Fit-for-Purpose Sample Preparation Options for Clinical Research …€¦ · Selecting the...

Fit-for-Purpose

Sample Preparation Options

for Clinical Research and Forensic Toxicology

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©2013 Waters Corporation 1

for Clinical Research and Forensic Toxicology

Nebila Idris

Senior Marketing Manager

Consumables Business Unit, Waters Corp.

For Research Use Only. Not for Use in Diagnostic Procedures.

Friendly Reminders…Friendly Reminders…

� Please use text chat functionality to submit your questions today.

� Jon Danaceau, Senior Applications Chemist, Waters Corp.

� “LIVE” Technical support during today’s event

� Upon conclusion, follow up information will be available:

� http://www.waters.com/May21

� Recorded version of today’s presentation


� Copies of today’s slides

� Product discount offers

� Product specific information

� Reference materials

� Goal of Sample Preparation

� Sample Preparation Options

– Protein Precipitation (PPT)

– Liquid-Liquid Extraction (LLE)

– Solid Phase Extraction (SPE)

� Selecting the Appropriate Sample Preparation Option

OverviewOverview



– Application Examples

o 25-OH Vitamin D2 and D3 in Serum

o Opiates (in Urine and Whole Blood)

o Benzodiazepines in Plasma

o Bath Salts in Urine

� Conclusion







OverviewOverview




� Conclusion

Where Do Samples Come From?Where Do Samples Come From?


Goal of Sample PreparationGoal of Sample Preparation

� Successful sample preparation for most analytical techniques

has a threefold objective:

– Provides the target analyte(s) in solution

– Removes interfering matrix elements

– Provides the analyte(s) at a concentration appropriate for detection

or measurement


� Having cleaner samples means:

– Better chromatography

– Lower limits of detection

– More confident analytical results

– Longer column lifetime

– Less instrument downtime

– Minimize costs in manpower and equipment maintenance

� Sample Prep makes your analytical lab more productive!

� Residual matrix components alter MS response

– Ion suppression (loss of signal) or ion enhancement

(gain in signal)

� Phospholipids are a major source of matrix

effects in biological samples

– Other matrix constituents (salts, proteins), dosing

media, formulation agents, mobile phase modifiers,

Matrix Effects: A Major ConcernMatrix Effects: A Major Concern


media, formulation agents, mobile phase modifiers,

plasticizers and release agents from labware and

blood collection devices

� Difficult to predict and control

� Can build up over time and lead to decreased

column lifetime, ion suppression, and decreased

sensitivity







OverviewOverview




� Conclusion

Sample Preparation OptionsSample Preparation Options

� Direct injection

� Protein precipitation (PPT)

� Liquid-liquid extraction (LLE)

� Solid-phase extraction (SPE)

– Reversed-phase SPE

– Mixed-mode SPE

Non-selective


– Mixed-mode SPEHighly selective

Classical Protein Precipitation (PPT)Classical Protein Precipitation (PPT)

� An organic solvent (e.g. acetonitrile) is added to the sample

matrix. Proteins are precipitated, and the precipitate is

removed by either filtration or centrifugation. The supernatant

is then analyzed.

� Pros:

– Simple & fast, minimal method development


– Simple & fast, minimal method development

– May be automated

� Cons:

– No selectivity

– “DIRTY” extracts

– Short column lifetime & frequent system shutdown

– No enrichment; may require solvent evaporation prior to

injection

Waters Solution for Waters Solution for PPT:PPT:SiroccoSirocco™™ 9696--Well PPT PlateWell PPT Plate

� Uses Filter Plate Technology

� Fast, easy in-well protein precipitation; precipitated proteins

are left behind in the wells (filters) and clean filtrates are

eluted

� Dramatically reduce the time and cost associated with

traditional PPT


traditional PPT


� Recovery


� Cleanliness


� Pros

– Precipitate-free extract; MS compatible

– Increased sample throughput; significant time

savings

– High recovery

– Suitable for limited sample volumes


– Special design to avoid cross-contamination and

leakage; no plugging

– No extractables from plate

� Cons

– No analyte enrichment

– Limited removal of matrix interferences

Liquid/Liquid Extraction (LLE)Liquid/Liquid Extraction (LLE)

� Pros:

– Removes proteins, provides some cleanup

– Easy to set up and perform when working with a few samples

� Involves mixing an aqueous sample solution with an immiscible

solvent; the organic layer containing the extracted analytes is

removed, dried, and reconstituted in an appropriate solvent for

LC/MS analysis


– Easy to set up and perform when working with a few samples

� Cons:

– Time/labor intensive; difficult to automate; may require multiple extraction

steps to improve recovery

– Final extract often not compatible with mobile phase

– Requires evaporation and reconstitution

– Does not enrich target analytes

– May not be ideal for polar drugs and metabolites

– Uses large volumes of costly hazardous organic solvents

– Emulsion formation

– Less selective than SPE; does not remove endogenous phospholipids

Waters Alternative to LLE:Waters Alternative to LLE:OstroOstro 9696--Well Sample Preparation PlateWell Sample Preparation Plate

� Designed for the cleanup of phospholipids and

proteins in plasma and serum

– Silica-based sorbent with C18 bonding retains

phospholipids

– Fast, easy in-well protein precipitation; precipitated

proteins and phospholipids are left behind in the wells

– Generic protocol; no method development


– Generic protocol; no method development

50

60

70

80

90

100

Ostro

LLE

% Recovery

Average Recovery:Average Recovery:OstroOstro vs. Traditional LLEvs. Traditional LLE


0

10

20

30

40

50 LLE

% Recovery

OstroOstro vs. Traditional LLEvs. Traditional LLE


Ostro provides a significant reduction in sample prep time relative to LLE in a 96-well format or in individual tubes; eliminates extract transfer and evaporation steps compared to traditional LLE

Ostro removes significantly more phospholipids for cleaner extracts; >95% of residual

phospholipids removed relative to LLE with MTBE

Comparison of Phospholipids Remaining after Comparison of Phospholipids Remaining after Various Sample Preparation TechniquesVarious Sample Preparation Techniques

