Fit-For-Purpose Sample Preparation Options for Clinical Research and Forensic Toxicology

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Fit-for-Purpose Sample Preparation Options for Clinical Research and Forensic Toxicology

Nebila Idris Senior Marketing Manager Consumables Business Unit, Waters Corp.

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 – 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

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, 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

Direct injection Non-selective Protein precipitation (PPT) Liquid-liquid extraction (LLE) Solid-phase extraction (SPE) – Reversed-phase SPE – Mixed -mode SPE Highly selective

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 – May be automated Cons: – No selectivity – “DIRTY” extracts – Short column lifetime & frequent system shutdown – No enrichment; may require solvent evaporation prior to injection

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

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

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

Pros : – Removes proteins, provides some cleanup – 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

Designed for the cleanup of phospholipids and proteins in plasma and serum

– Silica-based sorbent with C 18 bonding retains phospholipids – Fast, easy in-well protein precipitation; precipitated proteins and phospholipids are left behind in the wells – Generic protocol; no method development

100 90 80 70 60 Ostro 50 LLE

% Recovery Recovery %% 40 30 20 10 0

Ostro removes significantly more phospholipids for cleaner extracts; >95% of residual phospholipids removed relative to LLE with MTBE

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

MRM of m/z 184-184

100 184.4 > 184.4 (Lipid 184) 2.00e8

LLE with 5% 2.29 2.60 2.88 2.21 2.72 2.78

% 2.10 NH 4OH in MTBE 1.90

0 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

100 184.4 > 184.4 (Lipid 184) 2.00e8

LLE with MTBE 2.27 2.56 2.62 2.68 2.80

% 1.90

0 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

100 184.4 > 184.4 (Lipid 184) 2.00e8 Ostro™ %

1.90 1.77 1.96 0 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

100 184.4 > 184.4 (Lipid 184) PPT 2.00e8 1.38 1.42 1.63 1.75 1.96 2.21 2.84 1.51

% 1.32

0 Time 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

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 – High analyte recovery Cons – No sample enrichment – Does not remove all sources of matrix effects (salts, formulation agents, etc.)

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 endogenous compounds.

Reversed-phase ☑ most common – Polar mobile phase – Non-polar stationary phase Normal phase – Non-polar mobile 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

In reversed-phase chromatography, the stationary phase is non-polar and the mobile phase is polar.

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 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

NEW! SPE Textbook

N O

Hydrophilic Lipophilic monomer monomer

Retention of Polars Reversed-phase Retention

• Water wettable • Polar retention • Stable across pH 0-14 • No silanol interactions • High recoveries for acids, bases and neutrals

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

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

Selective for Selective for Basic Acidic Compounds Compounds

Selective for Selective for Strong Strong Acidic Basic Compounds Compounds For wide range of acidic, basic, and neutral compounds

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

A straightforward approach for selecting the right ion-exchange SPE sorbent 4 sorbents and 2 protocols

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 effects.

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

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

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 –No evaporative loss Speed –96-well plate in <30 min, <20 sec/sample Narrow and Tall bed –Eluates can be directly injected Compatible with most liquid handling robotic systems for automated high throughput SPE

0 % 0 % Recovery=98% 375 ElutionµLin 17X increase in area counts area in 17X increase S:Nin 11X increase Recovery98% = µL25 Elutionin .002 .004 .006 .008 .010 .012 1.3 1.20 1.10 1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 1.3 1.20 1.10 1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.83 0.83 (15X (15X concentration) plateµElutionMCX Oasis (No concentration)(No plate 10 MCX mg 1.40 0 1.40 0 411.2 411.2 191.2 > 411.2 191.2 > 2.17e6 2.17e6 Time 32 Overview

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) – 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.

