting for layers to separatebull Phase emulsions

sigma-aldrichcom

More Common Sample Prep ToolsTechnology

Protein Precipitationbull Advantages

- Requires little to no method development (universal)- Amenable to automation- Very simple (2-3 steps) and relatively inexpensive

bull Disadvantages- Sample dilution effect =gt requires concentration which is time

consuming due to aqueous portion of sample- Poor removal of matrix interferences =gt stress on analytical

system and increased ion-suppression resulting in poor reproducibility accuracy and sensitivity

sigma-aldrichcom

SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

sigma-aldrichcom

Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

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Overview of Solid Phase Extraction (SPE)

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Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

sigma-aldrichcom

Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

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SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

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Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

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Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

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Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

sigma-aldrichcom

SPE Bed WeightTube Size Selection

sigma-aldrichcom

SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

sigma-aldrichcom

Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

The Importance of Sample Preparation

sigma-aldrichcom

Real World amp Real Samples

0 2 4 6Time (min)

Urine Sample without SPE

0 2 4 6Time (min)

Urine Sample with SPE

sigma-aldrichcom

Why is sample preparation required

Current Sample = Unsuitable for further analysishellip Whybull Too dilute- analyte(s) not concentrated enough for quantitative detectionbull Too dirty- contains other sample matrix components that interfere with

the analysisbull Present sample matrix not compatible with or harmful to the

chromatographic columnsystem

Collected Sample GC HPLC or LC-MSMS Analysis

X

sigma-aldrichcom

Sources of Chromatographic Errors

sigma-aldrichcom

Time Spend on Analytical Process

sigma-aldrichcom

Why is sample prep especially important in bioanalysis

bull Due to many types of sample matrices encountered- Plasma- proteins lipids and other endogenous macromolecules- Urine- contains uric acid and many nitrogenous base products- Serum- Bile- Tissue Homogenates- Perfusates- Saliva- Seminal Fluid- Caco-2 buffer- Others

sigma-aldrichcom

Many ToolsTechnology for Sample Prep

bull Dilute and Shootbull Filtration]bull Protein Precipitationbull Equilibrium dialysis

ultrafiltrationbull Liquid Liquid Extractionbull Solid Phase Microextraction

(SPME)bull Solid Phase Extraction (off-

line and on-line)bull Turbulent Flow

Chromatographybull Monolithic Chromatographybull Immunoaffinity

Simpler Generic Methodology

More Complicated Requires Method

Dev

Less SelectiveMinimal Sample Cleanup amp

Concentration

Greater SelectivityOptimal Sample Cleanup amp

Concentration

sigma-aldrichcom

How to choose the right sample prep technology

bull Should depend on three specific criteria- Requirements of the assay- Time allowed to run sample prep method- Possible investment towards method development time

bull Example - Late DiscoveryEarly Development

bull Requires rapid sample turn aroundbull Higher limits of quantitationbull Very little method development time (1-2 days)bull Protein Precipitation may be ideal choice

- Development (pre-clinical and clinical)bull Drugs more potent and dosed at lower levelsbull Requires ultra-sensitivity great selectivity and rugged method developmentbull Greater method development time (3-5 days)bull SPE is more ideal choice

sigma-aldrichcom

Separatory FunnelsLLE = Old Technology

bull Large solvent consumptionbull Vigorous shakingmixingbull Waiting for layers to separatebull Phase emulsions

sigma-aldrichcom

More Common Sample Prep ToolsTechnology

Protein Precipitationbull Advantages

- Requires little to no method development (universal)- Amenable to automation- Very simple (2-3 steps) and relatively inexpensive

bull Disadvantages- Sample dilution effect =gt requires concentration which is time

consuming due to aqueous portion of sample- Poor removal of matrix interferences =gt stress on analytical

system and increased ion-suppression resulting in poor reproducibility accuracy and sensitivity

sigma-aldrichcom

SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

sigma-aldrichcom

Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

sigma-aldrichcom

Overview of Solid Phase Extraction (SPE)

sigma-aldrichcom

Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

sigma-aldrichcom

Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

sigma-aldrichcom

SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

sigma-aldrichcom

Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

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Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

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Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

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Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

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Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

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Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

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Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

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bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

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Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

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Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

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Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

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Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

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SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

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0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

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Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

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Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

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SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

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Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

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NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

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Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

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NEW Developments in Sample Prep

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SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

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SupelMIP Binding Site

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Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

sigma-aldrichcom

Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

sigma-aldrichcom

Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

sigma-aldrichcom

CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Real World amp Real Samples

0 2 4 6Time (min)

Urine Sample without SPE

0 2 4 6Time (min)

Urine Sample with SPE

sigma-aldrichcom

Why is sample preparation required

Current Sample = Unsuitable for further analysishellip Whybull Too dilute- analyte(s) not concentrated enough for quantitative detectionbull Too dirty- contains other sample matrix components that interfere with

the analysisbull Present sample matrix not compatible with or harmful to the

chromatographic columnsystem

Collected Sample GC HPLC or LC-MSMS Analysis

X

sigma-aldrichcom

Sources of Chromatographic Errors

sigma-aldrichcom

Time Spend on Analytical Process

sigma-aldrichcom

Why is sample prep especially important in bioanalysis

bull Due to many types of sample matrices encountered- Plasma- proteins lipids and other endogenous macromolecules- Urine- contains uric acid and many nitrogenous base products- Serum- Bile- Tissue Homogenates- Perfusates- Saliva- Seminal Fluid- Caco-2 buffer- Others

sigma-aldrichcom

Many ToolsTechnology for Sample Prep

bull Dilute and Shootbull Filtration]bull Protein Precipitationbull Equilibrium dialysis

ultrafiltrationbull Liquid Liquid Extractionbull Solid Phase Microextraction

(SPME)bull Solid Phase Extraction (off-

line and on-line)bull Turbulent Flow

Chromatographybull Monolithic Chromatographybull Immunoaffinity

Simpler Generic Methodology

More Complicated Requires Method

Dev

Less SelectiveMinimal Sample Cleanup amp

Concentration

Greater SelectivityOptimal Sample Cleanup amp

Concentration

sigma-aldrichcom

How to choose the right sample prep technology

bull Should depend on three specific criteria- Requirements of the assay- Time allowed to run sample prep method- Possible investment towards method development time

bull Example - Late DiscoveryEarly Development

bull Requires rapid sample turn aroundbull Higher limits of quantitationbull Very little method development time (1-2 days)bull Protein Precipitation may be ideal choice

- Development (pre-clinical and clinical)bull Drugs more potent and dosed at lower levelsbull Requires ultra-sensitivity great selectivity and rugged method developmentbull Greater method development time (3-5 days)bull SPE is more ideal choice

sigma-aldrichcom

Separatory FunnelsLLE = Old Technology

bull Large solvent consumptionbull Vigorous shakingmixingbull Waiting for layers to separatebull Phase emulsions

sigma-aldrichcom

More Common Sample Prep ToolsTechnology

Protein Precipitationbull Advantages

- Requires little to no method development (universal)- Amenable to automation- Very simple (2-3 steps) and relatively inexpensive

bull Disadvantages- Sample dilution effect =gt requires concentration which is time

consuming due to aqueous portion of sample- Poor removal of matrix interferences =gt stress on analytical

system and increased ion-suppression resulting in poor reproducibility accuracy and sensitivity

sigma-aldrichcom

SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

sigma-aldrichcom

Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

sigma-aldrichcom

Overview of Solid Phase Extraction (SPE)

sigma-aldrichcom

Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

sigma-aldrichcom

Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

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SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

sigma-aldrichcom

Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

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Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

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Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

sigma-aldrichcom

SPE Bed WeightTube Size Selection

sigma-aldrichcom

SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

sigma-aldrichcom

Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

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bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

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Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

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Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

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Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

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Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

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SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

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0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

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Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

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Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

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SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

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Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

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Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

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NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

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Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

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0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

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Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

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Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

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Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

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Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

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Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

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Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

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Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

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NEW Developments in Sample Prep

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SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

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SupelMIP Binding Site

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Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

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Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

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96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

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96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

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New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

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Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

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Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

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Why is sample preparation required

Current Sample = Unsuitable for further analysishellip Whybull Too dilute- analyte(s) not concentrated enough for quantitative detectionbull Too dirty- contains other sample matrix components that interfere with

the analysisbull Present sample matrix not compatible with or harmful to the

chromatographic columnsystem

Collected Sample GC HPLC or LC-MSMS Analysis

X

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Sources of Chromatographic Errors

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Time Spend on Analytical Process

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Why is sample prep especially important in bioanalysis

bull Due to many types of sample matrices encountered- Plasma- proteins lipids and other endogenous macromolecules- Urine- contains uric acid and many nitrogenous base products- Serum- Bile- Tissue Homogenates- Perfusates- Saliva- Seminal Fluid- Caco-2 buffer- Others