MRM of m/z 184-184

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

100 184.4 > 184.4 (Lipid 184)2.00e8

2.882.292.21

2.10

1.90

2.60 2.782.72

184.4 > 184.4 (Lipid 184)2.00e8

2.802.27 2.622.56 2.68LLE with MTBE

LLE with 5%NH4OH in MTBE


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

1.90

2.62 2.68

184.4 > 184.4 (Lipid 184)2.00e8

1.961.901.77

184.4 > 184.4 (Lipid 184)2.00e8

2.842.211.961.751.421.38

1.32

1.631.51

PPT

Ostro™

PhospholipidPhospholipid BuildBuild--upup


Waters Alternative to Waters Alternative to LLLE:LE:OstroOstro 9696--Well Sample Preparation PlateWell Sample Preparation Plate

� Pros

– No method development required; uses a simple protocol

for analytes with diverse chemical properties

– Reproducible phospholipid and protein removal

– Provides a significant reduction in sample prep time relative to LLE;

easy to automate

– Eliminates extract transfer and evaporation steps


– Eliminates extract transfer and evaporation steps

– High analyte recovery

� Cons

– No sample enrichment

– Does not remove all sources of matrix effects (salts, formulation

agents, etc.)

Solid Phase Extraction (SPE)Solid Phase Extraction (SPE)

� SPE is used to chemically separate the different components

of a sample.

� It’s the only technique that will clean up and concentrate the

final sample for further analysis.

� The only technique that can minimize matrix interferences

including proteins, phospholipids, salts, and other


including proteins, phospholipids, salts, and other

endogenous compounds.

SPE Retention MechanismsSPE Retention Mechanisms

� Reversed-phase ☑ most common – Polar mobile phase

– Non-polar stationary phase

� Normal phase

– Non-polar mobile phase

– Polar stationary phase


– Polar stationary phase

� Ion exchange

– Cationic/anionic exchanger stationary phase

– Ionization states of the analytes and the sorbents

� Mixed-mode

– Combination of reversed-phase plus ion exchange mechanisms

ReversedReversed--Phase SPE:Phase SPE:How Does It Work?How Does It Work?

� In reversed-phase chromatography, the stationary phase is

non-polar and the mobile phase is polar.


Solid Solid Phase Extraction (SPE)Phase Extraction (SPE)

� Pros

– Increases analyte concentration in the sample; helps achieve higher

detection sensitivity

– Minimizes matrix interferences that alter MS response

– Ability to simultaneously extract analytes of wide polarity range

– Highest recovery and reproducibility

– Washes and elution solvents can be manipulated for optimum recovery and


– Washes and elution solvents can be manipulated for optimum recovery and

cleanup

– Variety of device formats and sorbent chemistries

– Can be automated for high throughput analysis

– Lower solvent consumption; less exposure to toxic agents

– Increases column lifetime; less instrument downtime

� Cons:

– May require method development

– Perceived cost

� Waters has been at the forefront of SPE innovation since 1977

with the launch of Sep-Pak products (the first bonded silica

device for SPE)

� In 1996, Waters revolutionized SPE technology with the

introduction of Oasis HLB, the first water-wettable—yet

hydrophobic— polymeric sorbent

Waters and SPEWaters and SPE


hydrophobic— polymeric sorbent

NEW!SPE Textbook

Hydrophilic

monomer

Lipophilic

monomer

NO

Hydrophilic-Lipophilic BalancedCopolymer

Waters Waters SPE Products:SPE Products:OasisOasis®® HLB Sorbent ChemistryHLB Sorbent Chemistry


monomer monomer

Reversed-phase Retention

• Water wettable

• Polar retention

• Stable across pH 0-14

• No silanol interactions

• High recoveries for acids, bases and neutrals

Retention of Polars

Waters Waters SPE Products:SPE Products:OasisOasis®® HLB Sorbent ChemistryHLB Sorbent Chemistry


Oasis HLB: A Universal Sorbent for Acidic, Basic, and Neutral Compounds

OasisOasis®® Family of MixedFamily of Mixed--Mode Sorbents:Mode Sorbents:ReversedReversed--Phase Retention and Ion ExchangePhase Retention and Ion Exchange

Selective for Basic

Compounds

Selective for Acidic

Compounds

Sorbent ALWAYS Charged (-) Sorbent ALWAYS Charged (+)


Selective for Strong Basic

Compounds

Selective for Strong Acidic

Compounds

Sorbent charged (+) at Low pH; unionized at high pH

Sorbent charged (-) at high pH; unionized at low pH

For wide range of acidic, basic, and neutral compounds

OasisOasis®® 2x42x4 Method:Method:Streamlining Method DevelopmentStreamlining Method Development

Oasis® 2x4 Method:1. Characterize your analyte.2. Select 1 of the 4 Oasis sorbents.3. Apply the designated Protocol (1 of 2).4. Analyze SPE recoveries and matrix

� A straightforward approach for selecting the right ion-exchange

SPE sorbent

� 4 sorbents and 2 protocols


4. Analyze SPE recoveries and matrix effects.

Oasis sorbent selection tools are available in plate and cartridge formats for convenient method

development.

Waters SPE Device FormatsWaters SPE Device Formats

� Formats

– 96-well plates (with 5, 10, 30, 60

mg of sorbent)

– Syringe barrel cartridges

– Glass cartridges

– Online columns


– µElution plates

� How to process samples?