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

CH3

H3C CH3 H3C CH3

CH3 H CH3 CH3 CH3

H H

CH2 CH2

HO HO Ergocalciferol Cholecalciferol

Vitamin D 2 Vitamin D 3 MW 396.7 MW 384.6

Sample Pretreatment Add 20 µL of IS to 150 µL of serum; Perform protein precipitation with 150 µL of

aqueous 0.2M ZnSO 4 and 600 µL of MeOH; Centrifuge *All liquid handling steps Condition and Equilibrate Plate (except the centrifugation 200 µL MeOH then 200 µL 60% MeOH step) were executed using the Tecan Evo 100 robot. Load 600 µL of supernatant from pretreatment step Wash Wash1:200 µL of 5:95 MeOH:Water Wash2: 200 µL of 60:40 MeOH:Water

Elute Elute 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

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

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 – 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

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

Compound RT Formula

1 Morphine-3β-D-glucuronide 1.21 C23 H27 NO 9

2 Oxymorphone-3β-D-glucuronide 1.21 C23 H27 NO 10

3 Hydromorphone-3β-D- glucuronide 1.34 C23 H27 NO 9

4 Morphine-6β-D-glucuronide 1.47 C23 H27 NO 9

5 Morphine 1.50 C17 H19 NO 3

6 Oxymorphone 1.61 C17 H19 NO 4

7 Hydromorphone 1.76 C17 H19 NO 3

8 Codeine-6β-D-glucuronide 2.00 C24 H29 NO 9

9 Dihydrocodeine 2.07 C18 H23 NO 3

10 Codeine 2.14 C18 H21 NO 3

11 Oxycodone 2.37 C18 H21 NO 4

12 6-Acetylmorphone (6-AM) 2.41 C19 H21 NO 4

13 O-desmethyl Tramadol 2.46 C15 H23 NO 2

14 Hydrocodone 2.50 C18 H21 NO 3

15 Norbuprenorphine-glucuronide 2.83 C31 H43 NO 10

16 Norfentanyl 2.93 C14 H20 N2O

17 Tramadol 3.21 C16 H25 NO 2

18 Normeperedine 3.58 C14 H19 NO 2

19 Meperidine 3.60 C15 H21 NO 2

20 Buprenorphine-glucuronide 3.64 C35 H49 NO 10

21 Norbuprenorphine 3.77 C25 H35 NO 4

22 Fentanyl 4.29 C22 H28 N2O

23 Buprenorphine 4.55 C29 H41 NO 4 + 24 EDDP+ 4.79 C20 H24 N

25 Propoxyphene 5.18 C22 H29 NO 2

26 Methadone 5.25 C21 H27 NO

Morphine Morphine-3-glucuronide 6-monoacetylmorphine (Heroin metabolite) Morphine-6-glucuronide

Codeine Oxymorphone Oxymorphone-3-glucuronide

Codeine-6-glucuronide

Oasis MCX µElution Plate Protocol Sample Dilution Protocol Condition Plate 100 µL urine 200 µL MeOH then 200 µL Water Add 100 µL IS Sample Pretreatment (dissolved in water) 100 µL urine + 100 µL 4% H 3PO 4+ 100 µL IS Vortex

Load Inject 10 µL 300 µL pretreated sample

Wash 200 µL Water, then 200 µL MeOH

Elute 2 x 50 µL (60:40 ACN:MeOH + 5%

NH 4OH)

o Evaporate under N 2 @ 37 C Reconstitute in 50 µL of starting mobile phase (2% ACN/0.1% FA)

Inject 10 µL

8.71e6 Compound 1 Morphine-3β-D-glucuronide 2 Oxymorphone-3β-D-glucuronide 100 3 Hydromorphone-3β-D- glucuronide 4 Morphine-6β-D-glucuronide 5 Morphine 26 6 Oxymorphone 13,14 17 7 Hydromorphone 18,19 8 Codeine-6β-D-glucuronide 9 Dihydrocodeine 20 10 Codeine 11 Oxycodone 12 6-Acetylmorphone (6-AM) 13 O-desmethyl Tramadol 22 14 Hydrocodone

% 15 Norbuprenorphine-glucuronide 24 16 Norfentanyl 17 Tramadol 18 Normeperedine 19 Meperidine 20 Buprenorphine-glucuronide 25 11,12 21 Norbuprenorphine 22 Fentanyl 23 Buprenorphine 8 9,10 24 EDDP+ 6 23 3 7 25 Propoxyphene 1,2 15,16 21 4,5 26 Methadone 0 Time 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

