Transforming Sample Preparation - Waters Corporation Sample Preparation ... Competitor Sµ plate...

©2015 Waters Corporation 1

Transforming Sample Preparation

Euan Ross

Business Development Manager

Sample Preparation

European Headquarters


Agenda

The importance of sample preparation and industry trends

Simplifying Sample Preparation


History – Making SPE Simple

Since 1996, Waters goal was to simplify complex sample

preparation methods with generic sorbent & protocols

– Oasis HLB introduced in 1996 as the first water-wettable polymeric

SPE into the market

– Gold standard SPE sorbent in sample prep market for the last 18

years

– Sample preparation technique of choice for LC-MS due to cleanliness

of extracts and universal applicability.

– Well understood SPE technology

– 5 step generic protocol

Condition

Equilibrate

Wash

Elute

Load

• 200 µL Methanol (MeOH)

• 200 µL H2O

• 200 µL 5% MeOH in H2O

• 25 µL*2 MeOH

• 200 µL spiked dilute plasma (100 µL plasma + 100µL 4% H3PO4)


Oasis HLB: First Water-Wettable Sorbent

Hydrophilic

monomer

Lipophilic

monomer

N O

Reversed-phase retention

Hydrophilic-Lipophilic Balanced Copolymer

Retention of polar compounds

C18 silica-based sorbents and non water-wettable polymeric sorbents dewet if allowed to dry

Oasis sorbents do not exhibit this undesirable behavior


Oasis uElution Protocol Comparison: Standard vs. Simplified

Condition

Equilibrate

Wash


Elute

• 25 µL*2 MeOH

Load


• 200 µL Methanol (MeOH)

• 200 µL H2O


• 25 µL*2 MeOH


Standard Simplified


Oasis HLB vs. Competitor Recovery: Simplified Protocol

Oasis HLB works well with simplified 3-step (Load-Wash-Elute) protocol Average 3 Step Recoveries = 83±1.7% (Average 5 Step Recoveries = 90±4.5%)

Competitor Sµ plate does not work with simplified 3-step (Load-Wash-Elute)

protocol Comparative separations may not be representative of all applications.

0102030405060708090

100

µElution plate 3 step SOLAµ plate 3 stepCompetitor Sµ plate 3 step

Competitor: Polymeric sorbent


All Polymeric SPE Sorbents are NOT Created Equal

0

20

40

60

80

100

120

Oasis HLB Competitor S RP Competitor SX RP Competitor P RP

AZT

7 - Hydroxycoumarin

Phenacetin

Betamethasone

Protriptyline

Alprazolam

Methoxyverapamil

Terfenadine

Oasis AVG Recovery: 100% Oasis AVG RSD: 1%

Lower recoveries and higher variability with other sorbents

Comparative separations may not be representative of all applications.


History – Making SPE Simple

Mixed-mode ion exchangers

– Simplify the complex sample preparation methods to generic

procedures

– Simplified mixed mode ion exchange sample prep with 2 protocols x

4 sorbents

Will these work with a simplified protocol?

(no condition and equilibration)


Yes… 720005290EN


Moving Towards Easier Sample Preparation Techniques - Summary

Oasis HLB Simplified Protocol

Is it possible to enhance and improve the

performance of this simplified protocol?


Agenda



Introducing Oasis PRiME HLB


What is Oasis PRiME HLB?

PROCESS

ROBUSTNESS

improvements in…

MATRIX EFFECTS

EASE of USE

We’ll come back to this after we explain how…

What’s in a name (what does PRiME mean)?


Oasis PRiME HLB in 3 Words

SIMPLER

FASTER

CLEANER


Oasis PRiME HLB – What is it?

A reversed-phase solid phase extraction device

– PATENT PENDING

Designed to simplify solid phase extraction

– SIMPLER

o Easy protocols that result in cleaner samples

o No condition and equilibration steps are required

o Easy to implement into laboratory workflows without SPE

expertise


SIMPLER – In 3 Steps

3 Step Protocol – no SPE expertise required

Load:

Pre-Treated Sample

Wash:

5% MeOH

Elute:

90/10

Acetonitrile/MeOH

Does it work?

