Fit for Purpose Sample Preparation in Bioanalysisand the ...Introduction / Speaker Bio Erin E....

Fit for Purpose Sample Preparation in

Bioanalysis and the Role of

Phospholipid Removal Plates

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©2012 Waters Corporation 1

Phospholipid Removal Plates

October 9th, 2012

Erin E. Chambers, Principal Applications Chemist

Waters Corporation, Milford, MA USA

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Introduction / Speaker BioIntroduction / Speaker Bio

Erin E. Chambers, Principal Applications Chemist,

Waters Corporation, Milford, MA USA


Erin Chambers is a Principal Applications Chemist, providing

pharmaceutical applications development and support for Waters

Applied Technology Group. Erin's primary role is to support regulated

and discovery Bioanalysis, and develop bioanalytical methods for drug

molecules, both large and small. She is responsible for sample

preparation, mass spectrometry, and LC method development, and

also provides customer and in-house training on these topics. Her most

recent focus has been on peptide and protein bioanalysis as well as the

role of phospholipid removal plates in bioanalysis.

Our Approach to Sample Prep Choice in Our Approach to Sample Prep Choice in BioanalyticalBioanalytical Method DevelopmentMethod Development

� No “one size fits all”

� Different segments of drug development process

– Scientific and business drivers may be different

– Drivers may be the same but with varying degrees of risk

tolerance

� Use of scientifically appropriate criteria for final method


� Use of scientifically appropriate criteria for final method

choice

Segments Within BioanalysisSegments Within Bioanalysis

Discovery(Candidate Selection)

>Thousands of cmpds

>Average sensitivity

>Clear/Clean extract

>Easy to use

Development(Pre-clinical)

>Several cmpds

>Better sensitivity

>Clean/Pure extract

>Method development

Late Phase(Phase 1-3 Clinical)

>Targeted cmpds

>Highest sensitivity

>Pure extract

> Reliable and


>Easy to use

>Reliable

>Non-regulated

>Method development

>Reliable and inter/intra reproduc.

>Regulated

> Reliable and inter/intra reproducibility

>Regulated

Sometimes “methods” linked

“Methods” always linked

Leads to different testing patterns

Why Do Sample Prep?Why Do Sample Prep?Challenges in Bioanalytical MethodsChallenges in Bioanalytical Methods

� Robustness– Method must work equally well for spiked and incurred samples

– Must pass ISR

– Methods must transfer successfully across labs, analysts, matrix sources…

� Reproducibility

– Data quality must be sufficient to meet inter- and intra-day accuracy and

precision regulatory guidelines

o Must not exceed ±15%, except at LLOQ where it must not exceed ±20%


o Must not exceed ±15%, except at LLOQ where it must not exceed ±20%

– Must pass SOP-defined ISR criteria

� Speed

– 96-well format

– Validate more assays per unit time

– Higher quality information sooner for critical decisions

– Increase productivity

– Decreased time to market

Why Do Sample Prep? Why Do Sample Prep? Challenges in Challenges in BioanalyticalBioanalytical MethodsMethods

� Sensitivity– Need high sensitivity data to meet increasingly low detection limits

– Large molecule therapeutics or biomarkers

– Better S:N at lower levels improves consistency and reliability of quantitation

– Accurate representation of elimination time points

– Accommodate alternate routes of administration which yield lower plasma drug concentrations


plasma drug concentrations

o Intra-nasal

� Selectivity– Must be selective for analyte in multiple sources of complex biological matrices to meet matrix effects guidelines

– Analytes must be adequately separated from metabolites

– Historically heavy reliance on MS for selectivity

o New regulations require additional means of ensuring selectivity

Matrix Effects: A Major Concern Matrix Effects: A Major Concern

� Residual matrix components alter MS response

– Ion suppression (loss of signal)

– Ion enhancement (gain in signal)

� Phospholipids identified as a major source of

matrix effects in plasma

– Other plasma constituents (salts, proteins), dosing media,

formulation agents, mobile phase modifiers can also contribute


formulation agents, mobile phase modifiers can also contribute

– Other matrices, other interferences

� Difficult to predict and control

– Plasma/matrix variability

o Inter-subject variability (diet, protein levels, etc.)

o Species variability

o Dependent on concentration of lipids, specific proteins

– Extremely analyte specific

– Can be compounded by co-eluting analytes

Goals of Sample PreparationGoals of Sample Preparation

Minimize risk

� Minimize matrix effects

– Reduction of ion suppression/enhancement, interferences, background

� Eliminate sample to sample variability

– More reproducible quantitation

– More robust assays

– i.e., Plasma from different subjects or species

� Decrease assay variability


� Decrease assay variability

– Pass ISR

– Successful transfer across labs, analysts, sites

Increased sensitivity

� Sample concentration

� Removal of interferences

Cleaner Samples

� Increased instrument uptime

� Improved method robustness

Key Attributes of Key Attributes of Sample Preparation TechniquesSample Preparation Techniques


Different Methods for Different Different Methods for Different Purposes: Decision Making ProcessPurposes: Decision Making Process