– Gravity

– Pressure

– Vacuum

– Automation

Waters SPE Device Formats: Waters SPE Device Formats: Oasis µElution Plate TechnologyOasis µElution Plate Technology

� Patented plate design

� Ideal for SPE cleanup and analyte enrichment of

small sample volumes (10 µL to 375 µL)

� Elute in as little as 25µL; up to 15X concentration

� No evaporation and reconstitution required

–Saves time


–Saves time

–No evaporative loss

� Speed

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

–Eluates can be directly injected

� Compatible with most liquid handling robotic

systems for automated high throughput SPE

Narrow and Tall bed

%

100 411.2 > 191.22.17e6

0.83

Why Oasis µElution Format?Why Oasis µElution Format?Up to a 15X ConcentrationUp to a 15X Concentration

0.5 ng/mL risperidone

Elution in 25 µLRecovery = 98%11X increase in S:N17X increase in area counts

Oasis 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 > 191.22.17e6

0.83

Elution in 375 µLRecovery= 98%

MCX 10 mg plate(No concentration)







OverviewOverview




� Conclusion

� Look at various options to determine the best fit

– “Fit for purpose” rather than one-size-fits-all

� There are a number of factors that influence the selection of

a sample prep method:

– Analyte properties

– Analyte concentration level(s)

Selecting the Appropriate Sample Selecting the Appropriate Sample Preparation Option Preparation Option


– Analyte concentration level(s)

– Sample matrix

– Analytical technique

– Required throughput

– Regulatory requirements

� Business needs and degrees of risk tolerance

– Reliability of the assay, cost of re-test, column life time,

instrument down-time, etc.







OverviewOverview



– Application Example

o 25-OH Vitamin D2 and D3 in Serum

� Conclusion

Vitamin D in SerumVitamin D in Serum

Assay Use

Measurement of analytes in serum

Analytes

25-OH Vitamin D2 and D3


Assay Requirements

� A single extraction step for 25OHD2 and 25OHD3

� A robust, sensitive method

� High throughput; semi-automated sample prep protocol

� Suitable for small sample volume

� Must eliminate evaporation and reconstitution steps

Vitamin DVitamin D

CH3

CH3

CH3

CH3

CH3

H

CH3

CH3

CH3

CH3

H

H


Ergocalciferol

Vitamin D2

MW 396.7

Cholecalciferol

Vitamin D3

MW 384.6

CH2

OH

H

CH2

OH

H

SemiSemi--Automated Oasis HLB Automated Oasis HLB µElution µElution Extraction Procedure*Extraction Procedure*

Sample PretreatmentAdd 20 µL of IS to 150 µL of serum;

Perform protein precipitation with 150 µL of aqueous 0.2M ZnSO4 and 600 µL of MeOH;

Centrifuge

Condition and Equilibrate Plate200 µL MeOH then 200 µL 60% MeOH

Load 600 µL of supernatant from pretreatment step

*All liquid handling steps (except the centrifugation step) were executed using the Tecan Evo 100 robot.


600 µL of supernatant from pretreatment step

WashWash1:200 µL of 5:95 MeOH:WaterWash2: 200 µL of 60:40 MeOH:Water

EluteElute 1: 80 µL 95:5 MeOH:IPA

Elute 2: 50 µL Water

Inject 20 µL

� UPLC System: Waters ACQUITY UPLC System

� Column: ACQUITY UPLC BEH Phenyl Column, 2.1 x 50 mm, 1.7µm

� Mass Spectrometer: ACQUITY TQD system

Assay PerformanceAssay Performance

� Linearity

– >0.997 over the range of 2.5–220 ng/mL

� Intra-assay and inter-assay precision


� Accuracy determined by analysis of DEQAS samples; all results

were within 10.8% deviation of the expected value

� Recovery: >80%; minimal matrix effects

Chromatogram of 25Chromatogram of 25--OH Vitamin D3OH Vitamin D3


Benefits of the Benefits of the Oasis HLB Oasis HLB µElution µElution Plate for this AssayPlate for this Assay

� Simultaneous extraction and detection of 25(OH)D2 and

25(OH)D3 in serum

� Sensitive method for accurate and reliable measurement of low

levels

� Uses fast, high throughput protocol

– Semi-automated extraction procedure using Tecan Evo 100


– Semi-automated extraction procedure using Tecan Evo 100

– Can process and analyze up to 192 samples in 3 hours

� Suitable for small sample volume (150 µL of serum)

� No need for solvent evaporation and reconstitution steps; saves

time







OverviewOverview




o Opiates and Metabolites in Urine

� Conclusion

OOpiates and Metabolites in Urinepiates and Metabolites in Urine

Assay Use

Screening of analytes in urine

Analytes

26 natural opiate drugs, semi-synthetic opioids, and synthetic

narcotic analgesic compounds


Assay Requirements

� A single, robust sample preparation method

� High throughput

� High recovery

� No enzymatic hydrolysis

� Better linearity, accuracy, precision and reduced matrix effects

compared to “dilute and shoot” method

Retention Times and FormulaeRetention Times and Formulae

Compound RT Formula

1 Morphine-3β-D-glucuronide 1.21 C23H27NO9

2 Oxymorphone-3β-D-glucuronide 1.21 C23H27NO10

3 Hydromorphone-3β-D- glucuronide 1.34 C23H27NO9


5 Morphine 1.50 C17H19NO3

6 Oxymorphone 1.61 C17H19NO4

7 Hydromorphone 1.76 C17H19NO3

8 Codeine-6β-D-glucuronide 2.00 C24H29NO9

9 Dihydrocodeine 2.07 C18H23NO3

10 Codeine 2.14 C18H21NO3

11 Oxycodone 2.37 C H NO


11 Oxycodone 2.37 C18H21NO4

12 6-Acetylmorphone (6-AM) 2.41 C19H21NO4

13 O-desmethyl Tramadol 2.46 C15H23NO2

14 Hydrocodone 2.50 C18H21NO3

15 Norbuprenorphine-glucuronide 2.83 C31H43NO10

16 Norfentanyl 2.93 C14H20N2O

17 Tramadol 3.21 C16H25NO2

18 Normeperedine 3.58 C14H19NO2

19 Meperidine 3.60 C15H21NO2

20 Buprenorphine-glucuronide 3.64 C35H49NO10

21 Norbuprenorphine 3.77 C25H35NO4

22 Fentanyl 4.29 C22H28N2O

23 Buprenorphine 4.55 C29H41NO4

24 EDDP+ 4.79 C20H24N+

25 Propoxyphene 5.18 C22H29NO2

26 Methadone 5.25 C21H27NO

Opiate StructuresOpiate Structures

MorphineMorphine-3-glucuronide 6-monoacetylmorphine

(Heroin metabolite)