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Many ToolsTechnology for Sample Prep

bull Dilute and Shootbull Filtration]bull Protein Precipitationbull Equilibrium dialysis

ultrafiltrationbull Liquid Liquid Extractionbull Solid Phase Microextraction

(SPME)bull Solid Phase Extraction (off-

line and on-line)bull Turbulent Flow

Chromatographybull Monolithic Chromatographybull Immunoaffinity

Simpler Generic Methodology

More Complicated Requires Method

Dev

Less SelectiveMinimal Sample Cleanup amp

Concentration

Greater SelectivityOptimal Sample Cleanup amp

Concentration

sigma-aldrichcom

How to choose the right sample prep technology

bull Should depend on three specific criteria- Requirements of the assay- Time allowed to run sample prep method- Possible investment towards method development time

bull Example - Late DiscoveryEarly Development

bull Requires rapid sample turn aroundbull Higher limits of quantitationbull Very little method development time (1-2 days)bull Protein Precipitation may be ideal choice

- Development (pre-clinical and clinical)bull Drugs more potent and dosed at lower levelsbull Requires ultra-sensitivity great selectivity and rugged method developmentbull Greater method development time (3-5 days)bull SPE is more ideal choice

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Separatory FunnelsLLE = Old Technology

bull Large solvent consumptionbull Vigorous shakingmixingbull Waiting for layers to separatebull Phase emulsions

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More Common Sample Prep ToolsTechnology

Protein Precipitationbull Advantages

- Requires little to no method development (universal)- Amenable to automation- Very simple (2-3 steps) and relatively inexpensive

bull Disadvantages- Sample dilution effect =gt requires concentration which is time

consuming due to aqueous portion of sample- Poor removal of matrix interferences =gt stress on analytical

system and increased ion-suppression resulting in poor reproducibility accuracy and sensitivity

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SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

sigma-aldrichcom

Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

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Overview of Solid Phase Extraction (SPE)

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Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

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Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

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Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

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SPE Vacuum Manifold (cont)

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Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

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Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

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Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

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Basic SPE Steps amp Approaches

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General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

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Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

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Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

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Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

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Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

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Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

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Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

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Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

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Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

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Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

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Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

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bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

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Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

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Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

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Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

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Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

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SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

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0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

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Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

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Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

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SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

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Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

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Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

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NEW Developments in Sample Prep

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SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

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SupelMIP Binding Site

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Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

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96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Sources of Chromatographic Errors

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Time Spend on Analytical Process

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Why is sample prep especially important in bioanalysis

bull Due to many types of sample matrices encountered- Plasma- proteins lipids and other endogenous macromolecules- Urine- contains uric acid and many nitrogenous base products- Serum- Bile- Tissue Homogenates- Perfusates- Saliva- Seminal Fluid- Caco-2 buffer- Others

sigma-aldrichcom

Many ToolsTechnology for Sample Prep

bull Dilute and Shootbull Filtration]bull Protein Precipitationbull Equilibrium dialysis

ultrafiltrationbull Liquid Liquid Extractionbull Solid Phase Microextraction

(SPME)bull Solid Phase Extraction (off-

line and on-line)bull Turbulent Flow

Chromatographybull Monolithic Chromatographybull Immunoaffinity

Simpler Generic Methodology

More Complicated Requires Method

Dev

Less SelectiveMinimal Sample Cleanup amp

Concentration

Greater SelectivityOptimal Sample Cleanup amp

Concentration

sigma-aldrichcom

How to choose the right sample prep technology

bull Should depend on three specific criteria- Requirements of the assay- Time allowed to run sample prep method- Possible investment towards method development time

bull Example - Late DiscoveryEarly Development

bull Requires rapid sample turn aroundbull Higher limits of quantitationbull Very little method development time (1-2 days)bull Protein Precipitation may be ideal choice

- Development (pre-clinical and clinical)bull Drugs more potent and dosed at lower levelsbull Requires ultra-sensitivity great selectivity and rugged method developmentbull Greater method development time (3-5 days)bull SPE is more ideal choice

sigma-aldrichcom

Separatory FunnelsLLE = Old Technology

bull Large solvent consumptionbull Vigorous shakingmixingbull Waiting for layers to separatebull Phase emulsions

sigma-aldrichcom

More Common Sample Prep ToolsTechnology

Protein Precipitationbull Advantages

- Requires little to no method development (universal)- Amenable to automation- Very simple (2-3 steps) and relatively inexpensive

bull Disadvantages- Sample dilution effect =gt requires concentration which is time

consuming due to aqueous portion of sample- Poor removal of matrix interferences =gt stress on analytical

system and increased ion-suppression resulting in poor reproducibility accuracy and sensitivity

sigma-aldrichcom

SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

sigma-aldrichcom

Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

sigma-aldrichcom

Overview of Solid Phase Extraction (SPE)

sigma-aldrichcom

Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

sigma-aldrichcom

Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

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SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

sigma-aldrichcom

Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

sigma-aldrichcom

SPE Bed WeightTube Size Selection

sigma-aldrichcom

SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

sigma-aldrichcom

Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

sigma-aldrichcom

Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

sigma-aldrichcom

Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

sigma-aldrichcom

Discovery Ag-ION Protocol

sigma-aldrichcom

181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

sigma-aldrichcom

CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

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Time Spend on Analytical Process

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Why is sample prep especially important in bioanalysis

bull Due to many types of sample matrices encountered- Plasma- proteins lipids and other endogenous macromolecules- Urine- contains uric acid and many nitrogenous base products- Serum- Bile- Tissue Homogenates- Perfusates- Saliva- Seminal Fluid- Caco-2 buffer- Others

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Many ToolsTechnology for Sample Prep

bull Dilute and Shootbull Filtration]bull Protein Precipitationbull Equilibrium dialysis

ultrafiltrationbull Liquid Liquid Extractionbull Solid Phase Microextraction

(SPME)bull Solid Phase Extraction (off-

line and on-line)bull Turbulent Flow

Chromatographybull Monolithic Chromatographybull Immunoaffinity

Simpler Generic Methodology

More Complicated Requires Method

Dev

Less SelectiveMinimal Sample Cleanup amp

Concentration

Greater SelectivityOptimal Sample Cleanup amp

Concentration

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How to choose the right sample prep technology

bull Should depend on three specific criteria- Requirements of the assay- Time allowed to run sample prep method- Possible investment towards method development time

bull Example - Late DiscoveryEarly Development

bull Requires rapid sample turn aroundbull Higher limits of quantitationbull Very little method development time (1-2 days)bull Protein Precipitation may be ideal choice

- Development (pre-clinical and clinical)bull Drugs more potent and dosed at lower levelsbull Requires ultra-sensitivity great selectivity and rugged method developmentbull Greater method development time (3-5 days)bull SPE is more ideal choice

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Separatory FunnelsLLE = Old Technology

bull Large solvent consumptionbull Vigorous shakingmixingbull Waiting for layers to separatebull Phase emulsions

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More Common Sample Prep ToolsTechnology

Protein Precipitationbull Advantages

- Requires little to no method development (universal)- Amenable to automation- Very simple (2-3 steps) and relatively inexpensive

bull Disadvantages- Sample dilution effect =gt requires concentration which is time

consuming due to aqueous portion of sample- Poor removal of matrix interferences =gt stress on analytical

system and increased ion-suppression resulting in poor reproducibility accuracy and sensitivity

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SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

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Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

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Overview of Solid Phase Extraction (SPE)

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Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

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Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

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Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

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SPE Vacuum Manifold (cont)

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Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

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Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

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Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

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Basic SPE Steps amp Approaches

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General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

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Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

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Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

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Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

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Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

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Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

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Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

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Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

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96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

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Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Why is sample prep especially important in bioanalysis

bull Due to many types of sample matrices encountered- Plasma- proteins lipids and other endogenous macromolecules- Urine- contains uric acid and many nitrogenous base products- Serum- Bile- Tissue Homogenates- Perfusates- Saliva- Seminal Fluid- Caco-2 buffer- Others

sigma-aldrichcom

Many ToolsTechnology for Sample Prep

bull Dilute and Shootbull Filtration]bull Protein Precipitationbull Equilibrium dialysis

ultrafiltrationbull Liquid Liquid Extractionbull Solid Phase Microextraction

(SPME)bull Solid Phase Extraction (off-

line and on-line)bull Turbulent Flow

Chromatographybull Monolithic Chromatographybull Immunoaffinity

Simpler Generic Methodology

More Complicated Requires Method

Dev

Less SelectiveMinimal Sample Cleanup amp

Concentration

Greater SelectivityOptimal Sample Cleanup amp

Concentration

sigma-aldrichcom

How to choose the right sample prep technology

bull Should depend on three specific criteria- Requirements of the assay- Time allowed to run sample prep method- Possible investment towards method development time

bull Example - Late DiscoveryEarly Development

bull Requires rapid sample turn aroundbull Higher limits of quantitationbull Very little method development time (1-2 days)bull Protein Precipitation may be ideal choice