Wash and elution steps can be adjusted

if desired


3-Step Protocol Example: Test Analyte Properties

Name pKa Log P Comments

1B Azidothymidine (AZT) 9.68 0.05 Antiretroviral drug for HIV/AIDS

2B 7-Hydroxycoumarin 7.8 1.6 Gradient in sunscreen, absorb UV

3A Phenacetin 2.2 1.6 Pain, fever reducer

4N Betamethasone -- 1.1 Anti-inflammatory and

immunosuppressive

5B Protriptyline 8.2 4.4 Antidepressant

6A Alprazolam 2.4 4.9 Panic and anxiety disorders

7B Amitriptyline 9.7 4.8 Antidepressant

8A Naproxen 4.2 3.2 Pain, fever reducer

9B Propranolol 9.5 2.5 Hypertension

Drug Panel Mixture: Highly variable hydrophobicities, wide pKa

range and Log Ps


1 2

3

4 7

8

9

3-Step Protocol Example: Chromatogram

1. AZT (Azidothymidine)

2. Propranolol

3. 7-Hydroxycoumarin

4. Phenacetin

5. Protriptyline

6. Amitriptyline

7. Betamethasone

8. Alprazolam

9. Naproxen

5 6


3-Step Protocol Example: Recovery and Matrix Effects

-20

0

20

40

60

80

100

120

Luckycat 1cc plasma 500-500 Luckycat 1cc MERecovery Matrix Effects

3-Step Protocol CONCLUSION HIGH analyte recoveries (>80%) and

LOW (<15%) matrix effects


3-Step Protocol Example: Excellent Reproducibility

0

20

40

60

80

100

120

Batch #1 Batch #2 Batch #3 Batch #4 Batch #5

3-Step Protocol CONCLUSION Reproducible recoveries for all acidic, basic and neutral

compounds batch to batch, with an average recovery of 87%


SIMPLER – In 2 Steps

2 Step Protocol – Ideal for high organic samples, like meat or

milk extracts

Load – Collect

Matrix Interferences

Retained

Rinse – Collect

Analytes Pass Through

Unretained

Does it work?


Recovery of Multi-residue Veterinary from Milk (60 compounds in 9 drug classes)

One single method replaces 9 separate methods!!! Excellent recoveries ranging from 70% to 120% with precision (RSD) < 20% (n=5) for all compounds (Average recovery 91%, %RSD @ 6 (n=5)) Recovery values are a subject to the initial milk extraction efficiency

0

20

40

60

80

100

120

140

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rol

Cle

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rol

Racto

pam

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Salb

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Terb

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Tulo

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Zilpate

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Clindam

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Ery

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Lin

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Spir

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Sulfam

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sulfam

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izole

Sulfam

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oxypyridazi…

sulfanilaceta

mid

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sulfaphenazole

Sulfapyri

din

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sulfis

om

idin

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Tri

meth

opri

m

Cin

oxacin

Cip

rofloxacin

Danofloxacin

Diflo

xacin

Enoxacin

Enro

floxacin

Flu

mequin

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Lom

efloxacin

Marb

ofloxacin

Nalidix

ic a

cid

Norf

loxacin

Ofloxacin

Orb

iflo

xacin

Oxolinic

acid

Pefloxacin

Sara

floxacin

Chlo

ram

phenic

ol

florf

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Thia

mphenic

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penic

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Beta

meth

asone

Cort

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Dexam

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Hydro

cort

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Mepre

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Meth

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– SIMPLER

– FASTER

o Faster flows though the device

o More even flows across cartridges and plates with less plugging

o Faster overall processing with the elimination of condition and

equilibration steps combined faster flows (especially important with

cartridges)