Typical ProcessTypical Process

1. The simplest method which meets the assay needs is usually chosen• PPT or LLE are common starting points

2. For very challenging assays (low detection limits, closely related endogenous constituents, inhalation products, peptides, etc) SPE may be first choice


peptides, etc) SPE may be first choice

3. Exact technique chosen will depend on outcome of study and how much risk can be tolerated

In this seminar:

Multiple methods run to compare and contrast technique differences

Development of Bioanalytical AssaysDevelopment of Bioanalytical Assays


Example 1: Challenging AssayExample 1: Challenging AssayPeptides in PlasmaPeptides in Plasma

DesmopressinMW 1069pI = 8.6

Assay Use


Assay UseRegulated analysis of patient samples

Assay Requirements

� LLOQ 1-5 pg/mL

� Highest selectivity method possible

� Free from closely related endogenous interferences

� Challenging detection limits

� Need to concentrate without evaporation

Sample Preparation ComparisonSample Preparation Comparison

% Analyte Recovery % Matrix Effects

-10

0

10

20

30

40

PPT LLE RP SPE MM SPEDesmopressin

40

60

80

100

Desmopressin


-50

-40

-30

-20

1. Adequate recovery?

0

20

40

PPT LLE RP SPE MM SPE



-10

0

10

20

30

40


40

60

80

100

Desmopressin


-50

-40

-30

-20


0

20

40




-10

0

10

20

30

40


40

60

80

100

Desmopressin


-50

-40

-30

-20


2. Highest selectivity?

3. Minimizes matrix effects?

0

20

40




-10

0

10

20

30

40


40

60

80

100

Desmopressin


-50

-40

-30

-20


2. Highest selectivity

3. Minimizes matrix effects

0

20

40


Choice is mixed-mode SPE

Oasis SolidOasis Solid--Phase Extraction (SPE)Phase Extraction (SPE)


SPE Format:SPE Format:Enabling Technology for Peptide ExtractionEnabling Technology for Peptide Extraction

Oasis µElution plate technology

� Up to 15X concentration without evaporation

–Concentration often necessary to reach LOD’s with peptides

� No evaporation, analyte loss

–Thermally unstable peptides

–Peptides sticking to walls of collection plates


–Peptides sticking to walls of collection plates

� Speed

– 96-well plate in <30 min, <20 seconds/sample

PST peptide method development platesSmall molecule method development plates

%

100 411.2 > 191.22.17e6

Why Oasis µElution Format?Why Oasis µElution Format?Sample Enrichment: Up to a 15X ConcentrationSample Enrichment: Up to a 15X Concentration

0.5 ng/mL risperidone

MCX µElution plate

15X concentration


Time0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40

%

0

100

0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.400

411.2 > 191.22.17e6MCX 10 mg plate

No concentration

Representative Validation Results: Representative Validation Results: Oasis WCXOasis WCX

Baseline Magnification Factor 5x

Baseline Magnification Factor 25x

Compound name: Desmopressin (1)Correlation coefficient: r = 0.999622, r^2 = 0.999244

Calibration curve: 1.5469 * x + 0.000270265Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Re

sp

on

se

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

1pg/mL in plasma

5pg/mL in plasma

1 pg/mL

%

100

0.50 1.00 1.50 2.00

%

0

100 x251.04765

x5


Factor 5x

Sample Name Std. Conc Area IS Area Calc. Conc. %Dev

Blank human plasma 2.024 20334 0.0003

0.001 ng/mL 0.001 5.015 17062 0.0010 2.7

0.002 ng/mL 0.002 9.138 17886 0.0018 -9

0.005 ng/mL 0.005 22.187 16283 0.0049 -1.4

0.01 ng/mL 0.01 45.187 17035 0.0096 -3.6

0.02 ng/mL 0.02 113.447 17912 0.0231 15.4

0.05 ng/mL 0.05 240.559 18804 0.0467 -6.6

0.1 ng/mL 0.1 490.062 18654 0.0959 -4.1

1 ng/mL 1 4365.578 15747 1.0125 1.3

5 ng/mL 5 30420.492 20869 5.3239 6.5

10 ng/mL 10 48969.102 17701 10.1042 1

20 ng/mL 20 104231.141 19458 19.5643 -2.2

Desmopressin 1Desmopressin 1--10000pg/mL10000pg/mL

plasma

Blank

Time0.50 1.00 1.50 2.00

%

0

100

0.50 1.00 1.50 2.000

1.04138

1.050.83

Example 2: Routine Small Molecule AssayExample 2: Routine Small Molecule AssayTamsulosinTamsulosin and and DoxazosinDoxazosin in Plasmain Plasma

NHO

OCH3CH3

S

OO

NH2

TamsulosinMW 408.5

pKa = 8.1 and 10.2

DoxazosinMW 451.5pKa = 7

O

ON

N

N

N

O

O

CH3

CH3

H

NH2


Assay UseHigh throughput, discovery setting

Assay Requirements

� LLOQ 50-100 pg/mL

� Simplest sample preparation

� No time for method development

� Non-regulated though may be transferred to development setting

at later stage

O

O



20

30

40

50

60

Tamsulosin

Doxazosin40

50

60

70

80

90

100

Tamsulosin

Doxazosin


-20

-10

0

10

20

PPT PLR Plate LLE RP SPE MM SPE

Doxazosin

1. Simplest sample prep

2. No method development

0

10

20

30

40


Doxazosin

Benefits of PPT Plates Benefits of PPT Plates for this Applicationfor this Application