- Development (pre-clinical and clinical)bull Drugs more potent and dosed at lower levelsbull Requires ultra-sensitivity great selectivity and rugged method developmentbull Greater method development time (3-5 days)bull SPE is more ideal choice

sigma-aldrichcom

Separatory FunnelsLLE = Old Technology

bull Large solvent consumptionbull Vigorous shakingmixingbull Waiting for layers to separatebull Phase emulsions

sigma-aldrichcom

More Common Sample Prep ToolsTechnology

Protein Precipitationbull Advantages

- Requires little to no method development (universal)- Amenable to automation- Very simple (2-3 steps) and relatively inexpensive

bull Disadvantages- Sample dilution effect =gt requires concentration which is time

consuming due to aqueous portion of sample- Poor removal of matrix interferences =gt stress on analytical

system and increased ion-suppression resulting in poor reproducibility accuracy and sensitivity

sigma-aldrichcom

SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

sigma-aldrichcom

Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

sigma-aldrichcom

Overview of Solid Phase Extraction (SPE)

sigma-aldrichcom

Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

sigma-aldrichcom

Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

sigma-aldrichcom

SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

sigma-aldrichcom

Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

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0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

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0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

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0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

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bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

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Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

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Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

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Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

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Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

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600

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1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

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SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

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0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

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Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

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Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

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SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

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Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

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Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

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NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

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Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

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0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

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Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

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Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

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Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

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SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

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Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

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Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

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Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

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Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

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Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

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NEW Developments in Sample Prep

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SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

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SupelMIP Binding Site

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Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

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Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

sigma-aldrichcom

Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Many ToolsTechnology for Sample Prep

bull Dilute and Shootbull Filtration]bull Protein Precipitationbull Equilibrium dialysis

ultrafiltrationbull Liquid Liquid Extractionbull Solid Phase Microextraction

(SPME)bull Solid Phase Extraction (off-

line and on-line)bull Turbulent Flow

Chromatographybull Monolithic Chromatographybull Immunoaffinity

Simpler Generic Methodology

More Complicated Requires Method

Dev

Less SelectiveMinimal Sample Cleanup amp

Concentration

Greater SelectivityOptimal Sample Cleanup amp

Concentration

sigma-aldrichcom

How to choose the right sample prep technology

bull Should depend on three specific criteria- Requirements of the assay- Time allowed to run sample prep method- Possible investment towards method development time

bull Example - Late DiscoveryEarly Development

bull Requires rapid sample turn aroundbull Higher limits of quantitationbull Very little method development time (1-2 days)bull Protein Precipitation may be ideal choice

- Development (pre-clinical and clinical)bull Drugs more potent and dosed at lower levelsbull Requires ultra-sensitivity great selectivity and rugged method developmentbull Greater method development time (3-5 days)bull SPE is more ideal choice

sigma-aldrichcom

Separatory FunnelsLLE = Old Technology

bull Large solvent consumptionbull Vigorous shakingmixingbull Waiting for layers to separatebull Phase emulsions

sigma-aldrichcom

More Common Sample Prep ToolsTechnology

Protein Precipitationbull Advantages

- Requires little to no method development (universal)- Amenable to automation- Very simple (2-3 steps) and relatively inexpensive

bull Disadvantages- Sample dilution effect =gt requires concentration which is time

consuming due to aqueous portion of sample- Poor removal of matrix interferences =gt stress on analytical

system and increased ion-suppression resulting in poor reproducibility accuracy and sensitivity

sigma-aldrichcom

SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

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Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

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Overview of Solid Phase Extraction (SPE)

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Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

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Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

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SPE Vacuum Manifold (cont)

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Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

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Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

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Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

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Basic SPE Steps amp Approaches

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General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

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Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

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Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

sigma-aldrichcom

Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

sigma-aldrichcom

Discovery Ag-ION Protocol

sigma-aldrichcom

181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

sigma-aldrichcom

CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

How to choose the right sample prep technology

bull Should depend on three specific criteria- Requirements of the assay- Time allowed to run sample prep method- Possible investment towards method development time

bull Example - Late DiscoveryEarly Development

bull Requires rapid sample turn aroundbull Higher limits of quantitationbull Very little method development time (1-2 days)bull Protein Precipitation may be ideal choice

- Development (pre-clinical and clinical)bull Drugs more potent and dosed at lower levelsbull Requires ultra-sensitivity great selectivity and rugged method developmentbull Greater method development time (3-5 days)bull SPE is more ideal choice

sigma-aldrichcom

Separatory FunnelsLLE = Old Technology

bull Large solvent consumptionbull Vigorous shakingmixingbull Waiting for layers to separatebull Phase emulsions

sigma-aldrichcom

More Common Sample Prep ToolsTechnology

Protein Precipitationbull Advantages

- Requires little to no method development (universal)- Amenable to automation- Very simple (2-3 steps) and relatively inexpensive

bull Disadvantages- Sample dilution effect =gt requires concentration which is time

consuming due to aqueous portion of sample- Poor removal of matrix interferences =gt stress on analytical

system and increased ion-suppression resulting in poor reproducibility accuracy and sensitivity

sigma-aldrichcom

SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

sigma-aldrichcom

Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

sigma-aldrichcom

Overview of Solid Phase Extraction (SPE)

sigma-aldrichcom

Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

sigma-aldrichcom

Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

sigma-aldrichcom

SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

sigma-aldrichcom

Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

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Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

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bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

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Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

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Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

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Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

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Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

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SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

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0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

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Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

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Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

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SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

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Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

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Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

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Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

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0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

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Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

sigma-aldrichcom

Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

sigma-aldrichcom

Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Separatory FunnelsLLE = Old Technology

bull Large solvent consumptionbull Vigorous shakingmixingbull Waiting for layers to separatebull Phase emulsions

sigma-aldrichcom

More Common Sample Prep ToolsTechnology

Protein Precipitationbull Advantages

- Requires little to no method development (universal)- Amenable to automation- Very simple (2-3 steps) and relatively inexpensive

bull Disadvantages- Sample dilution effect =gt requires concentration which is time

consuming due to aqueous portion of sample- Poor removal of matrix interferences =gt stress on analytical

system and increased ion-suppression resulting in poor reproducibility accuracy and sensitivity

sigma-aldrichcom

SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

sigma-aldrichcom

Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

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Overview of Solid Phase Extraction (SPE)

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Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

sigma-aldrichcom

Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

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SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

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Basic SPE Steps amp Approaches

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General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

sigma-aldrichcom

181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

sigma-aldrichcom

CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

More Common Sample Prep ToolsTechnology

Protein Precipitationbull Advantages

- Requires little to no method development (universal)- Amenable to automation- Very simple (2-3 steps) and relatively inexpensive

bull Disadvantages- Sample dilution effect =gt requires concentration which is time

consuming due to aqueous portion of sample- Poor removal of matrix interferences =gt stress on analytical

system and increased ion-suppression resulting in poor reproducibility accuracy and sensitivity

sigma-aldrichcom

SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

sigma-aldrichcom

Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

sigma-aldrichcom

Overview of Solid Phase Extraction (SPE)

sigma-aldrichcom

Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

sigma-aldrichcom

Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

sigma-aldrichcom

SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

sigma-aldrichcom

Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

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Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

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Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

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Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

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bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

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Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

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Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

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Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

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Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

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SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

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0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

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Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

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Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

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SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

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Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

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Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

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NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

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Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

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0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

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Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

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Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

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Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

sigma-aldrichcom

Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

SPE Advantages amp Disadvantages

Disadvantages- Perceived difficulty to master its usage (method development)

bull Wide range of chemistries many choices for manipulating solvent and pH conditions make it difficult to grasp

- More steps and MD time required- Greater cost per sample

Advantages- Greater selectivity- paramount importance (eg bioanalysis (pgmL))- Wide variety of sample matrices- High recoveries amp good reproducibility- Amenable to automation- Low solvent volumes

sigma-aldrichcom

Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

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Overview of Solid Phase Extraction (SPE)

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Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

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Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

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SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

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Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

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Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

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Basic SPE Steps amp Approaches

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General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

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NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

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96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Prior to the actual analysis SPE is most commonly used tohellip

1 Clean Up - Strip the analyte(s) away from endogenous interferences

2 Concentrate analytes(s) for better sensitivity

3 Exchange sample environments for better chromatography -eg analytes in serum =gt analytes in

mobile phase

Purpose of Solid Phase Extraction (SPE)

sigma-aldrichcom

Overview of Solid Phase Extraction (SPE)

sigma-aldrichcom

Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

sigma-aldrichcom

Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

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SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

sigma-aldrichcom

Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

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bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

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Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

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Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

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Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Overview of Solid Phase Extraction (SPE)

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Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

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Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

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SPE Vacuum Manifold (cont)

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Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

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Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

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Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

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Basic SPE Steps amp Approaches

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General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

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Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

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Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

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NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

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SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

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96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

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96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Basic SPE Concept

bull Another form of chromatographybull Hardware = plastic (polypropylene) or

glassbull Sorbent held in place by two PE fritsbull Packing material is very similar to HPLC

- Often irregular shape vs spherical (HPLC)

- Much larger particle size (gt50um) vs HPLC (lt= 5um)

- SPE particle size distribution much broader than HPLC

bull Use it only once

Serological Grade Polypropylene

or Glass Tube

Polyethylene SS or Teflonreg Frits (20microm pores)