Morphine-6-glucuronide(Heroin metabolite)

Codeine

Codeine-6-glucuronide

Oxymorphone

Oxymorphone-3-glucuronide

Extraction MethodologiesExtraction Methodologies

Condition Plate200 µL MeOH then 200 µL Water

Sample Pretreatment100 µL urine + 100 µL 4% H3PO4+

100 µL IS

Load 300 µL pretreated sample

100 µL urine

Add 100 µL IS (dissolved in water)

Vortex

Oasis MCX µElution Plate Protocol Sample Dilution Protocol

Inject 10 µL


Wash200 µL Water, then

200 µL MeOH

Elute2 x 50 µL

(60:40 ACN:MeOH + 5% NH4OH)

Evaporate under N2 @ 37oC

Reconstitute in 50 µL of starting mobile phase (2% ACN/0.1% FA)

Inject 10 µL

100

8.71e6

Chromatogram of All Compounds Chromatogram of All Compounds

13,14 17

18,1920

26

Compound

1 Morphine-3β-D-glucuronide

2 Oxymorphone-3β-D-glucuronide

3 Hydromorphone-3β-D-

glucuronide


5 Morphine

6 Oxymorphone

7 Hydromorphone

8 Codeine-6β-D-glucuronide

9 Dihydrocodeine

10 Codeine

11 Oxycodone


Time1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

%

0

1,234,5

8 9,107

6

11,12

15,16 21

22

23

24

25

11 Oxycodone

12 6-Acetylmorphone (6-AM)

13 O-desmethyl Tramadol

14 Hydrocodone

15 Norbuprenorphine-glucuronide

16 Norfentanyl

17 Tramadol

18 Normeperedine

19 Meperidine

20 Buprenorphine-glucuronide

21 Norbuprenorphine

22 Fentanyl

23 Buprenorphine

24 EDDP+

25 Propoxyphene

26 Methadone

Average Recovery on the Average Recovery on the Oasis MCX Oasis MCX µµElutionElution PlatePlate

60%

80%

100%

120%


0%

20%

40%

Mean + S.D. for 6 Different Lots of Urine

Matrix Factors and %CV with Matrix Factors and %CV with Oasis MCX vs. DilutionOasis MCX vs. Dilution

0.60

0.80

1.00

1.20

1.40MCX

Dilution*

*

* *

*

*

** ** *

*


0.00

0.20

0.40

* Analytes for which matrix factors significantly different between the two protocols

Linearity Results Linearity Results for Oasis MCX for Oasis MCX µµElutionElution PlatePlate

Curve Point (ng/mL)

5 10 20 40 50 100 200 400 500

R2 % Acc %CV % Acc %CV % Acc %CV % Acc %CV % Acc %CV % Acc %CV % Acc %CV % Acc %CV % Acc %CV

Morphine-3-β-d-glucuronide 0.996 98.8 8.9% 99.0 7.9% 103.7 5.0% 103.2 4.7% 104.7 5.6% 99.5 1.1% 100.7 4.9% 95.9 3.4% 96.2 2.7%

Oxymorphone-3-b-d-glucuronide 0.997 101.7 0.1% 97.3 4.9% 97.6 3.6% 101.5 1.0% 103.3 7.6% 103.4 3.6% 101.2 2.3% 98.5 5.6% 95.7 7.1%

Hydromorphone-3-b-d-glucuronide 0.998 98.5 1.1% 100.7 2.9% 103.4 4.4% 102.3 1.0% 98.5 7.1% 102.9 3.0% 100.9 3.1% 98.7 4.7% 95.6 3.0%

Morphine-6-gluc 0.994 97.3 11.6% 104.0 7.3% 95.9 6.3% 107.5 2.2% 104.5 2.8% 104.4 2.2% 101.6 6.5% 94.1 5.8% 92.3 2.9%

Morphine 0.992 102.0 5.1% 93.9 11.3% 102.2 8.3% 107.0 9.9% 99.6 2.6% 99.0 4.9% 92.5 4.4% 104.8 9.7% 100.8 12.3%

Oxymorphone 0.998 99.7 0.7% 98.9 2.3% 103.1 2.9% 100.5 2.8% 101.1 3.2% 102.0 7.7% 102.0 1.3% 97.9 3.0% 95.9 3.3%

Hydromorphone 0.998 98.9 7.7% 101.3 2.9% 97.2 6.2% 106.2 0.9% 100.5 0.8% 101.3 2.5% 99.3 0.6% 98.7 3.1% 97.4 2.0%

Codeine-6-β-d-glucuronide 0.998 100.5 0.5% 100.9 4.7% 96.8 2.0% 102.1 0.4% 96.5 2.8% 99.1 6.6% 100.9 3.1% 100.9 2.3% 102.4 1.3%

Dihydrocodeine 0.997 96.7 6.4% 102.0 1.0% 101.5 0.2% 107.0 0.0% 103.5 0.7% 102.0 0.7% 100.6 1.8% 95.3 1.0% 93.1 1.5%

Codeine 0.995 95.6 4.1% 102.2 3.5% 105.8 0.9% 108.0 2.1% 101.4 1.5% 104.8 0.9% 100.3 2.2% 93.6 0.5% 91.4 2.3%


Codeine 0.995 95.6 4.1% 102.2 3.5% 105.8 0.9% 108.0 2.1% 101.4 1.5% 104.8 0.9% 100.3 2.2% 93.6 0.5% 91.4 2.3%

Oxycodone 0.996 96.9 4.4% 101.6 3.0% 101.7 5.0% 105.7 0.1% 104.8 1.0% 102.9 1.4% 100.1 1.2% 96.0 3.6% 91.8 4.2%

6-Acetylmorphone (6-AM) 0.997 95.5 4.9% 105.7 1.3% 99.5 3.1% 103.6 4.0% 100.1 2.4% 98.8 2.9% 101.6 0.9% 100.1 0.9% 94.7 4.5%

O-desmethyl Tramadol 0.999 99.2 3.3% 100.2 0.2% 99.1 0.2% 105.0 1.3% 101.0 1.6% 102.0 0.4% 100.4 0.5% 97.6 1.0% 96.6 0.5%