� If PPT works, it will be used

– Simple, generic, inexpensive, fastest workflow

– Dilute and inject

– Similar recovery for analyte and IS

� May opt for a PL removal plate for maximum uptime


Sirocco™ for 96Sirocco™ for 96--wellwellProtein PrecipitationProtein Precipitation


� Fast, easy in-well protein precipitation

� Clean filtrates

� No extractables from plate

� No cross-talk

� No leaking, no plugging

Representative Validation Results: Representative Validation Results: SiroccoSirocco

Compound name: Tamsulosin

Correlation coefficient: r = 0.994265, r^2 = 0.988563

Calibration curve: 0.195307 * x + 0.00616236

Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )

Curve type: Linear, Origin: Exclude, Weighting: 1/x̂ 2, Axis trans: None

Conc0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

Re

sp

on

se

0.0

50.0

Conc

Re

sid

ua

l

-10.0

0.0

10.0

Blank plasma

50 pg/mL Tamsulosin

in extracted plasma

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

2

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

2

MRM of 5 Channels ES+ 409.2 > 228 (Tamsulosin)

3.60e31.55

0.85

0.650.50

1.38

1.09

2.721.722.542.101.84

1.99 2.362.272.97

MRM of 5 Channels ES+ 409.2 > 228 (Tamsulosin)

3.60e3

0.81

0.71

1.571.40

1.08

1.47

1.69

2.672.161.79 2.03 2.52

2.27 2.39 2.762.90


Standard Tamsulosin IS Calc. conc.

conc. ng/mL Area Area Response ng/mL %Dev

Standard 0.05 145.54 9403.865 0.015 0.048 -4.6

Standard 0.1 233.068 8513.663 0.027 0.109 8.6

Standard 0.5 808.595 7329.04 0.11 0.533 6.7

Standard 1 1317.302 7247.719 0.182 0.899 -10.1

Standard 5 7259.513 6729.309 1.079 5.492 9.8

Standard 10 15302.424 7057.208 2.168 11.071 10.7

Standard 50 73504.031 7314.083 10.05 51.424 2.8

Standard 100 130508.695 7231.187 18.048 92.377 -7.6

Standard 500 545718.563 6679.502 81.7 418.287 -16.3

QC 0.075 122.762 6281.676 0.02 0.069 -8.7

QC 0.25 308.741 6385.332 0.048 0.216 -13.6

QC 2.5 2530.293 6298.24 0.402 2.025 -19

QC 25 28631.146 6289.577 4.552 23.276 -6.9

QC 250 204488.063 4589.559 44.555 228.097 -8.8

Fit for Purpose?Fit for Purpose?

Assay Requirements:


� No time for method development

�


� Non-regulated though may be transferred to

development setting at later stage

� Simplest sample preparation

Example 3: MultiExample 3: Multi--matrix, API and metabolitematrix, API and metaboliteOxycodoneOxycodone, metabolite and D6 IS in Plasma, metabolite and D6 IS in Plasma

O

O

CH3

O

N

CH3

OH

OxycodoneMW 315.4pKa = 8.5

Assay Use

IS:D-6 OxycodoneMW 321.4pKa = 8.5


Assay UseRoutine analysis or screening of patient samples, GLP labAssay Requirements

� LLOQ 50 pg/mL

� Simple method

� Method must work for urine too

� Must transfer across lab with varying levels of expertise

� Metabolites and related compounds need to be cleaned up and

quantitated also


10

20

30

40

50

60

70

80

90

100

Oxycodone

Oxycodone d6

% Analyte Recovery in plasma


0


% Matrix Effects in plasma

-100

-80

-60

-40

-20

0

20

40

PPTPLR

Plate

LLE RP SPE MM SPE

Oxycodone

Oxycodone d6

Method must work for Urine too!


10

20

30

40

50

60

70

80

90

100

Oxycodone

Oxycodone d6

% Analyte Recovery in plasma


0


% Matrix Effects in plasma

-100

-80

-60

-40

-20

0

20

40

PPTPLR

Plate

LLE RP SPE MM SPE

Oxycodone

Oxycodone d6

Method must work for Urine too!

Best recovery and lowest matrix effects on reversed-phase SPE

Oasis HLB Recovery: Oasis HLB Recovery: Metabolite and Urine Matrix DataMetabolite and Urine Matrix Data

60

80

100

urine matrix

Urine Matrix Recovery

40

60

80

100

Noroxycodone Recoveryand Matrix Effects

Metabolite Recovery? Second matrix?