SPE Packing

Luer Tip

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

sigma-aldrichcom

Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

sigma-aldrichcom

SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

sigma-aldrichcom

Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

sigma-aldrichcom

SPE Bed WeightTube Size Selection

sigma-aldrichcom

SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

sigma-aldrichcom

Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

sigma-aldrichcom

Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

sigma-aldrichcom

181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

sigma-aldrichcom

CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Discovery SPE 96-Well Plates

Upper Frit

Packed Bed

Lower Frit

Plate Descriptionbull Square well extraction plate

20-225 ml capacity polypropylene

bull Available for all Discovery SPE phases

bull Bed weight = 25 50 or 100mgwell

bull Compatible with most robotic systems and automated sample processing systems TomTec Quadra Gilson SPE 215 etc

High Throughput Solid Phase Extraction

sigma-aldrichcom

Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

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SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

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Basic SPE Steps amp Approaches

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General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

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Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

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Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

sigma-aldrichcom

Discovery Ag-ION Protocol

sigma-aldrichcom

181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

sigma-aldrichcom

CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Vacuum manifold

SPE Vacuum Manifold

SPE tubes

Vacuum line and gauge

Sample collection

tubes (volumetric

flasks)

Waste reservoir

Sample introduction

Indiv Port Valves

sigma-aldrichcom

SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

sigma-aldrichcom

Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

sigma-aldrichcom

SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

sigma-aldrichcom

Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

sigma-aldrichcom

Discovery Ag-ION Protocol

sigma-aldrichcom

181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

sigma-aldrichcom

CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

SPE Vacuum Manifold (cont)

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

sigma-aldrichcom

Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

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Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

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Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

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Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

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Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

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Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

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Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

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bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

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Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

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Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

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Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

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Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

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SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

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0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

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Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

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Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

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SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

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Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

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Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

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NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

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Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

sigma-aldrichcom

Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

sigma-aldrichcom

Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

sigma-aldrichcom

CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Most Common SPE Robots for Automated SPE

TomTec Quadra System

Gilson SPE 215 System Code 802 amp 803 ldquoTab-lessrdquo 1 amp

3mL racks

Zymark RapidTrace System

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

sigma-aldrichcom

Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

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SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

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0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

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Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

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0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

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Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

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Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

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Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

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SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

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Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

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Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

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Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

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Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

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Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

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NEW Developments in Sample Prep

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SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

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SupelMIP Binding Site

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Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

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Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

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96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

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96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

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New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

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Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

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Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Types of SPE TubesCartridges

SPE tubes are available in two materialsbull Polypropylene (serological grade)

- Most common- Suitable for most SPE applications- Inexpensive

bull Glass (serological grade)- Greater solvent resistance than plastic- No phthalates or plasticizers to leach into

sample- Can be heated - More expensive than plastic- Common in environmental analysis

An assortment of Supelco SPE tubes Second tubes in from

either side are glass

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

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Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

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Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

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bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

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Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

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Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

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Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

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Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

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Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Common SPE Hardware

Tubes

96-well plates

SPE DisksGlass or plastic tubes are the most common

SPE format

FunnelsBuumlchner formatideal for large

sample volumes

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Basic SPE Steps amp Approaches

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General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

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Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

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Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

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Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

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0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

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Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

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Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

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Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

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Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

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Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

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Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

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NEW Developments in Sample Prep

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SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

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SupelMIP Binding Site

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Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

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Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

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96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

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96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

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New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

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Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

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Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Basic SPE Steps amp Approaches

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

sigma-aldrichcom

SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

sigma-aldrichcom

Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

sigma-aldrichcom

Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

sigma-aldrichcom

Discovery Ag-ION Protocol

sigma-aldrichcom

181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

sigma-aldrichcom

CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

General Steps of an SPE Procedure

1) Sample Pre-treatment Dependent on compound of interest sample matrix and nature of retention chemistry involves pH adjustment centrifugation filtration dilution buffer addition etc

2) Conditioning Solvent is passed through the SPE material to wet the bonded functional groups =gt ensures consistent interaction

3) Equilibration Sorbent phase is treated with a solution that is similar (in polarity pH etc) to the sample matrix =gt maximizes retention

4) Sample Load Introduction of the sample = analytes of interest are bound extracted onto the phase sorbent

5) Washing Selectively remove unwanted interferences co-extracted with the analyte without prematurely eluting analytes of interest

6) Elution Removing analytes of interest with a solvent that overcomes theprimary and secondary retention interactions bw sorbent and analytes of interest

7) Evaporation of eluent reconstitution with mobile phase (optional)

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

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Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

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Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

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Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

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Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

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Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

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Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

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bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

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Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

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Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

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Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

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Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

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SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

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0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

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Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

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Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

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SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

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Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

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Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

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NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

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Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

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0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

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Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

sigma-aldrichcom

Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

sigma-aldrichcom

Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

sigma-aldrichcom

Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

sigma-aldrichcom

CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Three different SPE Strategies

There are 3 different elution strategies in SPE Which one to choose depends on the goal of the extraction

1 Bind-Elute Strategy bull Most commonbull Bind Analytes bind to tube unwanted matrix components are washed offbull Elute Eluant changed to remove analytes from tubebull Analytes are concentrated via evaporation prior to HPLC or GC analysis

2 Interference Removal Strategybull Bind all unwanted matrix components and allow analytes to pass through

during the sample loading stagebull Like chemical filtration

3 Fractionation Strategybull Retain and sequentially elute different classes of compounds by modifying

eluant pH or organic

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

sigma-aldrichcom

Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

sigma-aldrichcom

Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

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Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

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0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

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Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

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Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

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Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

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Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

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Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

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Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

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Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

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Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

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NEW Developments in Sample Prep

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SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

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SupelMIP Binding Site

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Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

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Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

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96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

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96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

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New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

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Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

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Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Bind-elute strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Matrix fraction= waste

2) Apply wash solvent

Analyte fraction

3) Apply elution solvent

Original sample (analytes amp IS

in a matrix)

1) Apply sample to SPE tube

HPLC orGC analysis

Purified amp concentratedanalytes amp IS

A dilute solution of

analytes amp ISin the

elution solvent

4) Evaporateelution solvent

reconstitute

Must first condition amp equilibrate SPE tube

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

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Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

sigma-aldrichcom

Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

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SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

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New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Interference removal strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

2) Apply elution solvent

3) Evaporateelution solvent reconstitute

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

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Normal-Phase SPE

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Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

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Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

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Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

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NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

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Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

sigma-aldrichcom

Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Fraction strategy diagram

Shown is a step-by-step bind-elute SPE extraction beginning with a filtered sample containing analytes and internal standard (IS) in a matrix and ending with purified and concentrated analytes and internal standard ready for HPLC or GC analysis

Original Sample(analyte amp ISIn matrix)

1) Apply sample to SPE tube

2) Apply wash solvent

3) Apply elution solvent 1

3) Apply elution solvent 2

A dilutesolution of analyte and ISin elution solvent

Fraction 2HPLC or

GC analysis

Fraction 1HPLC or

GC analysis

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Understanding Retention Mechanisms

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Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

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Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

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Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

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Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

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Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

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Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

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Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

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NEW Developments in Sample Prep

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SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

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SupelMIP Binding Site

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Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

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Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

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96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

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96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

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Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

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Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Understanding Retention Mechanisms

sigma-aldrichcom

Reversed-Phase SPE

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Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

sigma-aldrichcom

SPE Bed WeightTube Size Selection

sigma-aldrichcom

SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

sigma-aldrichcom

Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

sigma-aldrichcom

Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

sigma-aldrichcom

Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

sigma-aldrichcom

Discovery Ag-ION Protocol

sigma-aldrichcom

181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

sigma-aldrichcom

CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Reversed-Phase SPE

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

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Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

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Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

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SPE Method Development ndashSPE Selection amp The Role of pH

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Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

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Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

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Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

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Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

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SPE Phase Selection

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Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

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Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

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Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

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bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

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Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

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Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

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Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

sigma-aldrichcom

Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

sigma-aldrichcom

Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

sigma-aldrichcom

Discovery Ag-ION Protocol

sigma-aldrichcom

181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Example RP SPE Protocol1 Sample Pre-Treatment

bull Dilute samples 11 with buffer (10mM ammonium acetate)bull pH manipulation important for ionizable analytesbull Filter or centrifuge out particulates

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Critical for improving selectivitybull 5-20 MeOH commonbull Dilute MeOH in buffer used during sample load

5 Elute analytes of interestbull MeOH or MeCN most commonbull pH manipulation can improve recovery (adjust pH opposite to load

conditions)6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

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Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

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SPE Bed WeightTube Size Selection

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SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

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Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

sigma-aldrichcom

SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

sigma-aldrichcom

Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

sigma-aldrichcom

Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

sigma-aldrichcom

Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

sigma-aldrichcom

Dispersive SPE Product Line

sigma-aldrichcom

Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

sigma-aldrichcom

Discovery Ag-ION Protocol

sigma-aldrichcom

181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

sigma-aldrichcom

Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

sigma-aldrichcom

Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

sigma-aldrichcom

Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

sigma-aldrichcom

Useful RP SPE Tips

bull Drugs in biological fluids risk drug-protein binding effect- Disrupt during sample pre-treatment using 40uL 2 disodium EDTA

or 2 formic acid per 100uL plasma

bull Sorbent over drying only a concern during first conditioning stepbull If eluate evaporation necessary dry SPE tube with vacuum for