120%

100%

80%

60%

40%

20%

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

MCX 1.40 * Dilution * 1.20 * * * 1.00 ** * * ** 0.80

0.60 *

0.40

0.20

0.00

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

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% 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% 2 Norfentanyl 0.998 97.9 5.6% All 102.4 compounds 6.3% 99.9 3.5% 100.9showed 3.9% 101.8 excellent 0.5% 100.2 1.2linearity,% 101.7 1.3% with 98.0 2.1% R 96.8 1.1% Tramadol 0.995 95.0 0.1%values 103.0 0.4% of 104.0 >0.992. 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% All 100.8 calibration 1.3% 101.3 0.6% points 103.2 1.1% were 99.9 0.6% within 100.9 1.1 15%% 100.1 of 1.4% their 97.6 1.0% 98.5 1.2% Buprenorphine-gluc 0.997 104.1 2.2%expected 97.9 5.0% 94.5 values 0.8% 95.4 and 2.4% had 94.8 %CVs 2.4% 100.2 less 2.3% 1than01.5 2.3% 15% 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%

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 0.986 102.9 9.8% 91.2 14.9% 102.0 1.4% 111.2 0.8% 93.9 3.7% 106.9 5.7% 95.4 4.0% 102.5 8.2% 94.0 9.7% Oxymorphone-3-b-d-glucuronide 0.985 102.7 7.5% 100.2 3.1% 86.3 2.2% 105.7 11.0% 98.7 8.5% 100.0 6.9% 97.9 6.0% 102.5 7.9% 106.0 20.1% Hydromorphone-3-b-d-glucuronide 0.987 96.8 8.1% 100.8 4.0% 110.2 4.4% 109.1 8.1% 92.8 5.3% 101.3 6.5% 94.1 4.8% 101.9 12.9% 93.1 9.8% 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% Codeine-6-β-d-glucuronide 0.99 94.6 2.3% 107.8 15.2% 106.3 0.9% 104.2 5.8% 96.4 4.5% 98.0 7.5% 95.4 6.0% 98.9 3.2% 98.4 0.3% 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% 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% 6-Acetylmorphone (6-AM) 0.99 98.4 10.6% 105.1 11.4% 95.8 5.4% 106.9 2.9% 90.6 2.5% 105.2 6.8% 98.1 8.8% 101.9 6.5% 112.6 25.2% O-desmethyl Tramadol 0.997 96.8 9.0% 104.3 5.0% 102.4 4.1% 104.4 2.1% 100.1 1.0% 101.9 2.1% 94.8 3.8% 99.2 3.5% 96.1 3.0% 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%• Good 105.9 linearity 5.3% 105.8 5.4% and 102.9 accuracy 1.5% 108.0 6.6% for 103.7 most 1.6% compounds, 93.8 5.0% 93.9 2.8% but 91.5 1.4% Norfentanyl 0.995 95.6 4.1%8.6% 106.0 4.1%of calibration 102.9 6.4% 103.1 points 1.5% 102.5 exceeded 3.0% 104.2 2.8% the 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%recommended 102.8 3.8% 102.7 3.4% %CV 105.9 2.2%of 15%. 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%• Morphine 110.0 6.4% 103.4 shows 8.7% 103.9unacceptable 2.0% 105.8 5.1% 100.0precision 2.6% 97.4 throughout 5.2% 93.8 8.2% 92.4 1.7% Norbuprenorphine 0.995 95.4 5.5%the 104.8 calibration 1.4% 105.2 7.5% range. 105.2 3.9% 103.3 3.6% 102.5 2.7% 94.9 4.5% 94.7 3.8% 94.0 1.6% 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%

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

Assay Use Screening of compounds in whole blood

Analytes 22 natural opiate drugs, semi-synthetic opioids, and synthetic narcotic analgesic compounds