FASTER - Flows

Faster, more even sample flows across cartridges and plates

with less plugging

Loading compared to Oasis HLB with 4 inch Hg, N=4

Matrix Device Format Oasis PRiME HLB Speed Increase

1:1 Diluted Plasma µElution Plate 2 - 3X Faster

1:1 Diluted Plasma 1cc / 30mg Cartridge 4X Faster

1:1 Diluted Urine 30 mg Plate 6X Faster

1:1 Diluted Urine 10 mg Plate 2X Faster

1:1 Diluted Milk 3cc / 60 mg Cartridge 1 - 2X Faster

1:1 Diluted Milk 6cc / 200 mg Cartridge 2 - 3X Faster

FASTER flows across multiple devices and sample matrices


FASTER – Processing Time with a 96 Well Plate

Oasis PRiME HLB

Total processing time:

9 min 30 sec

Load:

Pre-treated Sample

(4 min)

Wash:

5% MeOH

(3.5 min)

Elute:

90/10

Acetonitrile/MeOH

(2 min)

Competitor SPE


13 min 30 sec

Condition:

MeOH (30 sec)

Equilibrate:

Water (2 min)

Load:

Pre-Treated Sample

(4.5 min)

Wash:

5% MeOH (4 min)

Elute:

90/10

Acetonitrile / MeOH

(2.5 min)

SSLE


28 – 33 min

Load sample, initiate (3 min)

Wait (5 – 10 min)

Add extraction solvent (2 min)

Wait (5 – 10 min)

Extract (1 min)

Evaporate (10 – 15 min)

Reconstitute (2 min )





– SIMPLER

– FASTER

– EVEN CLEANER

o Removes more than 95% of common matrix interferences, such as

salts, proteins and phospholipids, with the generic 3-step protocol

o Removes at least 70% more phospholipids than the generic

protocol with Oasis HLB


Phospholipids - A Brief Introduction

Lipids

Amphipathic, surfactant-like

property

Serve as a major structural

component of most biological

membranes

Form lipid bilayer in cell

membranes of organisms

Example: phosphatidylcholine

or lecithin

– Often found in animal and plant

tissues, such as egg yolks,

milk, soybeans, animal cells

Phosphate group

“hydrophilic head”

Fatty acids

“hydrophobic tails”


Phospholipid Build Up Can Lead to Matrix Effects and Unpredictable Results

Using protein precipitation, residual phospholipids build up on the column during the processing of one analytical batch (50 samples total) The bottom chromatogram shows the earliest and latest eluting parent compounds superimposed over the phospholipid trace. Note the elution of a significant phospholipid peak at the end of the gradient re-equilibration. This may cause matrix effects and a decrease in robustness.

Application Note: Rapid, Reliable Metabolite Ratio Evaluation for MIST Assessments in Drug Discovery and Preclinical Studies, 720004453en Danaceau et al. 2014 Bioanalysis 6(6) 761-771


CLEANER Eluates versus Competitors and PPT in Plasma

Oasis PRiME HLB 3-Step Protocol vs. Competitor 5-Step Protocol

CLEANER samples in fewer steps with Oasis PRiME HLB

Phospholipids Remaining in Final Eluate

Oasis PRiME HLB Competitor SX Competitor SO PPT 1:3 Plasma:ACN

Are

a C

ounts

n=5



CLEANER

What can this higher level of cleanliness do for your

analytical results?

Let’s look at a typical protocol for the analysis of

veterinary drug residues in meat…


CLEANER - Meat Matrices (Pork) Oasis PRiME HLB vs Silica C18

Oasis PRiME HLB (60 mg) vs. Silica C18 (100 mg)

Sample Pre-Treatment

– 5 g pork

– 10 mL of 0.2% formic acid 80:20 ACN:water

– Vortex, shake 30 min, centrifuge

Solid Phase Extraction Cleanup

– Silica C18 was conditioned first with 1 mL of 0.2 % formic acid in 80:20

ACN/water

– Oasis PRiME HLB was not conditioned

Load (Pass-Through) Step

– 0.5 mL for Oasis PRiME HLB (60 mg) vs. 1.0 mL for Silica C18 (100 mg)