10-15 min prior to elution to remove residual moisturebull Pass DCM through SPE before conditioning to remove SPE tube

impurities for highly sensitive analysesbull Reduce bed weight to minimize elution volumebull Increase bed weight to retain more polar compounds

sigma-aldrichcom

Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

sigma-aldrichcom

Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

sigma-aldrichcom

SPE Bed WeightTube Size Selection

sigma-aldrichcom

SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

sigma-aldrichcom

Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

sigma-aldrichcom

A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

sigma-aldrichcom

Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

sigma-aldrichcom

How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

sigma-aldrichcom

The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

sigma-aldrichcom

Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

sigma-aldrichcom

What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

sigma-aldrichcom

Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

sigma-aldrichcom

ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

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WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

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SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

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Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

sigma-aldrichcom

WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

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Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

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Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

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Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

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Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

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Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

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Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

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Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

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NEW Developments in Sample Prep

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SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

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SupelMIP Binding Site

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Extraction of ClenbuterolBeta-Agonists

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Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

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Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

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96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

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96-well MD Plate Generic Protocols

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Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

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Representative GC-MS C-gram for Pesticides in Orange

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Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

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New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

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Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

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Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips

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Normal-Phase SPE

sigma-aldrichcom

Example NP SPE Protocol1 Sample Pre-Treatment

bull Liq samples extracteddiluted with non-polar solvent (eg hexane DCM)bull Solid samples (soil sediment etc) extracted (soxhlet sonnication etc)

with non-polar solvent and concentratedbull Dry solvent extract with Na-sulfate or Ma-sulfate

bull Residual moisture can greatly affect analyte retention

2 Condition amp Equilibrate w 1-2 tube volumes non-polar solvent3 Load sample (consistent rate 1-2 drops per second)

bull Sample should not be in MeCN or MeOH

4 Wash sorbent (elutes co-retained interferences)bull Use a more polar solvent but not so polar as to elute analytes of interestbull Fractionation common in NP SPE

5 Elute analytes of interest with polar solventbull MeOH MeCN Acetone IPA are common

6 Evaporatereconstitute as necessary

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Common NP Solvents

sigma-aldrichcom

Ion-Exchange SPE

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Example IOX SPE Protocol1 Sample Pre-Treatment

bull Basic compounds dilute w 10-25mM buffer (eg potassium phosphate ammonium acetate) pH 3-6

bull Acidic compounds dilute with 10-25mM buffer (eg acetate) pH 7-9bull BOTH sorbent functional group amp analyte most be ionized

2 Condition amp Equilibratebull Condition with 1-2 tube volumes MeOH or MeCNbull Equilibrate with 1-2 tube volumes buffer (used during sample pre-treatment

3 Load sample (consistent rate 1-2 drops per second)4 Wash sorbent (elutes co-retained interferences)

bull Wash interferences with bufferbull Wash with 100 MeOH to remove hydrophobic interferences

5 Elute analytes of interestbull Adjust pH opposite to load conditions (eg 2-5 ammon hydroxide for

basic compounds)bull May require organic modifier (50-100 MeOH)

6 Evaporatereconstitute as necessary

sigma-aldrichcom

Useful IOX SPE Tips

bull IOX kinetics slower than RP amp NP =gt reduce flow ratebull Strong vs weak ion-exchangers

- Strong = sorbent functional group always ionized regardless of pH- Weak = sorbent functional group has controllable pKa

commonly used for extracting strong analytes

bull Counter-Ion Selectivity in IOX

sigma-aldrichcom

SPE Method Development ndashSPE Selection amp The Role of pH

sigma-aldrichcom

Critical Questions to Ask

bull What are requirements and goals to method development

bull What is known about the sample (sample matrix analyte Log Pow pKa(s) functional groups)

bull What investment in MD time can be made

bull Any known information from previous work with similar analytes

sigma-aldrichcom

Best practices for method development

bull Historically MD = hit or miss experiments in which many random variables evaluated at the same time

bull Results in user not knowing why a set of conditions worked or what type of leeway can be associated with operator variation changes in pH etc

bull Problems can easily arise during method transfer

bull Best method developers isolate one variable at a time and use 96-well technology to evaluate multiple variables in parallel

sigma-aldrichcom

Key to Successful SPE

bull Choose the appropriate SPE phase by understanding the sample matrix and identifying analyte(s) functional groups that influence its solubility polarity etc

bull Understand how the analyte(s) behaves on the sorbentin response to changing extraction conditions

bull Manipulate these conditions to meet the defined sample prep objectives

sigma-aldrichcom

Consider the analyte(s) of interest

What functional groups may influence the analytesrsquo solubility polarity ionization state (pKa) etc

Hydrophilic GroupsbullHydroxyl -OHbullAmino -NH2bullCarboxyl -COOHbullAmido -CONH2bullGuanidino -NH(C=NH)NH3

+

bull4deg Amine -NR3+

bullSulfate -SO3-

Hydrophobic GroupsbullCarbon-Carbon -C-CbullCarbon-Hydrogen -C-HbullCarbon-Halogen -C-ClbullOlefin -C=C

bullAromatic

Neutral GroupsbullCarbonyl -C=ObullEther -O-RbullNitrile -C=N

sigma-aldrichcom

Retention Mechanism Quick Look-Up

Reversed-Phase

Compoundssmall moleculesmoderately polar to non-polar

Sample matrixAqueous sampleseg biological fluids (urine plasma serum) water buffer

Ion-Exchange

CompoundsCharged ionizable compounds

Sample matrixAqueous or organic samplesLow salt concentration

Normal-Phase

CompoundsPolar to moderately non-polar compounds

Sample matrixOrganic samplesextractseg Hexane ethyl acetate dichloromethane

DPA-6S - Polyamide phase (nylon)-acid) Good for extracting

good for working with plant extracts (chlorophyl tannins humic phenolic compounds (multiple ndashOH groups) from aqueous samples

nt for extracting polar compounds from aqueous samples

superior selectivity for extracting basic compounds from biological

ENVI-Carb- Graphitized carbon excelle

DSC-MCAX- mixed-cation phase for fluids

sigma-aldrichcom

SPE Phase Selection

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry

Reversed-Phasebull C18 (18C)

- Less risk of silanol activity for predictable extractions- Broad affinity for a wide range of compounds- Potential use of stronger wash solvents- Greater risk of co-retention of matrix interferences- Extract many analytes with generic methodology

bull CN (Cyanopropyl 6C)- Weaker affinity to compounds- May retain compounds more selectivity than C18- Weaker wash solvents are required- Could yield weaker elution solvents- Could elute with smaller elution volumes- Increased risk of silanol activity (may not be bad though)

bull C8 (9C)- retains compounds with log Pow ge 1

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Ion-Exchangebull SCX amp SAX (strong ion exchange)

- Can be very selective- Elution typically done via pH manipulation to neutralize analytes- Always some mixed-mode properties (requires a combination of pH adjustment

and organic strength to elute compounds)

bull WCX amp NH2 (weak ion exchange)- Used for extracting strong bases and acids where elution cannot be done

through pH manipulation of analytes- Instead pH adjustment used to neutralize sorbent functional groups

sigma-aldrichcom

Choosing the Appropriate Phase Chemistry (cont)

Mixed-Mode SPEbull Dual mechanisms of attraction

- Reversed-phase + ion-exchange = broad affinity for a wide range of compounds

- Many compounds contain ionizable functional groups- Combination of hydrophobic and strong electrostatic

interactions allows researcher to use vigorous wash steps- Results in more selective extractions

sigma-aldrichcom

SPE Bed WeightTube Size Selection

sigma-aldrichcom

SPE is form of Chromatography

Analyte(s) of Interest

Solid Phase Chemistry

Mobile Phase Environment

sigma-aldrichcom

Ionization of Acidic amp Basic MoleculesAcids (eg carboxylic acids) (eg R-COOH R-COO-)

HA H+ + A-

(Un-ionized) (ionized)50 pKa 50

100 low pH 00 high pH 100

Bases (eg amines) (eg R-NH3+ R-NH2)

BH+ + OH- B

(Ionized) (Un-ionized)

50 pKa 50100 low pH 0

0 high pH 100

The Critical Role of pH in SPENeutral State (Blue) = promotes hydrophobic (RP) interaction

Ionized State (Green) = promotes electrostatic (IOX) interaction

pKa of most acids (eg ndashCOOH) is 3-5

bull Presence of halogen atom near a carboxy group strengthens acid effect (electron sink)

bull eg acetic acid (pKa 475) monochloro acetic acid (pKa 285) dichloroacetic acid (pKa 148)

pKa of most amines is 8-11

bull Aromatic (electron sink) amines have a lower pKa than aliphatic amines

bull eg Aromatic amines- aniline (pKa 46) pyridine (pKa 52) Aliphatic amines- (pKa97) dimethylamine (pKa 107)

sigma-aldrichcom

00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Hydrocortisone

WashElute Profile- Neutral Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

WashElute Profile- Basic Compounds

Alprenolol

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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00

