transforming sample preparation · protein precipitation(ppt), solid supported liquid extraction...

Transforming Sample Preparation

Introducing Waters Newest Solid Phase

Extraction Product

Thank you for joining us!

©2015 Waters Corporation 1

While you are waiting, please feel free to browse our library of

Webinar content: www.waters.com/meettheexperts

Thank you for joining us!

Our Webinar will begin shortly….

Click below to learn more about CORTECS, our newest Solid-Core

LC Column platform:www.waters.com/CORTECS

Review Recorded Versions of All 5 Events Here…


Events Here… www.waters.com/meettheexperts

Friendly Reminders…

� Please use text chat functionality to submit questions during the

Webinar.

� Upon conclusion, follow up information will be available:

� http://www.waters.com/Jun4

� Recorded version of today’s presentation


� PDF Copies of today’s slides

� Discount Offers on Oasis PRiME HLB Sample Preparation Products

� Product specific information

� Reference materials

Today’s Presenter – Xin Zhang

� Xin Zhang, Ph.D - Senior Research Chemist, Waters Corporation

� Today’s Webinar will be presented by Xin Zhang, Senior Research Chemist at Waters Corporation. She received her Ph.D in analytical chemistry from SUNY, Buffalo in 2007. At Waters Corporation, she has worked in Consumables R&D and the Technology


worked in Consumables R&D and the Technology Advancement Department.

� She has been focused on evaluating different sample prep techniques such as solid phase extraction(SPE), protein precipitation(PPT), solid supported liquid extraction (SSLE) as well as the Ostro Pass-through sample prepare technique. Recently, she has been working on simplified SPE procedures for the Oasis family. In addition, she also helps customers understand sample preparation by delivering educational seminars and trouble shooting.



Xin Zhang

Senior Research Chemist


When you think about doing sample preparation, would

you rather do this?

Start


End


Or this?

End


Start

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

Fewer columns need to be replaced


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 procedureCleanest extracts

“Good Enough” Sample Preparation

How Good is Good Enough?

Fast gradients are common and help increase throughput, but…

10 parent drugs 10 metabolites


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.

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.


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


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

Is it possible to solve this problem,


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.


problem, without a lot of extra work and

effort?

Agenda




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


– 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

NO

Hydrophilic-Lipophilic Balanced Copolymer


Reversed-phase retentionRetention 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

• 200 µL Methanol (MeOH)

• 200 µL H2O

Standard Simplified


Wash


Elute

• 25 µL*2 MeOH

Load

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


• 25 µL*2 MeOH

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

Oasis HLB vs. Competitor Recovery: Simplified Protocol

2030405060

708090

100

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


Oasis HLB works well with simplified 3-step (Load-Wash-Elute) protocolAverage 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.

01020

Competitor: Polymeric sorbent

All Polymeric SPE Sorbents are NOTCreated Equal

80

100

120

AZT

7 - Hydroxycoumarin

Phenacetin

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


0

20

40

60

Oasis HLB Competitor S RP Competitor SX RP Competitor P RP

Betamethasone

Protriptyline

Alprazolam

Methoxyverapamil

Terfenadine

Lower recoveries and higher variability with other sorbents


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?


performance of this simplified protocol?

Agenda





Introducing Oasis PRiME HLB


What is Oasis PRiME HLB?

PROCESS

ROBUSTNESS

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


R

improvements in…

MATRIX EFFECTS

EASE of USE

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

Oasis PRiME HLB in 3 Words

SIMPLER

FASTER


FASTER

CLEANER

Oasis PRiME HLB – What is it?

� A reversed-phase solid phase extraction device

– PATENT PENDING

�Designed to simplify solid phase extraction

– SIMPLER


– 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 -- 1.1Anti-inflammatory and immunosuppressive


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

8

9

3-Step Protocol Example: Chromatogram

1. AZT (Azidothymidine)

2. Propranolol

3. 7-Hydroxycoumarin

4. Phenacetin

5. Protriptyline


12

3

4 7

5. Protriptyline

6. Amitriptyline

7. Betamethasone

8. Alprazolam

9. Naproxen

5 6

3-Step Protocol Example: Recovery and Matrix Effects

40

60

80

100

120

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


-20

0

20

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

LOW (<15%) matrix effects

3-Step Protocol Example: Excellent Reproducibility

40

60

80

100

120

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


0

20

40

3-Step Protocol CONCLUSIONReproducible 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


Retained

Rinse – Collect

Analytes Pass Through

Unretained

Does it work?