– Pass through and collect

– Take 200 μL of load fraction, dilute with 250 μL of 25 mM of ammonium

formate (pH 4.5) in water and 300 μL water

– Inject 5 µL


Silica C18

Oasis PRiME HLB

CLEANER - Pork ACN Extract Phospholipids Remaining after Pass-Through

Time1.00 2.00 3.00 4.00 5.00 6.00

%

0

1.00 2.00 3.00 4.00 5.00 6.00

%

0

LC050615_3 1: MRM of 12 Channels ES+ TIC (Phospholipid)

1.87e8

4.58

4.33

LC050615_5 1: MRM of 12 Channels ES+ TIC (Phospholipid)

1.87e8

4.33

Lipid Removal from Acetonitrile-Based Meat

Extract RESULTS: Oasis PRiME HLB removes more than 90% of hexane-extractable total lipids (determined gravimetrically). Oasis PRiME HLB successfully removes both phospholipids and fats in pass through method. The silica C18 sorbent removes only fats, NOT phospholipids. Removal of both of these components results in fewer matrix effects and less column and/or instrument contamination.


Oasis PRiME HLB Summary

Oasis PRiME HLB is the next generation SPE device that sets the new performance standard for routine analyses

– Best choice for samples that contain proteins, fats, and/or lipids and can be prepared by reversed-phase ‘catch-and-release’ SPE or ‘pass-through’ SPE

SIMPLER: Streamlined protocols, no condition and equilibration steps, easy to implement into laboratory workflows without SPE expertise

FASTER: Faster flows through device, more even flows across cartridges and plates with less plugging, faster overall processing

CLEANER: Reduced matrix effects, phospholipid and fat removal in the pass-through method, less column and/or instrument contamination

Load:

Pre-Treated Sample

Wash:

5% MeOH

Elute:

90/10

Acetonitrile/MeOH

Load – Collect

Matrix Interferences

Retained

Rinse – Collect

Analytes Pass Through

Unretained

2-Step Pass-Through

3-Step Catch and Release


Agenda




Applications and comparisons

– Endogenous Steroids in Plasma

– Synthetic Cannabinoids in Whole Blood


Oasis PRiME HLB vs. Protein Precipitation (PPT): Endogenous Steroids in Plasma

Modified Oasis PRiME Protocol

Oasis PRiME HLB µElution plate, N=4

Protocol

150 μL plasma (10 ng/mL)

Precipitate with 300 μL of 4:1 MeOH:ZnSO4 (89g/L)

Spin @ 3220 rcf for 10’

Dilute supernatant with 900 μL 4% H3PO4

Load pretreated sample (2 aliquots)

Wash with 2 x 200 μL of 25% MeOH

Elute with 2 x 25 μL 90:10 ACN:MeOH

Dilute with 50 μL of 25% MeOH

Inject 7.5 μL


Recovery Matrix Effects

Mean S.D. %RSD Mean S.D.

Cortisol 72.7% 3.1% 4.2% -19.0% 3.1%

Adione 72.5% 1.9% 2.7% -6.9% 2.2%

17-OHP 71.5% 1.9% 2.6% -4.5% 1.3%

Mean -10.1%

Oasis PRiME HLB vs. PPT: Recovery and Matrix Effects

-40%

-20%

0%

20%

40%

60%

80%

Cortisol Adione 17-OHP

Recovery

Matrix Effects

Low standard deviations (3.1% or less) demonstrate the

consistency of the extraction and cleanup seen with Oasis PRiME HLB.

Endogenous Steroids in Plasma

Absolute average matrix effect is 10%


Total PL Area

PPT 22152370

PRiME HLB 690579

Oasis PRiME HLB superior performance: Removal of 97% of phospholipids vs. PPT

Oasis PRiME HLB vs. PPT Phospholipid Removal

Time2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.52

2.81

PPT

Oasis PRiME HLB

PPT Protocol:

150 μL plasma (10 ng/mL)

Precipitate with 300 μL of 4:1 MeOH:ZnSO4

Spin @ 3220 rcf for 10’