200

400

600

800

1000

1200

1400

0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100 0 5 10 20 40 60 80 100

MeOH

Rec

over

y (

)

MeOH in 2 CH3COOH MeOH in DI H2O MeOH in 2 NH4OH

Ibuprofen

WashElute Profile- Acidic Compounds

DSC-18 SPE DSC-8 SPE DSC-CN SPE

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A Systematic Approach to SPE Method Development ndash Case study examples in

pharma bioanalysis

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Specific Challenges for Bioanalyses

bull Determine analyte concentrations in biological fluids- Data used to understand time course of drug action and

pharmacokinetics of an in-vivo system- Data used to support epidemiological studies- Requires efficientadequate sample preparation good

chromatographic separation and sensitive detection techniquebull Difficulties in analyzing biological samples

- Many sample matrices encountered (eg plasma urine tissues etc)- Difficult Sample Matrices =gt Selectivity is Key- Must separate drugs metabolites andor other small molecules from

endogenous matrix interferences

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How are most SPE methods developed

Incorporate the sample matrix or real samples immediately andhellip

bull Choose a very generic or robust methodbull Duplicate an existingsimilar application from a previous

methodbull Copy an existing application from an SPE vendor or

literature referencebull Go to the local SPE ldquogururdquo for help

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The Problem with these approacheshellip

Example of Problems

bull Dealing with novel analytes

bull Poor Recovery Is it due tohellip- Poor retention- Pre-mature elution- Over retention

bull Poor Reproducibility Typically caused by one or more inadequate steps Which one

bull Insufficient clean-up Stronger wash solvent Maybe a different SPE phase

More often investigator will have more questions than answersrarr Leads to a Non-Systematic Approach to method development and optimization amp Variable MD Time

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Systematic SPE Method Development

Phase amp Hardware Selection

ExperimentationbullLoad OptimizationbullWash Elute Profile

Evaluation

Incorporate Sample MatrixTroubleshoot Method

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What is Systematic SPE MD all about

bull Maximizing Selectivity- The ability of the sorbent and extraction method to discriminate

between the analyte(s) of interest and endogenous interferences within the sample matrix

bull By employing two or three expts using standard solutions wo sample matrix

- systematically adjust the two main variables that control selectivity(pH amp organic strength)

bull Benefits- understand how the analytes interact with the sorbent under

specific conditions- allows for a systematic approach to finding the optimal sample prep

conditions with greater efficiency and confidence

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Example Tricyclic Antidepressants from Sheep Serum

O

CHCH2CH2NCH3

CH3

N

CH2CH2CH2NCH3

CH3CHCH2CH2N

CH3

CH3

Doxepin Imipramine Amitrypityline

Consider the Analytes of Interest

sigma-aldrichcom

bull Different ionic forms retain differently on a given sorbent bull pH manipulation can control retention and selectivity on a given

sorbent - At pH ge 11 the 2deg or 3deg amine functional group should be neutralized - At pH le 7 the amine group should be ionized

Dibenzocycloheptene skeleton = excellent hydrophobic foot print for potential reversed-phase interaction

2deg amine basic functional group w apKa of ~9 Very useful for controllinganalytersquos ionization state

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

Load Optimization- Ensures retention of the analytes of interest

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at

neutral (DI H2O) and basic pH (1 NH4OH)

4 Collect Load eluate and analyze via HPLC-UV

Note load concentration was increased to provide adequate signal response for detecting small analyte breakthrough percentages Also note that acidic load conditions were avoided

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Example Tricyclic Antidepressants from Sheep Serum

Load Optimization Evaluation bull A lack of analyte presence in the load eluate was found for

both pH conditionsrarr Indicates adequate retention for both neutral and basic load conditions

bull Basic pH was chosen to ensure maximum retention for the three basic analytes

bull Stronger retention permits the potential use of stronger wash solvents increasing overall sample clean-up

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile- Determine analyte retention and elution patterns as a function of pH amp Organic

1 Conditions DSC-18 wells with 1mL MeOH2 Equilibrate DSC-18 wells with 1mL DI H2O3 Load 1mL 5microgmL standard test mix prepared at basic

pH (1 NH4OH)4 WashElute with 1mL of a test solvent ranging from 0-

100 MeOH in 2 NH4OH (high pH) DI H2O and 2 CH3COOH (low pH)

5 Collect washelute eluate and analyze via HPLC-UV

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

WashElute Profile Evaluation-

At low pH complete elution occurs at 60MeOH

At neutral pH complete elution occurs at 80MeOH

Under basic pH complete elution occurs at 80MeOH

At low pH retention limit is 10 MeOH

At neutral pH retention limit is 20 MeOH

Under high pH retention limit is 40 MeOH

Low pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

Neutral pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

High pH WashElute Profile

0 20 40 60 80 100

Methanol

Peak

Are

a (m

AU

s)

sigma-aldrichcom

Example Tricyclic Antidepressants from Sheep Serum

bull Incorporate Sample MatrixTroubleshoot Method-- Obtain specific guidelines for defining optimizing and troubleshooting the extraction method

- For most applications recovery values observed for real-matrix based solutions will parallel values obtained with standard solutions

sigma-aldrichcom

Systematically Developed Method on DSC-18 SPE-96 Well Plate (100mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 40 MeOH in 2 NH4OH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Note 60 acidified MeOH may have been a potential elution eluant

Generic Method on Competitor Polymeric Phase (30mgwell)

1 ConditionEquilibrate w 1mL MeOH amp 1mL DI H2O

2 Load 025-20microgmL TCAs spiked in sheep serum diluted in 2 NH4OH (11 vv) n=3 for ea concentration

3 Wash w 1mL 5 MeOH4 Elute w 1mL MeOH5 Evaporate eluate with N-purge (30degC

~10min) and reconstitute in 300microL MP

Systematic Method on DSC-18 Well Plate vsGeneric Method on Polymer Phase

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Results

0 2 4 6 8 10Time (min)

0 2 4 6 8 10Time (min)

Ext Stds

1microgmL Spiked serum

Blank serum

Ext Stds

1microgmL spiked serum

Blank serum

High Background Misleading interfering responses

POS Method Using DSC-18 SPE-96 Well plate

Generic Method Using Competitor Polymeric Well Plate

HPLC MethodColumn Discovery C18 15cmx46mm 5microm

amp 2cm guard column amp 05microm frit filter Mobile Phase MeCN 25mM KH2PO4 pH 7 (4555)Flow Rate 14mLmin Temp 30degC Det UV 254nm Inj 100microL

1 Doxepin2 Imipramine3 Amitryptyline

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ResultsEfficiency of Absolute Recovery of Tricyclic Antidepressants on POS MethodUsing Discovery DSC-18 SPE Vs Generic Method Using Competitor PolymerPhase

Compound Concentration Recovery plusmn RSD(n=3) on Discovery

DSC-18

Recovery plusmn RSD (n=3)on Competitor Polymer

Phase1 Doxepin 10microgmL

05microgmL025microgmL

908 plusmn 12911 plusmn 16892 plusmn 22

1088 plusmn 821276 plusmn 1351678 plusmn 32

2 Impipramine 10microgmL05microgmL025microgmL

955 plusmn 25977 plusmn 06978 plusmn 37

884 plusmn 56982plusmn 147931plusmn 03

3 Amitryptyline 10microgmL05microgmL025microgmL

910 plusmn 20874 plusmn 14895 plusmn 35

924 plusmn 511049 plusmn 1261335 plusmn 14

sigma-aldrichcom

Summarybull In this study through systematic SPE method development we

were able to1 optimize the wash solvent to maximize sample clean-up

resulting in minimal background and more accurate resultsndash Determination of 40 MeOH in 2NH4OH as wash solventndash 60 MeOH in 2 CH3COOH

2 achieve high and reproducible recoveries at the spike levels tested (gt 90 recovery le 4 RSD)

3 High background observed on generic method on polymeric SPE well plate

Example Tricyclic Antidepressants from Sheep Serum

sigma-aldrichcom

Example Furosemide from Horse Serum

Consider the Analytes of Interest

Furosemide Indapamide (IS)

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Load OptimizationSPE Discovery DSC-18 SPE 96-well 50mgwell

1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Furosemide from Horse Serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL furosemide and

indapamide in 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via

HPLC-UV

sigma-aldrichcom

WashElute Profile for Furosemide amp Indapamide (IS) on Discovery DSC-18 SPE

00

200

400

600

800

1000

1200

1400

0 20 40 60 80 100

Methanol in Wash

R

ecov

ery

Recovery Furosemide

Recovery Indapamide

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SPE Discovery DSC-18 SPE 50mg1mL1 Condition with 1mL methanol 2 Equilibrate with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)3 Load 1mL sample4 Wash with 1mL 10mM KH2PO4 pH 3 (adjusted with H3PO4)5 Elute with 1mL 60 methanol in DI H2O6 Directly analyze eluate (no evaporationreconstitution) via HPLC-