0

20

40

oxycodone oxycodone d6 noroxycodone

urine matrix

-40

-20

0

20

Plasma Recovery Plasma Matrix Effects

Noroxycodone

Single method works equally well for metabolite, and for both plasma and urine matrices

Benefits of RP SPE for This AssayBenefits of RP SPE for This Assay

� Negligible matrix effects using generic protocol

� Can be used for all matrices

� Can be used for related compounds and metabolites

� Samples can be directly injected

– LLOQ met without evaporation and reconstitution due to SPE

format


format

Hydrophilicmonomer

Lipophilicmonomer

NO

Hydrophilic-Lipophilic Balanced Copolymer

RP SPE:RP SPE:Oasis HLB Sorbent ChemistryOasis HLB Sorbent Chemistry


monomer monomer

Reversed-phase Retention

• Water wettable• Polar retention• Stable across pH 1-14• No silanol interactions• High recoveries for acids, bases and neutrals

Retention of Polars

Oasis HLB:Oasis HLB:Importance of WettabilityImportance of Wettability

Procainamide

Acetaminophen (polar)

Ranitidine

Propranolol% S

PE

Re

co

ve

ry

C18 (1cc/100mg) HLB (1cc/30mg)

40

60

80

100

40

60

80

100

Acetaminophen

(Polar)


Doxepin

Bouvier, Caparella

Silica: pore dewetting results in breakthrough at load step and poor SPE recoveries

Oasis HLB: No impact of sorbent drying

0

20

0 4 8Drying Time (minutes)

0

20

0 5 10Drying Time (minutes)

• Sorbents conditioned with MeOH • Allowed to dry for allotted times• Equilibrated with water • Analytes applied

Representative Validation Results: Representative Validation Results: Oasis HLBOasis HLB

Compound name: Oxycodone




Curve type: Linear, Origin: Exclude, Weighting: 1/x̂ 2, Axis trans: None

ng/mL0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

Resp

on

se

0.0

20.0

40.0

60.0

ng/mL

Re

sid

ua

l

-5.0

0.0

5.0

Standard Oxycodone IS Calc. conc.


Standard 0.05 154.3 20459.4 0.008 0.047 -5

Standard 0.1 271.3 17595.4 0.015 0.110 9.6

Standard 0.5 1131.9 17532.5 0.065 0.497 -0.6

Standard 1 2440.3 18100.7 0.135 1.051 5.1

Standard 5 12431.3 18628.3 0.667 5.249 5

Standard 10 24624.5 19441.6 1.267 9.973 -0.3

Standard 50 115082.9 17982.5 6.400 50.441 0.9

Standard 100 214974.1 18094.1 11.881 93.653 -6.3

Standard 500 828731.8 14250.0 58.157 458.478 -8.3


All standards and QC samples easily meet regulatory criteria

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

MRM of 2 Channels ES+ 316.287 > 241.009 (Oxycodone)

8.93e31.62

1.241.160.75 0.88 1.071.04 1.441.35 1.46

1.652.041.79 2.16


8.93e3

1.160.740.85

0.92 1.11 1.25 1.34 1.631.541.36 1.831.81 2.00 2.242.10Time

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100


8.93e3

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100


8.93e31.62

1.241.160.75 0.88 1.071.04 1.441.35 1.46

1.652.041.79 2.16


8.93e3

1.62

1.241.160.75 0.88 1.071.04 1.441.35 1.46

1.652.041.79 2.16


8.93e3

1.160.740.85

0.92 1.11 1.25 1.34 1.631.541.36 1.831.81 2.00 2.242.10

Blank plasma

50 pg/mL oxycodone

in extracted plasma

QC 0.25 594.1 18144.6 0.033 0.246 -1.5

QC 0.75 1714.8 17840.9 0.096 0.746 -0.6

QC 7.5 17403.2 17705.8 0.983 7.737 3.2

QC 75 159931.8 16837.3 9.499 74.872 -0.2

QC 0.25 587.429 18774.037 0.031 0.245 -2

QC 0.75 1727.213 18001.096 0.096 0.777 3.6

QC 7.5 17599.27 18394.02 0.957 7.859 4.8

QC 75 159529.25 17495.1 9.119 75 0

QC 0.25 719.038 20666.336 0.035 0.266 6.3

QC 0.75 1914.771 19666.842 0.097 0.757 0.9

QC 7.5 18367.924 19519.512 0.941 7.373 -1.7

QC 75 168356.422 18418.785 9.14 71.683 -4.4


Assay Requirements:

� LLOQ 50 pg/mL

� Simple method

�


� Method must work for urine too

� Must transfer across lab with varying levels of

expertise

� Metabolites and related compounds need to be

cleaned up and quantitated also

Example 4: Regulated AnalysisExample 4: Regulated AnalysisClopidogrelClopidogrel and and TiclopidineTiclopidine (IS) in (IS) in PlasmaPlasma

