Xin Zhang Senior Research Chemist - Waters Corporation€¦ · ©2015 Waters Corporation 1 Transforming Sample Preparation . Xin Zhang . Senior Research Chemist

©2015 Waters Corporation 1

Transforming Sample Preparation

Xin Zhang Senior Research Chemist



When you think about doing sample preparation, would you rather do this?

Start

End



Or this?

Sta

rt

End


Agenda

The importance of sample preparation and industry trends

Simplifying Sample Preparation Introducing Oasis PRiME HLB

Applications and comparisons Conclusions


Why Do Sample Preparation?

Need to remove matrix interferences – Increase signal to noise by simplifying the chromatographic separation

o Enables better, more consistent quantitation – Reduce variability in analytical results due to matrix inconsistencies

o Higher, more consistent recovery o Minimize matrix effects o Less rework

– Increase column lifetime o Fewer columns need to be replaced

– Reduces system downtime o Less time spent with wrenches or waiting for service

Need to concentrate analyte of interest

– Present in low levels = difficult to quantitate

Need to transfer (extract) analytes of interest into a solution that can be tested – Meat, fish and milk

But How?


How can I turn this?

Into this?

Sample Preparation


Sample Preparation Techniques: Which One?

How do you choose a technique to clean up complex sample matrices? – Filtration / Dilution – Protein precipitation (PPT) – Liquid-liquid extraction (LLE) – Solid-supported liquid-liquid extraction (SSLE) – Solid-phase extraction (SPE)

Objectives: Simplest technique

Fastest preparation procedure Cleanest extracts

“Good Enough” Sample Preparation


How Good is Good Enough?

Fast gradients are common and help increase throughput, but…

It is important to be able to accurately quantify drugs and metabolites during the discovery stage and preclinical assessments of candidate drugs (eg. for MIST assessment). The method needs to be fast, robust and applicable to diverse drug candidates and metabolites.

10 parent drugs

10 metabolites


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, illustrating co-elution. This may cause matrix effects. Also 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

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


Phospholipid Build Up Can Lead to Matrix Effects and Unpredictable Results

Using protein precipitation, remaining phospholipids build up on the column during the processing of one analytical batch. The bottom chromatogram shows the earliest and latest eluting parent compounds superimposed over the phospholipid trace, illustrating co-elution. This may cause matrix effects. Also 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

Is it possible to solve this problem,

without a lot of extra work and

effort?


Agenda


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 • 200 µL 5% MeOH in H2O

Elute • 25 µL*2 MeOH

Load


• 200 µL Methanol (MeOH)

• 200 µL H2O

• 200 µL 5% MeOH in 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

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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

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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

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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

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A reversed-phase solid phase extraction device Designed to simplify solid phase extraction

– 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 Total processing time:

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 Total processing time:

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 )



A reversed-phase solid phase extraction device Designed to simplify solid phase extraction

– 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 90% more phospholipids than the generic protocol with Oasis HLB


CLEANER

How does it compare to other SPE products?


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 Cou

nts

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)

1.87e84.58

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

%

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100

2.00 4.00 6.00 8.00

%

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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’



Calibration Linearity and Quality Control Results from Extracted Plasma Samples

N=6 Accuracy

(ng/mL) Androstenedione

(R2=0.990, 0.05-25) 17α-OH progesterone (R2=0.993, 0.05-25)

Cortisol (R2=0.996, 1-500)

QC Level (ng/mL)

Mean S.D. %CV Mean S.D. %CV QC Level (ng/mL)

Mean S.D. %CV

0.15 94.3% 5.4% 5.7% 93.7% 6.1% 6.5% 3 92.3% 4.9% 5.4%

1.5 95.0% 3.4% 3.6% 92.3% 4.7% 5.6% 30 94.8% 2.9% 3.0%

15 95.4% 5.3% 5.5% 93.7% 6.1% 6.5% 300 94.9% 5.7% 6.0%

Mean 94.9% 92.6% Mean 94.0%

Linear responses over the entire calibration range R2 ≥0.99

All accuracies and %CVs are within 10%


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

%%

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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

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 - Recovery and Matrix Effects

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-60

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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 – Wash with 2 x 0.5 mL 25% MeOH – 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 – Wash with 2 x 0.5 mL 25% MeOH – 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

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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%

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

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

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

1002015_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

%

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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%



Matrix Effects Linked to Phospholipids

Phospholipids caused substantial ion suppression for JWH-203 with all sorbents except Oasis PRiME HLB.

-100

-50

0

50

100

Oasis PRiME HLB PLX STX EVA

Phospholipids Matrix Effect



High Recovery and Low Matrix Effects for JWH-203 with Oasis PRiME HLB

Highly reproducible recoveries were achieved with Oasis PRiME HLB compared to the other SPE devices. Phospholipids caused substantial ion suppression for JWH-203 with all sorbents except Oasis PRiME HLB.

-100

-50

0

50

100

Oasis PRiME HLB 10mg 3 step

Bond Elut Plexa RP 10mg 5 step

Strata X RP 10mg 5 step

Evolute ABN 10mg 5 step

Recovery Matrix Effect

PLX STX EVA



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, and salts)

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


Questions?


Appendix

Certificate of Analysis Additional Applications / Information


Oasis PRiME HLB Certificate of Analysis



Salt Removal Test

Phospholipid Removal Test

Protein Removal Test

Low UV




Solvent Gradient Time Flow %A %B

0.6 70 30

1.0 0.6 50 50

2.0 0.6 45 55

2.5 0.6 5 95

3.5 0.6 5 95

3.6 0.6 70 30

4.5 0.6 70 30


UPLC I-Class - FL

MPA 0.1% HCCOH in Water

MPB 0.1% HCOOH in ACN

Column HSS T3 1.8 μm; 2.1 x 50 mm

MS Xevo TQ-S

Time0.50 1.00 1.50 2.00

%

0

100

0.50 1.00 1.50 2.00

%

0

100

0.50 1.00 1.50 2.00

%

0

100 0.70

1.49

1.54

Cortisol

Androstenedione

17α-OH progesterone

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Xin Zhang Senior Research Chemist - Waters Corporation€¦ · ©2015 Waters Corporation 1 Transforming Sample Preparation . Xin Zhang . Senior Research Chemist