UV 7 Determine relative recovery and RSD against working calibration

standards not subjected to SPE sample preparation

Systematically Developed SPE Method For Furosemide amp Indapamide from Serum

sigma-aldrichcom

0 2 4Time (min)

3683 Furosemide

4586 Indapamide (IS)

5microgmL ext std

5microgmL spike

05microgmL spike

005microgmL spikeSerum blank

HPLC-UV Conditions

Example Chromatograms of Extracts Generated from the Systematically Developed Method on Discovery DSC-18 SPE

Column Discovery C18 15cm x 46mm ID 5microm particles

Mobile Phase 10mM KH2PO4 pH 3 (adjusted with H3PO4)MeCN (6040)

Flow Rate 10mLminTemperature 35degCDetection UV 234nmInjection 1microL

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Relative Recovery of Furosemide from Horse Serum Using Discovery DSC-18 SPE

Sample Furosemide Spike Concentration (microgmL)

Avg Response Factor

Recovery plusmn RSD (n=3)

A 1000 2307 993 plusmn 31B 500 1168 1008 plusmn 14C 050 0107 974 plusmn 28D 010 0065 1207 plusmn 13E 005 0009 1328 plusmn 83

sigma-aldrichcom

Summarybull Furosemide not strongly retained on C18 SPE under neutral conditionsbull Wash solvents stronger than DI water caused compound elutionbull Selectivity improved by eluting with weaker elution solvent (eg 60

MeOH)bull Weaker elution solvent = direct injection of the final eluate bull Average relative recovery and RSD = 1002 plusmn 34bull Note that the procedure is quantitative down to 05microgmL serum bull Below this level reasonable precision is evident but accuracy suffersbull Primarily to the detection limitations of UV absorbance for furosemide

Flourescence detection or mass spectrometry is likely to provide increased sensitivity

bull Decreasing SPE elution volume may also be a viable choice for improving sensitivity

Example Furosemide from Horse Serum

sigma-aldrichcom

Example Corticosteroids from Urine

Hydrocortisone Prednisilone

CorticosteronePredinisone

sigma-aldrichcom

Load OptimizationSPE Conventional C18 96-well SPE (100mgwell)

Discovery DSC-CN SPE 96-well (100mgwell)1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp

DI H2O2 Load 1mL standards containing 50microgmL of each of the four

corticosteroids in DI H2O3 Collect load flow-through eluate amp analyze for compound break

through via HPLC-UV

Example Corticosteroids from Urine

WashElute Profile 1 Condition amp equilibrate SPE wells (C18 amp CN) with 1mL methanol amp DI H2O2 Load 1mL standards containing 50microgmL of each of the four corticosteroids

in DI H2O3 Washelute with 1mL test solvents ranging from 0-100 methanol4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

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HPLC-UV ConditionsColumn Discovery HS F5 5cm x 46mm

ID 3microm particlesMobile Phase MethanolDI H2O (4060)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 5microL

Example Corticosteroids from Urine

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WashElute Profile for Corticosteroids on C18 amp CN SPE

Convential C18 WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

Methanol

Pea

k A

rea

(mA

Us

)

hydrocortisone

prednisilone

prednisone

corticosterone

Discovery DSC-CN WashElute Profile

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 90 100

MethanolP

eak

Are

a (m

AU

s)

hydrocortisone

prednisilone

prednisone

corticosterone

For CN Full elutionat 100 MeOH

For C18 Full elutionat 100 MeOH

Increased hydrophobic retention observed on C18 relative to CN

For CN Retention Limit is 20 MeOH

For C18 Retention Limit is 20 MeOH

sigma-aldrichcom

SPE Conventional C18 96-well SPE (100mgwell)Discovery DSC-CN SPE 96-well (100mgwell)

1 Condition amp equilibrate with 1mL methanol and 1mL DI H2O2 Load 05 amp 10microgmL corticosteroids spiked in human urine

diluted in DI H2O (11 vv) n=33 Wash with 1mL 20 methanol 4 Elute with 1mL 100 methanol5 Evaporate eluate with nitrogen purge (30degC ~10 min) and

reconstitute in 200microL HPLC mobile phase

Systematically Developed SPE Method For Steroids From Urine

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Chromatograms of Blank amp Spiked Urine Extracts Generated on C18 amp CN SPE

10ugmL spiked urine extract on DSC-CN

Blank urine extract on DSC-CN0 2 4 6 8 10 12

Time (min)

Blank urine extract on conventional C18

High level of interferences caused increased backpressure resulting in HPLC system failure early in run sequence

0 2 4 6 8 10 12Time (min)

Blank urine extract on DSC-CN

Blank urine extracts on C18 amp CN SPE

Blank amp spiked urine extracts on CN SPE

sigma-aldrichcom

Recovery of Steroidal Compounds from Urine on Discovery DSC-CN SPE

Absolute Recovery plusmn RSD (n=3) Compound 05ugmL spike

level 10ugmL spike

level 1 Hydrocortisone 1233 plusmn 14 959 plusmn 17 2 Prednisilone 1072 plusmn 11 919 plusmn 11 3 Prednisone 1032 plusmn 10 884 plusmn 18 4 Corticosterone 1020 plusmn 12 931 plusmn 56

sigma-aldrichcom

Summarybull Under identical SPE protocols C18 SPE eluate carried a yellow tint

=gt lead to system failure due to high back pressurebull Stronger wash solvents required but stronger wash solvents will lead

to premature analyte elutionbull In contrast improved selectivity was observed on DSC-CNbull Chromatograms were free of interfering components bull On DSC-CN SPE avg absolute recovery and RSD was 1006 plusmn

19 bull Recovery values for C18 SPE were not obtained due to HPLC

system failure caused by insufficient sample clean-up

Example Corticosteroids from Urine

sigma-aldrichcom

NH

O

NH

O

CH3

CH2

O

CH3

Secobarbital

O

O

OH

CH3

Ketoprofen

O

OH

O

CH3

CH3

Naproxen

NHCH3

Nortriptyline

NCH3

CH3

Amitriptylin

Example Five pharmaceutical compounds from human urine

sigma-aldrichcom

Table 1 Fractionation Protocol Used for Discovery DSC-MCAX SPE 1g6mL1

Condition6mL methanol6mL 10mM acetic acid pH 3

Load1mL spiked urine sample Urine samples diluted 11 with 10mM potassium phosphate pH 3 prior to loading

bullLow sample pH neutralizes all acidic compounds inducing reversed-phase retentionbullLow sample pH ionizes basic compounds inducing ionic-exchange retention

WashWash with 6mL 10mM acetic acid pH 3The low pH aqueous wash solvent removes all non-basic

hydrophilic interferences and also locks basic compounds onto the sorbent reinforcing both ionic and reversed-phase retention

EluteElute basic compounds with 6mL 1TEA in methanol or 5 ammonium hydroxide in methanolTheincrease in elution pH neutralizes basic compounds disrupting ionic interactions between compound andsorbent Favorable hydrophobic interactions disrupted via the presence of methanol

Eluate EvaporationEluate fractions were evaporated to dryness at 37degC under N2-purge and reconstituted with 1mL mobile phase

sigma-aldrichcom

0 2 4 6Time (min)

Fraction 1 Neutral amp Acidic Compounds

Fraction 2 Basic Compounds

Spiked Urine Samples before DSC-MCAX SPE

SPE Tube Discovery DSC-MCAX 1g6mLHPLC Col Discovery C8

15cm x 46mm 5microm particles

Mobile Phase 1 (Pre-SPE amp Fraction 1)01 TFAMeOH (5050)

Mobile Phase 2 (Fraction 2)10mM ammonium acetateMeOH (4555)

Flow Rate 2mLminTemp AmbientDet 214nm UVInj Vol 10microL

sigma-aldrichcom

Example Five pharmaceutical compounds from human urine using DSC-MCAX

Compound Description Recovery plusmn RSD (n=3)

Neutral 1 Secobarbital (10microgmL spike) 1058 plusmn 21

Acidic 2 Ketoprofen (50microgmL spike) 1017 plusmn 13

Acidic 3 Naproxen (25microgmL spike) 1015 plusmn 08

Basic 4 Notriptyline (50microgmL spike) 1003 plusmn 05

Basic 5 Amitriptyline (50microgmL spike) 1033 plusmn 17

sigma-aldrichcom

Example Diazepammetabolites from porcine serum

diazepam

temazepam

oxazepam

desmethyl diazepam

sigma-aldrichcom

Load OptimizationSPE Discovery DSC-8 SPE 96-well (100mgwell)

1 Prepare standards containing 25microgmL diazepam and metabolites in neutral (10mM ammonium formate pH 71) and basic (1 NH4OH) solutions