Endogenous Steroids in Plasma


Synthetic Cannabinoids in Whole Blood

RCF: relative centrifugal force

Modified Oasis PRiME Protocol

Device: Oasis PRiME HLB 30 mg plate

Replicates: 6

Protocol

– Add 100 µL spiked blood to vial

– Add 100 µL (0.1M ZnSO4/NH4CH3COO), vortex for 5 seconds

– Add 400 µL ACN, vortex for 10 seconds then centrifuge for 5 minutes

at 7000 RCF

– Take supernatant, add 1200 µL water, vortex 5 seconds to mix

– Load above solution

– Wash with 2 x 0.5 mL 25% MeOH

– Elute with 2 x 0.5 mL 90/10 ACN/MeOH

– Evaporate and reconstitute with 100 µL of 30% ACN


Synthetic Cannabinoids in Whole Blood – Complex Panel of Analytes

No. Compound Mol.

Formula

Cone Voltage

(V)

1˚MRM Transitions

Collision Energy

(eV)

1 AM2233 C22H23IN2O 40 459.2→98.05 34

2 RCS-4, M10 C20H21NO3 40 324.2→121.0 22

3 RCS-4, M11 C20H19NO3 36 322.2→121.0 22

4 AM 1248 C26H34N2O 56 391.4→135.1 28

5 JWH-073 4-COOH C23H19NO3 50 358.2→155.1 26

6 JWH-073 4-OH met. C23H21NO2 50 344.2→155.1 22

7 JWH-018 5-COOH C24H21NO3 46 372.2→155.1 24

8 JWH-073 3-OH met. C23H21NO2 44 344.2→155.1 26

9 JWH-018 5-OH met. C24H23NO2 40 358.2→155.1 24

10 JWH-018 4-OH met. C24H23NO2 40 358.2→155.1 24

11 JWH-015 C23H21NO 42 328.2→155.1 24

12 RCS-4 C21H23NO2 44 322.2→135.1 26

14 JWH-022 C24H21NO 50 340.2→155.1 26

13 JWH-073 C23H21NO 48 328.2→155.1 26

15 XLR-11 C21H28FNO 48 330.3→125.1 26

16 JWH-203 C21H22ClNO 46 340.2→125.0 26

17 JWH-018 C24H23NO 44 342.2→155.1 26

18 RCS-8 C25H29NO2 42 376.3→121.1 26

19 UR-144 C21H29NO 46 312.3→125.1 24

20 JWH-210 C26H27NO 48 370.2→183.1 26

21 AB 001 C24H31NO 52 350.3→135.1 30

22 AKB 48 C23H31N3O 38 366.3→135.1 22

Analyte concentrations: 100 ng/mL


Time

Time

0.50 1.00 1.50 2.00 2.50 3.00 3.50

4.00 4.50 5.00 5.50 6.00 6.50 7.00

%%

0

0

100

100

Chromatogram with CORTECS C18 1) AM 2223 2) RCS4, M10

3) RCS-4, M11 4) AM 1248

5) JWH-073 4-COOH met. 6) JWH-073 4-OH met.

7) JWH-018 5-COOH met. 8) JWH-073 (+/-) 3-OH

met. 9) JWH-018 5-OH met. 10)JWH-018 (+/-) 4-OH

met. 11) JWH-015 12) RCS-4 13) JWH-073 14) JWH-022 15) XLR-11 16) JWH-203 17) JWH-018 18) RCS-8 19) UR-144 20) JWH-210 21) AB 001 22) AKB 48

3

2 1

4

5

6

7

8 9, 10

13, 14

11, 12 21

15 16

17 18

20

19

22


Synthetic Cannabinoids in Whole Blood - Recovery and Matrix Effects

-80

-60

-40

-20

0

20

40

60

80

100

120

3. elute with 90/10 ACN/MeOH MERecovery ME

Excellent recoveries obtained with an average RSD of 5%

Absolute average matrix effect: 17%


What would this synthetic cannabinoid

application look like on other SPE sorbents?