O

S

N

O

CH3

Cl

ClopidogrelMW 321.8pKa = 4.5

IS TiclopidineMW 263.8pKa = 7.6 S

N

Cl


Assay Use

Routine analysis or screening of patient samples, GLP lab

Assay Requirements


� Simple method

� Must transfer across lab with varying levels of expertise

� Lab is very concerned about assay robustness and build up of

phospholipids on LC columns and in MS source


% Analyte Recovery

10

20

30

40

50

60

70

80

90

Clopidogrel

Ticlopidine


% Matrix Effects0

10


-100

-80

-60

-40

-20

0

20

40


Clopidogrel

Ticlopidine



% Analyte Recovery

10

20

30

40

50

60

70

80

90

Clopidogrel

Ticlopidine


% Matrix Effects0

10


-100

-80

-60

-40

-20

0

20

40


Clopidogrel

Ticlopidine


2. Very simple method?


% Analyte Recovery

10

20

30

40

50

60

70

80

90

Clopidogrel

Ticlopidine


% Matrix Effects0

10


-100

-80

-60

-40

-20

0

20

40


Clopidogrel

Ticlopidine


2. Very simple method?

3. Phospholipid removal?

Only the phospholipid removalPlate meets all criteria

Choice of Technique: Lipid LevelsChoice of Technique: Lipid Levels

LLE w/ 5% NH4OH

LLE%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

1001.91e8

2.161.92

1.38

1.96 2.27 2.882.782.642.582.43

1.91e82.18

1.902.33 2.82

2.622.562.45 2.72

MRM 184 -> 184


PLR plate

RP SPE

PPT

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.800

1.91e8

1.901.77

1.96

1.91e8

1.90

1.77

2.191.96

1.91e8

1.90

1.421.38

1.28

1.612.16

1.942.21

2.86

Benefits of Phospholipid Removal Benefits of Phospholipid Removal Plates for this ApplicationPlates for this Application

� Very simple protocol

– Easily transferred from lab to lab

� Removes vast majority of phospholipids

– Improved instrument uptime

– More robust methods

� High recovery


� High recovery

– Helps meet detection limits

Ostro 96Ostro 96--well Plate well Plate for Phospholipid Removalfor Phospholipid Removal

Simple Pass-through Method

Best Recovery for Diverse Analytes

(6 replicates in data set)


Simple Pass-through Method

0

10

20

30

40

50

60

70

80

90

100

N-a

cety

lpro

cain

amid

e(b

)Pro

pran

olol

(b)

Pyren

esul

foni

cAci

d (s

a)

T6692

(qa)

Ketop

rofe

n(a

)N

iflum

icAci

d (z

)Bro

maz

epam

(b)

Ostro

LLE

SSLE% Recovery

Ostro 96Ostro 96--well Plate well Plate for Phospholipid Removalfor Phospholipid Removal

6000000

8000000

10000000

12000000

14000000

16000000

18000000

70

80

90

100

70

80

90

100

Level of Phospholipids Remaining

Reproducibility of PL Removalin 6 Lots of Human Plasma


0

2000000

4000000

Ostro Competitor 1 Competitor 2 PPT

0

50000

100000

150000

200000

250000

300000

350000

400000

Ostro LLE SSLE0

50000

100000

150000

200000

250000

300000

350000

400000

Ostro LLE SSLE0

50000

100000

150000

200000

250000

300000

350000

400000

Ostro LLE SSLE

12 3

45

6

Competitor 2

Competitor 1

Ostro0

10

20

30

40

50

60

70

12 3

45

6

Competitor 2

Competitor 1

Ostro0

10

20

30

40

50

60

70

Representative Validation Results: Representative Validation Results: OstroOstro

Compound name: Clopidogrel




Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Re

sid

ua

l

0.0

5.0

10.0

Standard Clopidogrel IS Calc. conc.


Standard 0.01 157.3 122606.4 0.001 0.009 -11.8

Standard 0.05 426.5 118007.4 0.004 0.056 11.1

Standard 0.1 560.7 104434.5 0.005 0.091 -9.2

Standard 0.5 3013.1 112846.8 0.027 0.518 3.7

Standard 1 6155.2 114711.6 0.054 1.059 5.9

Standard 5 28044.7 114521.0 0.245 4.894 -2.1

Standard 10 60742.4 115491.1 0.526 10.529 5.3

Standard 50 274997.2 115583.9 2.379 47.690 -4.6

Standard 100 590079.4 116193.5 5.078 101.814 1.8


ng/mL0 10 20 30 40 50 60 70 80 90 100

Re

sp

on

se

0.00

1.00

2.00

3.00

4.00

5.00

ng/mL

Re

sid

ua

l

-10.0

-5.0

0.0Standard 100 590079.4 116193.5 5.078 101.814 1.8

QC 0.075 432.0 109556.5 0.004 0.062 -17.1

QC 0.75 4382.0 113846.2 0.038 0.755 0.7

QC 7.5 42802.8 113869.8 0.376 7.520 0.3

QC 75 447395.8 110303.7 4.056 81.314 8.4

QC 0.075 355.6 112084.7 0.003 0.047 -37.7

QC 0.75 4944.7 121039.7 0.041 0.802 7

QC 7.5 39973.1 110003.2 0.363 7.270 -3.1

QC 75 403244.8 108186.3 3.727 74.722 -0.4

QC 0.075 534.3 123902.3 0.004 0.070 -7.2

QC 0.75 5479.0 126105.7 0.043 0.854 13.9

QC 7.5 46733.5 118830.3 0.393 7.869 4.9

QC 75 317429.4 93421.1 3.398 68.116 -9.2


Assay Requirements:


� Simple method

�


� Must transfer across lab with varying levels of

expertise

� Lab is very concerned about build up of


PhospholipidPhospholipid Removal Plates for Removal Plates for Discovery ScreensDiscovery Screens