Hydrocodone 0.999 99.4 0.6% 101.5 2.5% 96.7 1.1% 103.5 1.4% 98.8 0.6% 101.7 1.5% 101.2 0.5% 98.0 1.5% 99.1 1.2%

Norbuprenorphine-glucuronide 0.998 99.6 7.7% 100.6 2.1% 98.6 2.5% 103.1 1.9% 100.7 3.4% 96.8 3.6% 101.0 5.8% 101.0 1.1% 99.0 1.3%

Norfentanyl 0.998 97.9 5.6% 102.4 6.3% 99.9 3.5% 100.9 3.9% 101.8 0.5% 100.2 1.2% 101.7 1.3% 98.0 2.1% 96.8 1.1%

Tramadol 0.995 95.0 0.1% 103.0 0.4% 104.0 1.8% 109.7 0.1% 104.4 0.9% 103.3 1.0% 99.0 0.5% 92.6 1.0% 92.1 0.7%

Normeperedine 0.997 97.0 1.9% 101.2 1.7% 102.1 3.4% 107.4 0.7% 104.3 1.1% 102.8 0.5% 99.7 2.3% 94.2 0.9% 93.5 1.2%

Meperidine 1.000 98.7 0.1% 100.8 1.3% 101.3 0.6% 103.2 1.1% 99.9 0.6% 100.9 1.1% 100.1 1.4% 97.6 1.0% 98.5 1.2%

Buprenorphine-gluc 0.997 104.1 2.2% 97.9 5.0% 94.5 0.8% 95.4 2.4% 94.8 2.4% 100.2 2.3% 101.5 2.3% 105.2 2.4% 104.5 1.0%

Norbuprenorphine 0.997 96.5 0.7% 102.0 1.8% 102.7 1.7% 109.3 1.6% 102.7 2.1% 99.6 2.4% 101.2 3.1% 95.8 0.6% 93.1 3.0%

Fentanyl 0.998 98.1 1.8% 100.9 1.6% 100.7 1.0% 105.8 1.1% 102.0 0.9% 102.7 0.4% 101.1 0.4% 95.9 1.8% 94.4 0.5%

Buprenorphine 0.998 100.9 0.5% 98.1 2.6% 98.3 1.5% 105.1 1.1% 101.4 0.8% 103.7 0.9% 101.8 1.3% 97.4 0.5% 94.9 1.0%

EDDP+ 0.999 99.5 0.2% 100.6 0.9% 98.2 0.8% 104.2 0.8% 99.6 1.1% 101.3 0.3% 101.8 0.9% 97.9 0.3% 97.6 0.4%

Propoxyphene 0.996 96.9 2.6% 101.0 0.8% 102.2 0.0% 108.7 0.3% 105.2 0.4% 103.0 0.9% 100.2 1.6% 94.6 1.1% 90.8 1.2%

Methadone 0.998 99.3 1.8% 99.3 1.5% 100.0 0.2% 106.5 2.0% 102.8 0.9% 102.4 1.8% 100.4 1.6% 97.4 0.4% 93.9 1.0%

�All compounds showed excellent linearity, with R2

values of >0.992.

�All calibration points were within 15% of their expected values and had %CVs less than 15%

Curve Point (ng/mL)

5 10 20 40 50 100 200 400 500





Morphine-6-gluc 0.979 94.8 18.4% 109.9 3.2% 96.7 10.5% 110.7 16.3% 100.5 3.3% 98.7 6.5% 91.2 4.3% 100.4 2.9% 97.1 16.8%

Morphine 0.954 89.5 29.2% 98.6 18.9% 119.2 28.6% 92.3 15.4% 97.5 29.7% 93.0 10.8% 115.7 20.5% 99.7 16.3% 100.0 27.5%

Oxymorphone 0.989 89.4 2.5% 95.0 8.7% 96.3 8.3% 109.3 3.2% 100.5 11.1% 98.4 2.4% 94.5 9.7% 99.5 12.7% 97.3 17.1%

Hydromorphone 0.996 97.2 1.2% 110.8 8.4% 114.4 14.2% 102.8 3.6% 98.1 9.1% 100.0 1.8% 98.8 6.4% 97.3 1.6% 98.5 4.9%


Dihydrocodeine 0.997 97.6 1.7% 102.3 6.6% 105.1 6.6% 102.0 2.0% 97.3 2.6% 100.3 4.4% 95.9 4.1% 100.1 5.2% 99.3 5.4%

Codeine 0.99 93.4 11.3% 109.7 2.9% 104.4 8.4% 108.2 10.3% 99.7 5.8% 97.3 5.1% 94.8 6.1% 97.1 3.6% 95.3 2.8%

Linearity Results for Dilution ProtocolLinearity Results for Dilution Protocol


Codeine 0.99 93.4 11.3% 109.7 2.9% 104.4 8.4% 108.2 10.3% 99.7 5.8% 97.3 5.1% 94.8 6.1% 97.1 3.6% 95.3 2.8%

Oxycodone 0.993 98.6 8.2% 104.1 8.3% 98.0 11.6% 98.3 3.5% 99.4 4.1% 104.6 9.6% 97.0 0.7% 100.7 3.2% 99.3 8.6%



Hydrocodone 0.995 95.1 0.4% 113.3 6.0% 103.6 3.7% 105.6 6.0% 100.4 2.1% 99.0 2.1% 96.7 4.8% 97.4 6.8% 95.3 3.7%

Norbuprenorphine-glucuronide 0.992 94.6 13.4% 105.9 5.3% 105.8 5.4% 102.9 1.5% 108.0 6.6% 103.7 1.6% 93.8 5.0% 93.9 2.8% 91.5 1.4%

Norfentanyl 0.995 95.6 4.1% 106.0 4.1% 102.9 6.4% 103.1 1.5% 102.5 3.0% 104.2 2.8% 95.8 4.3% 95.8 5.7% 94.1 3.4%