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

Compound RT Formula

1 Morphine-3β-D-glucuronide 1.21 C23 H27 NO 9

2 Oxymorphone-3β-D-glucuronide 1.21 C23 H27 NO 10

3 Hydromorphone-3β-D- glucuronide 1.34 C23 H27 NO 9

4 Morphine 1.50 C17 H19 NO 3

5 Oxymorphone 1.61 C17 H19 NO 4

6 Hydromorphone 1.76 C17 H19 NO 3

7 Codeine-6β-D-glucuronide 2.00 C24 H29 NO 9

8 Dihydrocodeine 2.07 C18 H23 NO 3

9 Codeine 2.14 C18 H21 NO 3

10 Oxycodone 2.37 C18 H21 NO 4

11 6-Acetylmorphone (6-AM) 2.41 C19 H21 NO 4

12 O-desmethyl Tramadol 2.46 C15 H23 NO 2

13 Hydrocodone 2.50 C18 H21 NO 3

14 Norbuprenorphine-glucuronide 2.83 C31 H43 NO 10

15 Tramadol 3.21 C16 H25 NO 2

16 Normeperedine 3.58 C14 H19 NO 2

17 Meperidine 3.60 C15 H21 NO 2

18 Norbuprenorphine 3.77 C25 H35 NO 4

19 Fentanyl 4.29 C22 H28 N2O

20 Buprenorphine 4.55 C29 H41 NO 4

21 Propoxyphene 5.18 C22 H29 NO 2

22 Methadone 5.25 C21 H27 NO

Morphine Morphine-3-glucuronide 6-monoacetylmorphine (Heroin metabolite) Morphine-6-glucuronide

Codeine Oxymorphone Oxymorphone-3-glucuronide

Codeine-6-glucuronide

Add 150 µL of aqueous 0.1M

ZnSO 4/NH 4CH 3COOH 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

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

Evaporate to dryness under N 2 Reconstitute in 50 µL starting mobile phase (2% ACN/0.1% FA)

Inject 10 µL

1.46 e6 100 Compound 1 Morphine-3β-D-glucuronide 2 Oxymorphone-3β-D-glucuronide 3 Hydromorphone-3β-D- glucuronide 4 Morphine 15 5 Oxymorphone 6 Hydromorphone 7 Codeine-6β-D-glucuronide 12,13 8 Dihydrocodeine 9 Codeine 10 Oxycodone 11 6-Acetylmorphone (6-AM) 12 O-desmethyl Tramadol

% 16,17 13 Hydrocodone 14 Norbuprenorphine-glucuronide 15 Tramadol 16 Normeperedine 17 Meperidine 18 Norbuprenorphine 7,8,9 19 Fentanyl 20 Buprenorphine 21 Propoxyphene 14 21 3 6 10,11 22 Methadone 1,2 20 4,5 18 19 22 0 Time 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

180%

160%

140%

120%

100%

80%

60%

40%

20%

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 0.985 95.3 7.6% 108.4 5.7% 99.3 7.2% 98.1 15.7% 102.9 17.9% 104.8 6.4% 95.0 7.0% 99.6 12.4% 94.1 6.1% Oxymorphone-3-b-d-glucuronide 0.983 98.8 16.8% 111.0 2.5% 97.4 6.6% 103.1 5.2% 95.3 21.1% 102.5 3.1% 101.3 3.1% 100.7 6.3% 96.1 11.0% Hydromorphone-3-b-d-glucuronide 0.986 92.4 5.0% 115.2 3.9% 105.2 8.4% 108.6 6.0% 94.7 8.5% 99.0 13.7% 91.6 14.8% 105.8 5.0% 94.4 8.3% 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% Codeine-6-β-d-glucuronide 0.973 101.3 5.7% 103.8 3.3% 85.3 6.5% 82.9 2.0% 94.9 14.6% 87.9 10.5% 104.3 8.8% 121.8 5.2% 116.2 2.0% 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% 6-Acetylmorphone (6 -AM) 0.984 96.4 17.0% 113.9 8.6% 88.5 9.8% 100.2 14.5% 106.1 10.8% 96.3 14.3% 100.1 13.7% 99.1 6.4% 103.4 10.5% O-desmethyl Tramadol 0.990 99.7 4.2% 112.8 6.3% 96.5Most 4.6% compounds 109.6 1.2% 94.4 3.4%demonstrated 101.4 8.7% 95.4 9.3% good 98.5 linearity, 5.8% 93.9 3.5% 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% 2 Norbuprenorphine-glucuronide 0.989 93.9 23.9% 106.9 92.5with 5.8% all 97.7 R 11.3%values 113.6 > 0.98. 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% 97.693% 2.7% of 104.6 the 5.6% calibration 99.4 3.4% 102.3 points 8.0% 96.1 were 8.5% 99.5 within 2.3% 94.315% 2.5% Meperidine 0.994 102.4 5.3% 115.0 2.8% 98.1of their 1.4% 104.8 expected 5.5% 95.9 2.5% values 98.4 7.4% 96.6 7.5% 97.1 2.0% 94.6 3.6% Norbuprenorphine 0.989 96.9 2.6% 115.9 6.0% 97.8 1.8% 105.3 6.1% 103.6 3.6% 103.8 12.9% 95.0 14.1% 97.1 8.4% 89.5 3.6% Fentanyl 0.992 97.8 3.8% 112.4 5.1% 98.594% 3.2% of 103.0 calibration 7.9% 98.2 6.2% points 102.3 6.4% have95.3 6.9%%CV 96.6 values 0.5% 92.1 6.6% Buprenorphine 0.994 93.0 2.9% 108.9 4.8% 99.3<15% 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%