Synthetic Cannabinoids in Whole Blood - Procedures

Oasis PRiME HLB

– Load pre-treated sample


– Elute with 2 x 0.5 mL 90/10

ACN/MeOH

– Evaporate and reconstitute in 100 µL

30% ACN

All other SPE devices

– Condition with 1 mL MeOH

– Equilibration with 1 mL water

– Load pre-treated sample


– Elute with 2 x 0.5 mL 90/10 ACN/MeOH

– Evaporate and reconstitute in 100 µL 30% ACN

Blood sample pre-treatment

•Add 100 µL spiked blood to vial

•Add 100 µL (0.1M ZnSO4/NH4CH3COO), vortex for 5 seconds

•Add 400 µL ACN, vortex for 10 seconds then centrifuge for 5 minutes at 7000 RCF

•Take supernatant, add 1200 µL water, vortex 5 seconds to mix

SPE procedure, N=5

Required Steps


0

20

40

60

80

100

120

Oasis PRiME HLB 10mg 3 step Evolute ABN 10mg 5 step

Strata X RP 10mg 5 step Bond Elut Plexa RP 10mg 5 step

EVA

PLX

High Recovery and Low Variability with Oasis PRiME HLB

10mg Plate, N=5 % Recovery

Range Average % Recovery

% RSD Range

Average % RSD

Oasis PRiME HLB 90-110%

(AM2233=71%) 100 3-7 4

EVA 60-97% 85 1-17 7

STX 59-92% 80 2-27 11

PLX 46-84% 73 3-41 11

STX



-100

-80

-60

-40

-20

0

20

40

60

80

100 Oasis PRiME HLB 10mg 3 step

Evolute ABN 10mg 5 step

Strata X RP 10mg 5 step

Bond Elut Plexa RP 10mg 5 step

Low Matrix Effects with Oasis PRiME HLB

Oasis PRiME HLB: Most matrix effects within 20%, maximum 43%

JWH-203 has large matrix effects with all sorbents except Oasis PRiME HLB (-11% ME)

For the last 5 compounds, large variability was observed with all competitors SPE

EVA

STX

PLX

Why is there so much ion suppression?



Ion Suppression Due to Co-Elution of Phospholipid and JWH-203

Evolute ABN 10mg

Time5.40 5.60 5.80 6.00 6.20

%

0

100

5.40 5.60 5.80 6.00 6.20

%

0

100

2015_0514_05 8: MRM of 11 Channels ES+ 524.4 > 184.4 (PL 524.4)

1.60e7

100_Cann_Test_150514_8 4: MRM of 10 Channels ES+ 340.2 > 125 (JWH-203)

4.51e6

Phospholipid 524

JWH-203

JWH-203 (1-pentyl-3-(2-

chlorophenylacetyl)indole) is a

synthethic cannabinoid

JWH-203 coelutes with phospholipid

524 (Lysophosphatidylcholine) at 5.74

minutes

JWH-203

Lysophosphatidylcholine


Ion Suppression Due to Phospholipid Co-elution with JWH-203

Time 5.60 5.80 6.00 6.20

%

0

100

5.60 5.80 6.00 6.20

%

0

100

5.60 5.80 6.00 6.20

%

0

100

5.60 5.80 6.00 6.20

%

0

100 2.51e7

Lyso-PL (524.4 > 184.4)

Time 5.60 5.80 6.00 6.20

%

0

100

5.60 5.80 6.00 6.20

%

0

100

5.60 5.80 6.00 6.20

%

0

100

5.60 5.80 6.00 6.20

%

0

100

JWH-203 (340.2 > 125)

2.06e6

EVA

STX

PLX

Oasis PRiME HLB ME = -11%

ME = -83%

ME = -88%

ME = -75%



Agenda




Applications and comparisons

Conclusions


Oasis PRiME HLB will result in…

PROCESS (no condition and equilibration steps/faster

flows/less clogging)

ROBUSTNESS (removes variability due to sample

matrix)

improvements in…

MATRIX EFFECTS (removes phospholipids, proteins, fats

and salts)

EASE of USE (simple methods, fewer steps, and faster

flows)


Questions?

[email protected]

Transforming Sample Preparation - Waters Corporation Sample Preparation ... Competitor Sµ plate...

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