Tramadol 0.996 95.9 1.6% 104.6 3.0% 103.5 0.8% 107.4 1.4% 101.6 1.1% 101.6 1.4% 95.7 2.1% 96.3 0.4% 93.4 1.8%

Normeperedine 0.996 97.0 3.6% 102.8 3.8% 102.7 3.4% 105.9 2.2% 101.7 1.9% 104.5 1.7% 97.5 3.1% 96.0 1.6% 91.9 5.2%

Meperidine 0.997 96.5 1.5% 105.7 6.0% 100.4 3.1% 104.8 1.6% 100.0 2.0% 100.9 2.9% 96.2 1.8% 98.8 1.8% 96.6 3.9%

Buprenorphine-gluc 0.991 93.3 13.3% 110.0 6.4% 103.4 8.7% 103.9 2.0% 105.8 5.1% 100.0 2.6% 97.4 5.2% 93.8 8.2% 92.4 1.7%


Fentanyl 0.997 97.2 0.4% 102.9 3.9% 101.9 4.8% 105.9 0.6% 102.6 1.0% 101.1 3.2% 96.0 3.5% 97.4 5.6% 95.1 1.6%

Buprenorphine 0.994 97.2 8.6% 102.8 9.4% 102.0 8.8% 102.9 0.9% 105.6 4.9% 102.2 2.9% 100.1 5.6% 94.7 7.9% 92.3 1.0%

EDDP+ 0.998 97.3 1.2% 103.5 4.3% 101.3 1.2% 104.2 0.8% 101.4 0.9% 100.8 1.7% 97.2 3.2% 98.3 1.1% 95.9 1.7%

Propoxyphene 0.995 95.8 1.0% 105.3 3.0% 101.1 1.1% 105.9 1.7% 105.7 1.0% 102.2 3.1% 99.7 2.7% 94.8 0.8% 89.4 2.4%

Methadone 0.997 98.8 0.9% 101.1 2.1% 98.5 3.4% 105.1 0.5% 103.1 2.5% 102.8 4.0% 101.0 3.0% 98.0 6.4% 91.6 1.2%

Highlighted cells exceed recommended values for intra-assay precision (15%) or deviate from expected values by >15%

• Good linearity and accuracy for most compounds, but 8.6% of calibration points exceeded the recommended %CV of 15%.

• Morphine shows unacceptable precision throughout the calibration range.

Benefits of the Oasis MCX µElution Plate Benefits of the Oasis MCX µElution Plate for this Assayfor this Assay

� Single extraction method for 26 opioid drugs and metabolites

� Rapid and simple sample preparation

– No enzymatic hydrolysis needed

– 96-well plates utilized

� Recoveries >90% for most compounds

� Ability to process small sample volume (100 µL)



� Substantially improved linearity, accuracy, precision and

reduced matrix effects with the Oasis MCX µElution vs. sample

dilution







OverviewOverview




o Opiates and Metabolites in Whole Blood

� Conclusion

OOpiates and Metabolites in Whole Bloodpiates and Metabolites in Whole Blood

Assay Use

Screening of compounds in whole blood

Analytes

22 natural opiate drugs, semi-synthetic opioids, and synthetic




Assay Requirements

� A single-step, robust sample preparation method

� Fast, high throughput protocol

� Must work with a small sample volume (50 µL of whole blood)

� No enzymatic hydrolysis

Retention Times and FormulaeRetention Times and Formulae

Compound RT Formula


2 Oxymorphone-3β-D-glucuronide 1.21 C23H27NO10

3 Hydromorphone-3β-D- glucuronide 1.34 C23H27NO9

4 Morphine 1.50 C17H19NO3

5 Oxymorphone 1.61 C17H19NO4

6 Hydromorphone 1.76 C17H19NO3

7 Codeine-6β-D-glucuronide 2.00 C24H29NO9

8 Dihydrocodeine 2.07 C18H23NO3


18 23 3

9 Codeine 2.14 C18H21NO3

10 Oxycodone 2.37 C18H21NO4

11 6-Acetylmorphone (6-AM) 2.41 C19H21NO4

12 O-desmethyl Tramadol 2.46 C15H23NO2

13 Hydrocodone 2.50 C18H21NO3

14 Norbuprenorphine-glucuronide 2.83 C31H43NO10

15 Tramadol 3.21 C16H25NO2

16 Normeperedine 3.58 C14H19NO2

17 Meperidine 3.60 C15H21NO2

18 Norbuprenorphine 3.77 C25H35NO4

19 Fentanyl 4.29 C22H28N2O

20 Buprenorphine 4.55 C29H41NO4

21 Propoxyphene 5.18 C22H29NO2

22 Methadone 5.25 C21H27NO

Opiate StructuresOpiate Structures

MorphineMorphine-3-glucuronide 6-monoacetylmorphine

(Heroin metabolite)


Morphine-6-glucuronide(Heroin metabolite)

Codeine

Codeine-6-glucuronide

Oxymorphone

Oxymorphone-3-glucuronide

Whole Blood Whole Blood Extraction Methodology Extraction Methodology with with OstroOstro PlatePlate

Add 150 µL of aqueous 0.1M ZnSO4/NH4CH3COOH to each well

Add 50 µL of whole blood and vortex briefly (5 sec.) to lyse the cells

Add 600 µL of ACN containing internal standards to the prepared samples


standards to the prepared samples

Vortex for 3 minutes and elute into a 96-well collection plate

Evaporate to dryness under N2

Reconstitute in 50 µL starting mobile phase (2% ACN/0.1% FA)

Inject 10 µL

100

Chromatogram of All CompoundsChromatogram of All Compounds

12,13

15

1.46 e6

Compound


2 Oxymorphone-3β-D-glucuronide

3 Hydromorphone-3β-D- glucuronide

4 Morphine

5 Oxymorphone

6 Hydromorphone

7 Codeine-6β-D-glucuronide

8 Dihydrocodeine

9 Codeine

10 Oxycodone

11 6-Acetylmorphone (6-AM)