Assay UseDiscovery screening

Analytes26 compounds: benzodiazepines, metabolites and internal standards


Discovery screening

Assay Requirements

� Very high throughput

� Detection limits not challenging

� Lab is concerned about system robustness and build up of


� Direct injection to speed up workflow

� Simplest sample prep possible

—Lab technicians doing sample prep

Chromatographic ResultsChromatographic Results

1. 7-aminonitrazepam2. 7-aminoclonazepam3. 7-aminoflunitrazepam4. Clozapine5. Midazolam6. Chlordiazepoxide7. Alpha-Hydroxymidazolam8. Bromazepam9. n-Desmethylflunitrazepam10. Nitrazepam

110, 11, 12, 13

14, 15, 16, 17, 18, 19, 20, 21

100

0.80


10. Nitrazepam11. Clonazepam d412. Clonazepam13. Flunitrazepam14. Triazolam15. 2-Hydroxyethylflurazepam16. Hydroxyalprazolam d517. Alpha-Hydroxyalprazolam18. Alprazolam19. Alprazolam d520. Oxazepam21. Clobazam22. Estazolam23. Desalkylflurazepam24. Temazepam25. Nordiazepam26. Prazepam

2

3

4 6

5

7

8

9 22, 23, 24

25

26

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

Benefits of Phospholipid Removal Benefits of Phospholipid Removal Plates for this ApplicationPlates for this Application

� Very simple protocol

– Easily transferred from lab to lab

� Direct injection of eluate

– Streamlines workflow

– Answers faster


� Removes vast majority of phospholipids

– Improved instrument uptime

– More robust methods

� High recovery for analogs and metabolites

– No method development

Obvious Choice for Sample Preparation is Phospholipid Removal Plate

Recovery Using Ostro Phospholipid Recovery Using Ostro Phospholipid Removal PlateRemoval Plate

60

80

100

Average recovery = 84%


0

20

40

Tria

zola

m (T

-910)

alph

a-Hyd

roxy

mid

azola

m (H

-902

)

2-hyd

roxy

ethylf

lura

zepam

(F-9

01)

Hyd

roxy

alpr

azol

am d

5 (A

-904)

Clo

zapin

e (C-0

59)

Mid

azol

am (M

-908)

Praze

pam

(P-9

06)

alph

a-Hyd

roxy

alpr

azola

m (A

-905)

Clo

nazep

am d

4 (C

-905

)

Brom

azep

am (B

-903

)

Clo

nazep

am (C

-907)

Fluni

traze

pam

(F-9

07)

Alpra

zola

m d

5 (A-9

02)

Alpra

zola

m (A

-903)

Temaz

epam

(T-9

07)

Clo

bazam

(C-9

09)

n-D

esm

ethyl

fluni

traze

pam

(D-9

19)

Chl

ordia

zepo

xide

(C-0

22)

Estaz

olam

(E-9

01)

Des

alky

lflur

azepa

m (D

-915

)

Oxa

zepam

(O-9

02)

7-am

inoc

lonaz

epam

(A-9

15)

7-am

inof

luni

traze

pam

(A-9

12)

Nitr

azepam

(N-9

06)

Nor

diaz

epam

(N-9

05)

7-am

inon

itraz

epam

(A-9

14)

Linearity and AccuracyLinearity and Accuracy

� Standard curves were

run from 1 – 500

ng/mL

� Samples were extracted

using Ostro plate,

diluted, and directly

injected

Compound

Average %

Deviation r^2 value

Triazolam 16.1 0.940

Alpha-hydroxymidazolam 13.0 0.957

2-hydroxyethylflurazepam 8.3 0.986

Clozapine 10.3 0.974

Midazolam 14.7 0.953

Prazepam 25.2 0.907

Alpha-hydroxyalprazolam 13.0 0.964

Bromazepam 6.0 0.991

Clonazepam 12.4 0.966


injected Clonazepam 12.4 0.966

Flunitrazepam 7.7 0.991

Alprazolam 23.3 0.878

Temazepam 13.9 0.968

Clobazam 17.4 0.934

n-Desmethylflunitrazepam 12.9 0.959

Chlordiazepoxide 10.4 0.974

Estazolam 3.1 0.997

Desalkylflurazepam 9.7 0.979

Oxazepam 7.1 0.987

7-aminoflunitrazepam 7.2 0.987

Nitrazepam 16.1 0.948

Nordiazepam 6.2 0.992

7-aminonitrazepam 7.0 0.987

7-aminoclonazepam 10.4 0.978


Assay Requirements:

� Fast

� High throughput

� Simple method


� Simple method

� No method development

� Quantification is adequate

� Lab is very concerned about build up of


PhospholipidPhospholipid Removal Plates for Dried Removal Plates for Dried Blood Spot AnalysisBlood Spot Analysis

Analytes

N

CH3

O N

F N

N

N

O

CH3

OH

9-OH Risperidone MW 426.48 pKa 7.86


O N

F N

N

N

O

CH3

NH

N

N

Clozapine MW 326.82 pKa 7.6

Risperidone MW 410.48 pKa 8.24

DBS Processing: Extract Cleanliness DBS Processing: Extract Cleanliness with and without with and without OstroOstro PlatesPlates