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

0

20

40

60

80

100

120

140

Cim

ate

rol

Cle

nbute

rol

Racto

pam

ine

Salb

uta

mol

Terb

uta

line

Tulo

bute

rol

Zilpate

rol

Clindam

ycin

Ery

thro

mycin

kitasam

ycin

Lin

com

ycin

Spiram

ycin

Tilm

icosin

Tylo

sin

Sulfachlo

rpyridazin

e

Sulfaclo

zin

e

Sulfadim

eth

oxin

e

Sulfaguanid

ine

Sulfam

era

zin

e

sulfam

ete

r

Sulfam

eth

azin

e

sulfam

eth

izole

Sulfam

eth

oxypyridazi…

sulfanilaceta

mid

e

sulfaphenazole

Sulfapyridin

e

sulfis

om

idin

e

Trim

eth

oprim

Cin

oxacin

Cip

rofloxacin

Danofloxacin

Diflo

xacin

Enoxacin

Enro

floxacin

Flu

mequin

e

Lom

efloxacin

Marb

ofloxacin

Nalidix

ic a

cid

Norf

loxacin

Ofloxacin

Orb

iflo

xacin

Oxolinic

acid

Pefloxacin

Sara

floxacin

Chlo

ram

phenic

ol

florf

enic

ol

Thia

mphenic

ol

penic

illin V

Beta

meth

asone

Cort

isone

Dexam

eth

asone

Hydro

cort

isone

Mepre

dnis

one

Meth

ylp

rednis

olo

ne

Pre

dnis

olo

ne

Triam

cin

olo

ne

Triam

cin

olo

ne

…

Cefo

taxim

e

Ceft

iofu

r

cephapirin


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

Cim

ate

rol

Cle

nbute

rol

Racto

pam

ine

Salb

uta

mol

Terb

uta

line

Tulo

bute

rol

Zilpate

rol

Clindam

ycin

Ery

thro

mycin

kitasam

ycin

Lin

com

ycin

Spiram

ycin

Tilm

icosin

Tylo

sin

Sulfachlo

rpyridazin

e

Sulfaclo

zin

e

Sulfadim

eth

oxin

e

Sulfaguanid

ine

Sulfam

era

zin

e

sulfam

ete

r

Sulfam

eth

azin

e

sulfam

eth

izole

Sulfam

eth

oxypyridazi

sulfanilaceta

mid

e

sulfaphenazole

Sulfapyridin

e

sulfis

om

idin

e

Trim

eth

oprim

Cin

oxacin

Cip

rofloxacin

Danofloxacin

Diflo

xacin

Enoxacin

Enro

floxacin

Flu

mequin

e

Lom

efloxacin

Marb

ofloxacin

Nalidix

ic a

cid

Norf

loxacin

Ofloxacin

Orb

iflo

xacin

Oxolinic

acid

Pefloxacin

Sara

floxacin

Chlo

ram

phenic

ol

florf

enic

ol

Thia

mphenic

ol

penic

illin V

Beta

meth

asone

Cort

isone

Dexam

eth

asone

Hydro

cort

isone

Mepre

dnis

one

Meth

ylp

rednis

olo

ne

Pre

dnis

olo

ne

Triam

cin

olo

ne

Triam

cin

olo

ne

Cefo

taxim

e

Ceft

iofu

r

cephapirin



� Designed to simplify solid phase extraction

– SIMPLER

– FASTER

o Faster flows though the device


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

Matrix Device Format Oasis PRiME HLB

Speed Increase

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


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

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)

Competitor SPE


13 min 30 sec

Condition:

MeOH (30 sec)

SSLE


28 – 33 min

sample, initiate Load sample, initiate (3 min)

Wait (5 – 10 min)


(4 min)

Wash:

5% MeOH

(3.5 min)

Elute:

90/10

Acetonitrile/MeOH

(2 min)

Equilibrate:

Water (2 min)

Load:

Pre-Treated Sample

(4.5 min)

Wash:

5% MeOH (4 min)

Elute:

90/10

Acetonitrile / MeOH

(2.5 min)

Wait (5 – 10 min)

extraction solvent Add extraction solvent (2 min)

Wait (5 – 10 min)

Extract (1 min)

Evaporate (10 – 15 min)

Reconstitute (2 min )



� Designed to simplify solid phase extraction

– SIMPLER

– FASTER

– EVEN CLEANER

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


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 versusCompetitors and PPT in Plasma

Phospholipids Remaining in Final Eluate

Are

a C

ounts


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

CLEANER samples in fewer steps with Oasis PRiME HLB

Oasis PRiME HLB Competitor SX Competitor SOPPT 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


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

CLEANER - Pork ACN ExtractPhospholipids Remaining after Pass-Through

%

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

1.87e84.58

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


Oasis PRiME HLB

Time1.00 2.00 3.00 4.00 5.00 6.00

%

0

1.00 2.00 3.00 4.00 5.00 6.000

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

1.87e8

4.33

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

Load:

Pre-Treated Sample

Wash:

5% MeOH

3-Step Catch and Release


� 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

Elute:

90/10

Acetonitrile/MeOH

Load – Collect

Matrix Interferences

Retained

Rinse – Collect

Analytes Pass Through

Unretained

2-Step Pass-Through

Agenda






– Endogenous Steroids in Plasma

– Synthetic Cannabinoids in Whole Blood

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

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’


ModifiedOasis PRiME Protocol

� 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

RecoveryMatrix

Effects

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

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

Oasis PRiME HLB vs. PPT:Recovery and Matrix Effects

20%

40%

60%

80%Endogenous Steroids in Plasma


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%

-40%

-20%

0%

20%

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.