Time1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

%

0

1,23

4,5

7,8,9

6 10,11 14 21

16,17

22201918

12 O-desmethyl Tramadol

13 Hydrocodone

14 Norbuprenorphine-glucuronide

15 Tramadol

16 Normeperedine

17 Meperidine

18 Norbuprenorphine

19 Fentanyl

20 Buprenorphine

21 Propoxyphene

22 Methadone

% Recovery% Recovery

80%

100%

120%

140%

160%

180%


0%

20%

40%

60%

80%

Accuracy and %CV for Calibration Accuracy and %CV for Calibration Curves from 5Curves from 5--500 500 ngng//mLmL

Curve Point (ng/mL)

5 10 20 40 50 100 200 400 500





Morphine 0.986 96.7 5.5% 111.8 10.6% 99.2 5.5% 111.4 9.3% 99.9 11.1% 106.3 13.1% 98.9 14.0% 95.0 11.1% 89.6 9.7%

Oxymorphone 0.989 83.9 7.5% 102.2 10.1% 96.1 6.8% 103.9 1.5% 100.9 0.1% 105.5 7.6% 99.5 8.1% 102.3 3.4% 96.0 6.6%

Hydromorphone 0.988 99.0 3.1% 115.6 1.5% 101.6 6.1% 107.3 6.3% 98.8 3.8% 98.2 7.0% 92.9 7.4% 92.3 2.3% 89.1 4.0%


Dihydrocodeine 0.984 103.8 6.9% 108.1 5.8% 112.4 2.5% 110.8 6.0% 100.8 9.0% 94.1 9.6% 88.1 8.3% 80.2 7.5%

Codeine 0.979 116.0 3.2% 107.4 2.6% 116.1 3.4% 106.7 4.4% 106.7 6.6% 92.5 7.6% 85.5 5.7% 78.0 1.0%

Oxycodone 0.986 93.6 12.2% 116.7 0.1% 103.9 3.9% 109.5 5.6% 102.3 6.8% 107.2 2.1% 95.7 2.4% 83.1 4.8% 78.7 2.1%



�Highlighted cells differ from nominal values by >15% or have %CV values greater than 15%



Hydrocodone 0.990 102.2 0.3% 115.1 7.5% 100.3 1.6% 103.7 5.1% 93.8 1.1% 102.6 10.3% 95.1 11.2% 98.9 1.1% 92.6 11.9%

Norbuprenorphine-glucuronide 0.989 93.9 23.9% 106.9 92.5 5.8% 97.7 11.3% 113.6 105.8 12.6% 97.0 13.8% 106.9 7.1% 94.5 2.9%

Tramadol 0.988 102.2 0.6% 118.3 3.3% 102.2 2.1% 109.5 2.5% 102.1 5.1% 101.6 5.4% 94.1 5.8% 89.7 1.3% 84.4 4.5%

Normeperedine 0.995 99.1 3.5% 110.8 1.1% 97.6 2.7% 104.6 5.6% 99.4 3.4% 102.3 8.0% 96.1 8.5% 99.5 2.3% 94.3 2.5%

Meperidine 0.994 102.4 5.3% 115.0 2.8% 98.1 1.4% 104.8 5.5% 95.9 2.5% 98.4 7.4% 96.6 7.5% 97.1 2.0% 94.6 3.6%


Fentanyl 0.992 97.8 3.8% 112.4 5.1% 98.5 3.2% 103.0 7.9% 98.2 6.2% 102.3 6.4% 95.3 6.9% 96.6 0.5% 92.1 6.6%

Buprenorphine 0.994 93.0 2.9% 108.9 4.8% 99.3 3.2% 105.6 5.4% 96.9 6.8% 105.9 8.7% 101.0 9.2% 97.9 3.4% 94.2 4.8%

Propoxyphene 0.990 101.6 5.0% 113.5 2.8% 99.8 1.9% 108.2 3.3% 95.5 3.7% 103.6 6.7% 99.1 7.0% 93.8 3.7% 88.9 3.8%

Methadone 0.994 99.9 3.4% 113.0 2.4% 101.0 3.1% 107.0 2.9% 96.0 2.9% 99.3 2.8% 95.3 3.0% 99.3 2.2% 92.0 2.6%

�Most compounds demonstrated good linearity, with all R2 values > 0.98.

�93% of the calibration points were within 15% of their expected values

�94% of calibration points have %CV values <15%

Benefits of the Benefits of the OstroOstro Sample Preparation Sample Preparation Plate for this AssayPlate for this Assay

� Simultaneous extraction of 22 analytes from whole blood


– No enzymatic hydrolysis needed


� Reduces interferences such as proteins and phospholipids

� Uses a single-well protocol


� Uses a single-well protocol

– Significantly reduces sample preparation time (vs. classical PPT and

LLE)

– Eliminates potential analyte loss due to extract transfer








OverviewOverview



– Application Example

o Benzodiazepines and Metabolites in Plasma

� Conclusion

Screening for BScreening for Benzodiazepines and enzodiazepines and Metabolites in PlasmaMetabolites in Plasma

Assay UseScreening

Analytes26 compounds: benzodiazepines, metabolites and internal standards


Assay Requirements

� High recovery

� High throughput

� Detection limits not challenging

� Lab is concerned about system robustness and build up of phospholipids on

LC columns and in MS source

� Wants direct injection to speed up workflow

� Wants simplest sample prep possible

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

Recovery Using Recovery Using OstroOstro PlatePlate

40

60

80

100


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)

Average recovery = 84%

Linearity and AccuracyLinearity and Accuracy

� Standard curves were

run from 1–500 ng/mL

� R2 value was > 0.965

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


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

Benefits of Benefits of OstroOstro Plates for this AssayPlates for this Assay

� High recovery for analogs and metabolites

– No method development

� Very simple protocol

� Was able to meet the detection limits for the assay

� Direct injection of eluate

– Streamlines workflow


– Streamlines workflow

� Removes vast majority of phospholipids

– Improved instrument uptime

– More robust methods







OverviewOverview




o Bath Salts in Urine

� Conclusion

Bath Salts in UrineBath Salts in Urine

Assay Use

Screening of analytes in urine

Analytes

10 compounds in “Bath Salts”