DBS in-well Ostro

1st injection

DBS in-well Ostro

Last injection%

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100758.4 > 184.4 (Lipid 758)

4.88e6

758.4 > 184.4 (Lipid 758)4.88e6

%

100

%

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100758.4 > 184.4 (Lipid 758)

4.88e6

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100758.4 > 184.4 (Lipid 758)

4.88e6

758.4 > 184.4 (Lipid 758)4.88e6

758.4 > 184.4 (Lipid 758)4.88e6


Last injection

Traditional DBS1st injection

Traditional DBS

Last injection

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

758.4 > 184.4 (Lipid 758)4.88e6

2.20

758.4 > 184.4 (Lipid 758)4.88e6

2.212.16

3.37Time

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

%

0

758.4 > 184.4 (Lipid 758)4.88e6

758.4 > 184.4 (Lipid 758)4.88e6

2.20

758.4 > 184.4 (Lipid 758)4.88e6

2.20

758.4 > 184.4 (Lipid 758)4.88e6

2.212.16

3.37

OstroOstro for DBS Analysisfor DBS Analysis

100

0.50 1.00 1.50 2.00 2.50 3.00

%

0

100 427.1 > 109.8 (9-OH Risperidone)4.71e4

2.232.041.871.29

0.54

0.300.030.78 0.81 1.16

1.851.68

1.58

2.30

3.442.39

2.522.63 3.292.95 3.14

427.1 > 109.85 (9-OH Risperidone)4.71e4

1.30984

Blank Whole Blood Spot

Whole Blood Spiked at 50


Time0.50 1.00 1.50 2.00 2.50 3.00

%

0

Whole Blood Spiked at 50 pg/mL with 9-OH risperidone

Name Type

Std.

Conc Area

IS

Area Response Conc. %Dev

50 pg/mL Standard 0.05 945 37691 0.025 0.05 4.0

100 pg/mL Standard 0.10 1146 37531 0.031 0.10 -4.5

250 pg/mL Standard 0.25 1682 35798 0.047 0.23 -9.3

500 pg/mL Standard 0.50 3392 40329 0.084 0.52 4.5

1 ng/mL Standard 1.00 5114 39457 0.130 0.88 -11.5

5 ng/mL Standard 5.00 28760 38637 0.744 5.78 15.7

10 ng/mL Standard 10.00 57317 42923 1.335 10.49 4.9

25 ng/mL Standard 25.00 156098 51934 3.006 23.80 -4.8

50 ng/mL Standard 50.00 289427 45519 6.358 50.51 1.0

75 pg/mL QC QC 0.075 1467 51641 0.028 0.08 4.8

750 pg/mL QC QC 0.75 4473 44994 0.099 0.64 -14.1

7.5 ng/mL QC QC 7.50 48110 49944 0.963 7.53 0.4

15 ng/mL QC QC 15.00 96139 42200 2.278 18.00 20.0

Benefits of Benefits of PhospholipidPhospholipid Removal Removal Plates for DBSPlates for DBS

� In-well spot extraction

� Cleaner Extract than traditional DBS

� Simple method


� No method development

� Accurate quantification

� Eliminates phospholipids

PhospholipidPhospholipid Removal Plates for MIST Removal Plates for MIST AssessmentAssessment

� FDA recommends that the safety of drug metabolites be

evaluated if they are present in human plasma at

concentrations greater that 10% of the parent drug (or API) at

steady state

– Relies on accurate, reliable quantification

� 10 parent/metabolite pairs were spiked into human plasma and


extracted using the generic Ostro protocol

– Metabolites spiked at 10 and 30% of parent concentration

� Ratios calculated and compared to expected

� Matrix effects assessed

� Phospholipid accumulation assessed

PhospholipidPhospholipid Removal Plates for Removal Plates for MIST Analysis: MIST Analysis: AnalytesAnalytes

100

11: MRM of 1 Channel ES+

4.12e7

1

1. Risperidone2. Clozapine3. Bupropion4. Quetiapine5. Midazolam6. Fentanyl

7. Amitriptyline8.Terfenadine9.Testosterone

1. Norfentanyl2. OH-Bupropion3. Cloidogrel COOH

4. N-desmethyl Clozapine5. OH-Risperidone6. Norquetiapine7. α-OH Midazolam8. Fexofenadine9. Nortriptyline


Time0.80 1.00 1.20 1.40 1.60

%

0

2,3

4 6

10

7

8

9.Testosterone10. Clopidogrel

9

5

9. Nortriptyline10. DHT

Elution Profile for Phospholipids in Elution Profile for Phospholipids in PPT SamplesPPT Samples

A

Time0.50 1.00 1.50 2.00

%

0

100

1: MRM of 6 Channels ES+ TIC

1.42e8

2.471.751.67 1.92 1.96

2.01

First PPTinjection


Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25

%

0

100

1: MRM of 6 Channels ES+ TIC

3.22e8Risperidone

Clopidogrel

B

0.50 1.00 1.50 2.00

Last PPTinjection

PhospholipidPhospholipid Accumulation with and Accumulation with and without without OstroOstro