2 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O3 Load 1mL of each standard test mix (neutral and high pH)4 Collect load eluate amp analyze for compound break through via HPLC-UV

Example Diazepammetabolites from porcine serum

WashElute Profile 1 Condition amp equilibrate SPE wells with 1mL methanol amp DI H2O2 Load 1mL standards containing 25microgmL diazepam in 25mM ammonium

formate pH 713 Washelute respective wells with 1mL test solvents ranging from 0-100

methanol in 1 NH4OH pH 11 (high pH) 10mM ammonium formate pH 71 (neutral pH) and 10mM ammonium formate pH 27 (low pH)

4 Collect washelute eluate amp analyze for compound elution via HPLC-UV

sigma-aldrichcom

WashElute Profile for Diazepam amp Metabolites on Discovery DSC-8 SPE

At low pH complete elution occurs at 60 methanol

Low pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

Neutral pH WashElute Profile

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

High pH WashElute Profile

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100

Methanol

Peak

Are

a (m

AU

s)

oxazepamtemazepamdesmethyl diazepam

At low pH retention limit is 20 methanol

At neutral pH complete elution occurs at 80 methanol

At high pH complete elution occurs at 80 methanol

At neutral pH retention limit is 20 methanol

At high pH retention limit is 20 methanol

sigma-aldrichcom

SPE Methods Employed for Extracting Diazepam and Metabolites from Serum

Systematically Developed Method on C8

SPE Discovery DSC-8 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 20 methanol in 1 NH4OH pH 11

4 Elute with 1mL 60 methanol in 25mM ammonium formate pH 275

Generic Method on C18

SPE Conventional C18 SPE 96-well Plate (100mgwell)

1 Condition amp equilibrate each well with 1mL methanol amp DI H2O

2 Load 1mL 05microgmL diazepam and metabolites spiked in goat serum diluted in 10mM ammonium formate pH 71 (11 vv)

3 Wash with 1mL 5 methanol4 Elute with 1mL methanol5 Evaporate eluate with nitrogen purge

(30degC ~15 min) and reconstitute with 200microL HPLC mobile phase

sigma-aldrichcom

Example Chromatograms of Blank amp Spiked Serum Extracts Generated on C8 SPE

Ext Stds

Blank serum

0 2 4 6Time (min)

Low backgroundminimal interferences for optimal sensitivity and resolution at reduced run times

05microgmL spiked serum

Excellent Peak Shape

HPLC-UV ConditionsColumn Discovery C18

5cm x 46mm ID 5microm particlesMobile Phase Methanol10mM ammonium acetate pH 45

(4555)Flow Rate 15mLminTemperature 35degCDetection UV 240nmInjection 25microL

sigma-aldrichcom

Absolute Recovery of 05microgmL Diazepam amp Metabolites on Systematically Developed Method Using C8 vs Generic Method

on C18

Recovery plusmn RSD (n=3) Compound Developed Method

on Discovery DSC-8

Generic Method On Conventional C18

1 Oxazepam 947 plusmn 12 828 plusmn 40 2 Temazepam 999 plusmn 11 891 plusmn 40

3 Nordiazepam 942 plusmn 18 824 plusmn 50

4 Diazepam 900 plusmn 34 685 plusmn 91

sigma-aldrichcom

Summarybull Weaker eluent (60 methanol in low pH buffer) was determined for

DSC-8 allowing for direct analysis of the SPE eluate bull Provided good selectivity signified by chromatograms with low

background (Figure C) - allowed for minimal run times (6 min) resulting in faster and more

accurate resultsbull Average absolute recovery and RSD on C8 via the developed

method was 947 plusmn 19bull In contrast the generic method on C18 yielded an average absolute

recovery and RSD of 807 plusmn 53 (Table 3)

Example Diazepammetabolites from porcine serum

sigma-aldrichcom

Important SPE Tips

bull Drug Protein Binding Effects - must be disrupted during sample pre-treatment- 40microL 2 disodium EDTA per 100microL mouse plasma- 40microL 2 formic acid per 100microL mouse plasma- Other possible reagents (per 100microL matrix) 40microL 2 TCA

40microL 2 acetic acid 40microL 2 TFA 40microL 2 phosphoric acid or 200microL MeCN (protein ppt)

bull Sorbent over drying- Only critical with C18 amp only critical in first conditioning step- Phase just needs to be moist during sample addition- All other steps non-critical

sigma-aldrichcom

Important SPE Tips

Wash Stepbull Water wash step alone does not provide a clean eluate

Need some sort of organic modifier bull For IOX SPE MeOH or MeCN may be used as a wash

solventSorbent Drying prior to elutionbull Important to dry sorbent prior to elution otherwise

subsequent eluate evaporation will take a real long timeCompound volatility during evaporationbull Lower heat during evaporation or Use a keeper solvent

(eg dodecane)

sigma-aldrichcom

NEW Developments in Sample Prep

sigma-aldrichcom

SupelMIP (Molecular Imprinted Polymers) SPE

bull Highly cross-linked polymer preprsquod in presence of template molecule (analyte of interest or analog of analyte of interest)

bull Functional monomers interact with template non-covalently prior to or during polymerization

bull After polymerization using a cross linker the template is removed through exhaustive wash steps

bull Leaves specific cavities or imprints in the polymer that are chemically and sterically complementary to the template

bull Benefit = High selectivity =gt lower LLOQs when extracting analyte of interest from difficult sample matrices

sigma-aldrichcom

SupelMIP Binding Site

sigma-aldrichcom

Extraction of ClenbuterolBeta-Agonists

sigma-aldrichcom

Clenbuterol from Urine ndash SupelMIP vs Mixed-Mode

sigma-aldrichcom

Application Examples using MIP SPE

bull Beta-agonists from urine and tissue samplesbull Riboflavin (vitamin B2) from aqueous samples (milk)bull Triazine herbicides from water soil and food productsbull Chloramphenicol from biological matricesbull Beta Blockers from water and biological samplesbull NNAL (tobacco specific nitrosamines) and nicotine from

nictonine gum and biological samples

sigma-aldrichcom

96-well SPE Method Development Plates

Discovery 96-well SPE MD Plate

- Courtesy of Biotage

sigma-aldrichcom

96-well MD Plate Generic Protocols

sigma-aldrichcom

Dual-Layer SPE Products for Multi-Residue Pesticide Analysis in Foods

sigma-aldrichcom

Representative GC-MS C-gram for Pesticides in Orange

sigma-aldrichcom

Dispersive SPE Products for Multi-Residue Pesticide Analysis in Foods

1 Food initially extracted with aq miscible solvent (eg MeCN)

2 High amounts of salts (NaCl Mg-sulfate) and buffering agents added to induce phase separation and stabilize acidbase labile pesticides

3 Shakecentrifuge Isolate aliquot of sup for SPE clean-up

4 SPE done with bulk sorbents and salts not SPE tubes

Dispersive SPE Products for the ldquoQuEChERSrdquo Method

Supelco carries a line of centrifuge tubes containing pre-determined salts and sorbents to support most common method configurations used today

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Dispersive SPE Product Line

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Discovery Ag-ION SPE

Discovery Ag-Ion (silver-ion) SPE

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Discovery Ag-ION Protocol

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181 c

181 t

Fractionation of the standard FAME mixtureStandard sample total FAMEs at 1 mgml

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

6 8 10 12 14 16 18 20Time (min)

SPE fraction 4

SPE fraction 2

SPE fraction 3

SPE fraction1

Standard Mixture

160 180

181182 183

140

70 80 90 100 110 120 130 140 150 160 170 180 190 200Time (min)

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CisTrans Fractionation of FAMEs using Discovery Ag-ION SPE

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Hydrophilic Polymer SPE

bull Water wettablebull Broad affinity for a wide range of

compoundsbull Generic methodology = high

recoveriesbull Drawback = Possible poor selectivity

- Courtesy of Waters

Similar Hydrophilic PolymersCo-polymersbull Varian Focus ndash amide functionalized

PSDVBbull Phenomenex Strata X bull BiotageArgonautIST Evolutebull 3M Universal Resin

0 2 4 6 8 10Time (min)

Blank serum

High Background Misleading interfering responses

0 2 4 6 8 10Time (min)

C18

Polymer SPE

sigma-aldrichcom

New or Unique SPE Phase Chemistries (cont)Surface Driven LLE or Supported LE

- Courtesy of BiotageArgnoautIST

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Alternative SPE Formats

Reversible SPE Tubes

Rezorian SPE Cartridges

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Alt SPE Formats (cont)

bull Originally designed for proteomics applications- Peptide desaltingdetergent removal- Small volume applications (lt 10uL applictation)

bull Expanding to drugsmetabolites in biological fluidsbull Bi-directional flow amenable to some x-y-z robotic liquid handlersbull MiniTipstrade also available through Sigma-Aldrich (developed by Supelco)

ZipTip Pipette Tips- Courtesy of Millipore

Omix Tips- Courtesy of Varian Inc - Courtesy of ChromBA

BioTips