Absolute average matrix effect is 10%

Oasis PRiME HLB vs. PPT Phospholipid Removal

%

100

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


Total PL Area

PPT 22152370

PRiME HLB 690579

Oasis PRiME HLB superior performance:

Removal of 97% of phospholipids vs. PPT

Time2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.000

2.52

2.81

PPT

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. CompoundMol.

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


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

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50

%

0

100

Chromatogram with CORTECS C181) AM 22232) RCS4, M103) RCS-4, M114) AM 12485) 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-01512) RCS-4

3

21

4

5

6

7

89, 10


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

100

12) RCS-413) JWH-07314) JWH-02215) XLR-1116) JWH-20317) JWH-01818) RCS-819) UR-14420) JWH-21021) AB 00122) AKB 48

13,14

11,12 21

1516

17 18

20

19

22

Synthetic Cannabinoids in Whole Blood

� 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


RCF: relative centrifugal force

ModifiedOasis PRiME Protocol

– 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

-20

0

20

40

60

80

100

120

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


-80

-60

-40

Excellent recoveries obtained with an average RSD of 5%

Absolute average matrix effect: 17%

What would this synthetic cannabinoid


What would this synthetic cannabinoid

application look like on other SPE sorbents?

Synthetic Cannabinoids in Whole Blood - Procedures

Oasis PRiME HLB All other SPE devices

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


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

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

STX


0

10mg Plate, N=5% Recovery

RangeAverage % Recovery

% RSD Range

Average % RSD

Oasis PRiME HLB90-110%

(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 11Comparative separations may not be representative of all applications.

-40

-20

0

20

40

60

80

100Oasis 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

EVA

STX

PLX

Why is there so much ion suppression?


-100

-80

-60

-40

� 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)


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

Evolute ABN 10mg

%

100

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

1.60e7

Phospholipid 524

� JWH-203 (1-pentyl-3-(2-

chlorophenylacetyl)indole) is a

synthethic cannabinoid

� JWH-203 coelutes with phospholipid

524 (Lysophosphatidylcholine) at 5.74

minutes


Time5.40 5.60 5.80 6.00 6.20

%

0

100

5.40 5.60 5.80 6.00 6.200

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

4.51e6

JWH-203

JWH-203

Lysophosphatidylcholine

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

%

100

5.60 5.80 6.00 6.20

%

0

1002.51e7

Lyso-PL (524.4 > 184.4)

%

100

5.60 5.80 6.00 6.20

%

0

100

JWH-203 (340.2 > 125)

2.06e6

PLX

Oasis PRiME HLB ME = -11%


Time5.60 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

Time5.60 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

EVA

STX

PLX ME = -83%

ME = -88%

ME = -75%


Matrix Effects Linked to Phospholipids

0

50

100

Phospholipids Matrix Effect


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

-100

-50

Oasis PRiME HLB PLX STX EVA


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

0

50

100

Recovery Matrix Effect


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

Oasis PRiME HLB

10mg 3 step

Bond Elut Plexa

RP 10mg 5 step

Strata X RP 10mg

5 step

Evolute ABN

10mg 5 stepPLX STX EVA


Agenda






� Conclusions

Oasis PRiME HLB will result in…

PROCESS (no condition and equilibration steps/faster

flows/less clogging)

ROBUSTNESS (removes variability due to sample

matrix)


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

Low UV


Phospholipid Removal Test

Protein Removal Test

Solvent Gradient

Endogenous Steroids in Plasma

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

100

0.50 1.00 1.50 2.00

%

0

1000.70

1.49

Cortisol


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

Time0.50 1.00 1.50 2.00

%

0

100

0.50 1.00 1.50 2.00

%

0

1.54

Androstenedione

17α-OH progesterone

Synthetic Cannabinoids in Whole Blood - Conditions

Conditions ACQUITY UPLC I-Class/MS

Column ACQUITY UPLC CORTECS C18, 2.1 x 100 mm, 1.6 µm

Column Temperature 30°C

Sample Temperature 10°C

Detection MS: Xevo TQ-D

Injection Volume 5 µL/10 µL loop

Run Time (Flow Rate) 8.0 min (0.6 mL/min)


Run Time (Flow Rate) 8.0 min (0.6 mL/min)

Mobile Phase A Water with 0.1% formic acid

Mobile Phase B ACN with 0.1% formic acid

Strong wash solvent 40:40:20 ACN:IPA:Water

Weak wash solvent 10% ACN

Seal wash 50% MeOH

Thank You for Attending!

� Post-Event Home Page: http://www.waters.com/Jun4

� Introductory Offer on Oasis PRiME HLB Products

– Buy 1 Get 1 Free *

– Full Webinar Recording of Today’s Session w/PDF Slide

Deck

– Compilation of TODAY’S KEY Literature, Brochures etc…


– Compilation of TODAY’S KEY Literature, Brochures etc…

� For Questions and to Submit your Ideas for our Next Topic

– Please eMail - [email protected]

*(Limit 3 Free Boxes)

transforming sample preparation · protein precipitation(ppt), solid supported liquid extraction...

Documents