Assay Requirements


Assay Requirements

� A single, robust sample preparation method

� High throughput

� High recovery and sensitivity

� Low matrix interferences

StructuresStructures

Methedrone α-PPP

C11H15NO2 C13H17NO

α-PVP

C H NO


C15H21NO

Mephedrone

C11H15NO

α-PVP metabolite

C15H23NO

StructuresStructures

C11H13NO3

Ethylone

C12H15NO3C12H15NO3

ButyloneMethylone


MDPPP MDPV

C16H21NO3C14H17NO3

Extraction MethodologyExtraction Methodology

Condition Plate200 µL MeOH then 200 µL Water

Sample Pretreatment100 µL pooled urine + 100 µL 4% H3PO4

Load 200 µL pretreated sample

Wash


Wash200 µL 2% HCOOH, then 200 µL MeOH

Elute2 x 50 µL

(60:40 ACN:IPA + 5% NH4OH)

Neutralize with 5 µL of concentrated HCOOH; then dilute with 100 µL of water

Inject 10 µL

Retention Times and Formulae for Retention Times and Formulae for Bath Salt CompoundsBath Salt Compounds

Drug Alt Name RT Formula Mass

Cone

Voltage

MRM

Transitions

Coll.

Energy

1 Methylone3,4-methylenedioxy-N-

methylcathinone0.75 C11H13NO3 207.23

28

28

208.2→132.0

208.2→160.0

28

18

2 Ethylone MDEC, bk-MDEA 0.83 C12H15NO3 221.2630

30

222.3→174.1

222.3→204.1

20

14

3 Methedrone 4-methoxymethcathinone 0.84 C11H15NO2 193.2528

28

194.2→161.0

194.2→146.0

22

30

42 204.3→105.0 24


4 α-PPP Alpha-Pyrrolidinopropiophenone 0.86 C13H17NO 203.2842

42

204.3→105.0

204.3→98.0

24

28

5 MDPPP3',4'-Methylenedioxy-α-

pyrrolidinopropiophenone0.92 C14H17NO3 247.29

42

42

248.3→98.0

248.3→147.0

26

24

6 Butylone Bk-MBDB 0.92 C12H15NO3 221.2632

32

222.3→174.1

222.3→204.1

18

15

7 Mephedrone 4-methylmethcathinone, 4-MMC 1.02 C11H15NO 177.2426

26

178.2→145.0

178.2→91.0

22

34

8 α-PVP alpha-Pyrrolidinopentiophenone 1.66 C15H21NO 231.3438

38

232.4→91.0

232.4→105.0

26

28

9 MDPV Methylenedioxypyrovalerone 1.78 C16H21NO3 275.3538

38

276.4→175.0

276.4→205.0

22

20

10 α-PVP Met 1α-Pyrrolidinopentiophenone

metabolite 12.00 C15H23NO 233.35

30

30

234.4→72.0

234.4→173.0

20

24

Chromatography of All CompoundsChromatography of All Compounds% 4

2,3

5

6

8

910

1. Methylone

2. Ethylone

3. Methedrone

4. α-PPP

5. MDPPP

6. Butylone

7. Mephedrone


Time0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

1

4

7

6 7. Mephedrone

8. α-PVP

9. MDPV

10. α-PVP Met 1

LC System: ACQUITY UPLCColumn: ACQUITY BEH C18 1.7 µm, 2.1 x 100 mmMass spectrometer: XEVO® TQD

Recovery and Matrix EffectsRecovery and Matrix Effects

50.0%

70.0%

90.0%

110.0%

130.0%

Recovery


� Replacing MeOH with IPA in final elution step improves recovery

and eliminates most of the matrix effects

� All matrix effects <12.5%

-10.0%

10.0%

30.0%

Matrix Effects

Linearity and SensitivityLinearity and Sensitivity

Concentration (ng/mL)

1 5 10 50 100 500 R2

Methylone -3.80 9.85 10.13 2.83 -7.43 -15.87 0.990

Ethylone -2.60 8.13 10.43 2.20 -8.37 -9.80 0.990

Methedrone -3.80 9.85 10.13 2.83 -7.43 -15.87 0.987

α-PPP -1.37 4.33 5.67 -0.40 -6.80 -1.43 0.997

MDPPP -1.70 7.33 3.30 -0.93 -6.53 -1.43 0.996

Butylone -2.07 8.90 13.85 2.47 -6.13 -9.43 0.989


� Calibration curves from 1-500 ng/mL

� Good linearity and sensitivity for all compounds

� Nearly all calibration points were within 15% of their expected values

Butylone -2.07 8.90 13.85 2.47 -6.13 -9.43 0.989

Mephedrone -1.53 7.90 5.23 1.60 -6.33 -4.20 0.994

α-PVP -1.70 4.60 8.20 -3.80 -9.27 0.80 0.994

MDPV -1.20 3.60 3.20 -1.37 -9.23 2.33 0.997

α-PVP Met1 4.15 -9.60 -30.70 -4.97 9.47 11.07 0.983

Benefits of the Oasis MCX µElution Plate Benefits of the Oasis MCX µElution Plate for this Assayfor this Assay

� Successful analysis of a panel of 10 synthetic cathinone drugs



� Achieved excellent recovery and sensitivity, while virtually

eliminating matrix effects

� No evaporation and reconstitution steps necessary


� No evaporation and reconstitution steps necessary

� Possibility of using the same technique for related compounds







OverviewOverview




� Conclusion

ConclusionsConclusions

� Sample preparation is necessary for the best results

� Waters provides various sample prep strategies that combine

sorbents, formats, and methodologies for best results

� The application examples demonstrate a “fit-for-purpose”

approach to choosing the appropriate sample prep method

� Methods are not one-size fits all; the simplest method that


� Methods are not one-size fits all; the simplest method that

meets the assay’s needs should be chosen

� SPE offers the best cleanup option

– It concentrates the sample to achieve higher detection sensitivity

and removes the majority of matrix interferences that alter MS

response

Thank You! Thank You!

�Questions?

� Landing Page…

�http://www.waters.com/May21

–Promotional Offers on Sample Preparation

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