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

Simultaneous extraction of 22 analytes from whole blood Rapid and simple sample preparation – No enzymatic hydrolysis needed – 96-well plates utilized Reduces interferences such as proteins and phospholipids Uses a single -well protocol – Significantly reduces sample preparation time (vs. classical PPT and LLE) – Eliminates potential analyte loss due to extract transfer Ability to process small sample volume (50 µL)

Goal of Sample Preparation Sample Preparation Options – Protein Precipitation (PPT) – Liquid-Liquid Extraction (LLE) – Solid Phase Extraction (SPE) Selecting the Appropriate Sample Preparation Option – Application Example o Benzodiazepines and Metabolites in Plasma Conclusion

Analytes 26 compounds: benzodiazepines, metabolites and internal standards

Assay Use Screening

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

14, 15, 16, 17, 18, 19, 20, 21 100

1. 7-aminonitrazepam 2. 7-aminoclonazepam 3. 7-aminoflunitrazepam 4. Clozapine 1 10, 11, 12, 13 5. Midazolam 0.80 6. Chlordiazepoxide 7. Alpha-Hydroxymidazolam 8. Bromazepam 9. n-Desmethylflunitrazepam 10. Nitrazepam 26 11. Clonazepam d4 12. Clonazepam 13. Flunitrazepam 14. Triazolam % 15. 2-Hydroxyethylflurazepam 9 22, 23, 24 16. Hydroxyalprazolam d5 25 17. Alpha-Hydroxyalprazolam 18. Alprazolam 19. Alprazolam d5 20. Oxazepam 3 21. Clobazam 22. Estazolam 2 7 23. Desalkylflurazepam 4 6 24. Temazepam 25. Nordiazepam 26. Prazepam 5 8

0 Time 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

100

) 2) 4) ) ) 01) 3) 2) 06) 03) 09) 905) -910 -059) -90 -9 -919) -02 -902) F-907) (A-902) A-9 (A-90 (C-905) (C-907( (T-907) (E-901) (A-914) m (F-9 e (C m (M-908) d4 am (A-915) la m d5 pam p pam (A-912 pam (N- epam (P-9 epam (D epam (O azolam (H-90 azolam riazolamid (T azepam (B zepoxide (C traze T azolamClozapin d5 nazepam lflurazepa MidazoPraz Alprazolam ( Clobazam (C Est Oxaz Nitrazepamordiaze (N-906) rom Clo Temaze unitrazrdia N yalpr lonazepamB FlunitrazepamAlprazola inonitrazepam yethy C m Chlo aminoclonaze Desalkylflurazepam7- (D-915)aminofluni 7-a ha-Hydroxym Hydrox smethylfl 7- alp alpha-Hydroxyalprazolam (A-905) 2-hydrox n-De

Average recovery = 84%

Average % Standard curves were Compound Deviation r^2 value run from 1–500 ng/mL Triazolam 16.1 0.940 Alpha-hydroxymidazolam 13.0 0.957 R2 value was > 0.965 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 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