1.00E+08

1.20E+08

1.40E+08

Summed PL Area

Total PL Area


0.00E+00

2.00E+07

4.00E+07

6.00E+07

8.00E+07

0 10 20 30 40 50 60

Summed PL Area

Injection

PPT

Ostro

10%

12%

14%

16%

Calculated Ratio @ 10%

Measured response ratios for metabolitesspiked at 10% vs. 100% of parent drug concentrations

Red line = Expected response ratio

Accurate Assessment of Ratios When Accurate Assessment of Ratios When Metabolites Present at 10% of ParentMetabolites Present at 10% of Parent


0%

2%

4%

6%

8%

10%


Ostro Sample Preparation

Accurate Assessment of Ratios When Accurate Assessment of Ratios When Metabolites Present at 30% of ParentMetabolites Present at 30% of Parent

25%

30%

35%

40%


Measured response ratios for metabolitesspiked at 30% vs. 100% of parent drug concentrations

Red line = Expected response ratio


0%

5%

10%

15%

20%


Ostro Sample Preparation

Matrix Effects for Matrix Effects for OstroOstro versus PPT versus PPT Prepared SamplesPrepared Samples

-20.0%

0.0%

20.0%

40.0%

60.0%

80.0%

Ostro

PPT-40.0%

-20.0%

0.0%

20.0%

40.0%

Ostro

PPT

Parent Compounds Metabolites


-80.0%

-60.0%

-40.0%

PPT

* Paired t-tests reveal statistically significant differences between Ostro and PPT prepared samples

-80.0%

-60.0%

-40.0%PPT

Benefits of Benefits of PhospholipidPhospholipid Removal Plates Removal Plates for MIST Assessment Experimentsfor MIST Assessment Experiments

� Simple, generic protocol

� High recovery for parent compounds and metabolites

� Accurate quantification of metabolite to parent ratio


� Reduction of matrix effects

� Eliminates phospholipids

PhospholipidPhospholipid Removal Plates for Removal Plates for LipidomicsLipidomics

� Reseach on cellular lipid molecular species and interaction with

other lipids, proteins and metabolites is critical to understanding

mechanisms of disease

� Typical Bligh-Dyer extraction time consuming and uses a lot of

environmentally unfriendly solvent

� Ostro plates can be used for both selective enrichment of


� Ostro plates can be used for both selective enrichment of

specific classes of phospholipids and/or for total phospholipid

extraction and quantitiation

� Modified version of Ostro protocol allows both flow through and

elution enrichment of lipids

Efficiency of Lipid Extraction: Efficiency of Lipid Extraction: OstroOstroversus Traditional Bligh and Dyerversus Traditional Bligh and Dyer

Ostro Plate Compared to Traditional Met hods of P hosp holipid Quantitation

Lipid Class

Number of Lipids Detected Extraction Efficiency

of Ostro vs BD

BD Ostro E Ostro FT Ostro E Ostro FT

SM 17 17 0 1.00 0.00

L PC 9 9 0 1.00 0.00

PC 47 47 0 1.01 0.00

Cer 13 3 13 0.03 0.95

PG 15 5 15 0.06 1.37

L PE 10 6 10 0.07 0.60


L PE 10 6 10 0.07 0.60

PE 28 23 28 0.14 0.99

L PI 0 4 5 n/c n/c

PI 22 18 22 0.16 1.14

KeyBD: Bligh and Dyer method 1

Ostro E: Eluate fraction from the Ostro PlateOstro FT: Flow Through fraction from the Ostro Plate

Reproducibility of Lipid Extraction Reproducibility of Lipid Extraction using using OstroOstro


Benefits of Benefits of PhospholipidPhospholipid Removal Plates Removal Plates for for LipidomicsLipidomics ExperimentsExperiments

� Simple protocol

� Equally or more efficient than traditional lipid

extraction methods

� Reproducible lipid extraction


� Reproducible lipid extraction

� Enriches specific lipid classes or total lipids

� Faster and uses less solvent than traditional methods

ConclusionsConclusions

� Methods are not one-size fits all, choice is based on assay

need

� New sample prep techniques provide more options for the

bioanalytical chemist and their fit-for-purpose was

assessed

� Mixed-mode SPE facilitates routine achievement of low


� Mixed-mode SPE facilitates routine achievement of low

pg/mL LLOQ’s for challenging assays such as peptide

quantification

� Phospholipid removal plates provide a significantly

simpler, cleaner, and faster option for routine bioanalysis,

DBS, MIST assessments, and lipidomics research

� Joe Arsenault

� Erin Chambers

� Jonathan Danaceau

� Diane Diehl

� Kenneth Fountain

� Greg Lawless

AcknowledgementsAcknowledgements

� Gary Mantha

� John Martin

� Joanne Mather

� Rob Plumb

� Paul Rainville

� Jessalynn Wheaton


� Greg Lawless

Thank You! Thank You!

� Questions?

� Post Session Landing Page…www.waters.com/Fit

– Promotional Offers

– PDF Slide Deck

– Full Webinar Recording of Today’s Session

– Compilation of Literature, White Papers, Brochures


– Compilation of Literature, White Papers, Brochures

� General Questions – eMail: [email protected]

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