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 Removes vast majority of phospholipids – Improved instrument uptime – More robust methods

Assay Use Screening of analytes in urine

Analytes 10 compounds in “Bath Salts”

Assay Requirements A single, robust sample preparation method High throughput High recovery and sensitivity Low matrix interferences

Methedrone α-PPP α-PVP

C11 H15 NO 2 C13 H17 NO

C15 H21 NO

Mephedrone α-PVP metabolite

C H NO C11 H15 NO 15 23

Methylone Ethylone Butylone

C H NO C H NO 11 13 3 C12 H15 NO 3 12 15 3

MDPPP MDPV

C14 H17 NO 3 C16 H21 NO 3

Condition Plate 200 µL MeOH then 200 µL Water

Sample Pretreatment

100 µL pooled urine + 100 µL 4% H 3PO 4 Load 200 µL pretreated sample

Wash 200 µL 2% HCOOH, then 200 µL MeOH

Elute 2 x 50 µL

(60:40 ACN:IPA + 5% NH 4OH)

Neutralize with 5 µL of concentrated HCOOH; then dilute with 100 µL of water Inject 10 µL

Cone MRM Coll. Drug Alt Name RT Formula Mass Voltage Transitions Energy 3,4-methylenedioxy-N- 28 208.2→132.0 28 1 Methylone 0.75 C H NO 207.23 methylcathinone 11 13 3 28 208.2→160.0 18 30 222.3→174.1 20 2 Ethylone MDEC, bk-MDEA 0.83 C H NO 221.26 12 15 3 30 222.3→204.1 14 28 194.2→161.0 22 3 Methedrone 4-methoxymethcathinone 0.84 C H NO 193.25 11 15 2 28 194.2→146.0 30 42 204.3→105.0 24 4 α-PPP Alpha-Pyrrolidinopropiophenone 0.86 C H NO 203.28 13 17 42 204.3→98.0 28 3',4'-Methylenedioxy-α- 42 248.3→98.0 26 5 MDPPP 0.92 C H NO 247.29 pyrrolidinopropiophenone 14 17 3 42 248.3→147.0 24 32 222.3→174.1 18 6 Butylone Bk-MBDB 0.92 C H NO 221.26 12 15 3 32 222.3→204.1 15 26 178.2→145.0 22 7 Mephedrone 4-methylmethcathinone, 4-MMC 1.02 C H NO 177.24 11 15 26 178.2→91.0 34 38 232.4→91.0 26 8 α-PVP alpha -Pyrrolidinopentiophenone 1.66 C H NO 231.34 15 21 38 232.4→105.0 28 38 276.4→175.0 22 9 MDPV Methylenedioxypyrovalerone 1.78 C H NO 275.35 16 21 3 38 276.4→205.0 20 α-Pyrrolidinopentiophenone 30 234.4→72.0 20 10 α-PVP Met 1 2.00 C H NO 233.35 metabolite 1 15 23 30 234.4→173.0 24

1. Methylone 2. Ethylone 2,3 3. Methedrone 5 4. α-PPP 9 10 5. MDPPP 6. Butylone

%% 4 6 7. Mephedrone 8. α-PVP 9. MDPV 10. α-PVP Met 1

0 Time 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

LC System: ACQUITY UPLC

Column: ACQUITY BEH C 18 1.7 µm, 2.1 x 100 mm Mass spectrometer: XEVO ® TQD

130.0%

110.0%

90.0%

70.0%

50.0% Recovery Matrix Effects

30.0%

10.0%

-10.0%

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 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

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

Successful analysis of a panel of 10 synthetic cathinone drugs Rapid and simple sample preparation – 96-well plates utilized Achieved excellent recovery and sensitivity, while virtually eliminating matrix effects No evaporation and reconstitution steps necessary Possibility of using the same technique for related compounds

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 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

Questions? Landing Page… http://www.waters.com/May21 – Promotional Offers on Sample Preparation Products –Full Webinar Recording of Today’s Session –PDF Slide Deck –Compilation of KEY Application Notes, Literature, White Papers, Brochures etc… Questions and Submit your Ideas for our Next Topic – [email protected]