Thank you for joining us! Our session will begin shortly ...€¦ · Successful Sample Preparation...

Successful Sample Preparation Strategies for

LC/MS/MS Analysis of Drugs

in Complex Biological Matrices for Forensic

Toxicology Applications

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©2013 Waters Corporation 1

Toxicology Applications

Jonathan Danaceau, Ph.D.

Senior Applications Chemist

Waters Corporation

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Today’s SpeakerToday’s Speaker

Jonathan Danaceau, Ph.D.Senior Applications ChemistConsumable Business UnitWaters Corporation, Milford, MA USA

� Dr. Jonathan Danaceau is a Senior Applications Chemist at Waters


� Dr. Jonathan Danaceau is a Senior Applications Chemist at Waters Corp. He received his B.S. in Biology from Allegheny College (Meadville, PA) and his Ph.D. in Neuroscience from the University of Utah (Salt Lake City, UT). Jon has an extensive background in bioanalysis including experience in the pharmaceutical industry, forensic toxicology, and anti-doping analysis. Jon joined Waters’ Chemistry Applied Technology group in 2011 and currently focuses on sample preparation and chromatography solutions for various applications, including forensic toxicology and clinical research. He will highlight a number of methods today that he has developed directly.

OverviewOverview

� Goal of Sample Preparation

� Sample Preparation Options

� Application Examples– Opioids and metabolites in urine, whole blood and oral fluid

– Synthetic cannabinoids in urine and whole blood

– Synthetic cathinones “Bath Salts” in urine



– THC and metabolites in whole blood

� Summary

OverviewOverview



� Application Examples– Opioids and metabolites in urine, whole blood and oral fluid

– Synthetic cannabinoids in urine and whole blood




– THC and metabolites in whole blood

� Summary

Goal of Sample PreparationGoal of Sample Preparation

� Provides the target analyte(s) in solution

� Provides the analyte(s) at a concentration appropriate for detection or measurement– Concentrating the analyte helps increase sensitivity and achieve lower limits of detection

� Removes interfering matrix elements (such as phospholipids,


� Removes interfering matrix elements (such as phospholipids, salts, proteins, nucleic acids, sugars, etc.) that alter the MS response or co-elute with the target analyte– Matrix effects result in ion suppression (loss of signal) or ion enhancement (gain in signal)

– Matrix effects have a negative impact on the accuracy, precision, and robustness of the method; add to method variability

Importance of Clean SamplesImportance of Clean Samples

� Having cleaner samples means:– Better chromatography

– Lower limits of detection

– Decreases assay variability; more robust assay

o Reduced matrix effects

o Fewer reanalyses


o Fewer reanalyses

o Less chance of false positives/negatives

– Longer column lifetime

– Less instrument downtime

– Minimize costs in manpower and equipment maintenance

� Sample Prep makes your analytical lab more productive!

Sample Preparation: A Major BottleneckSample Preparation: A Major Bottleneck

� Typically the most difficult and time-consuming step

� It is the single biggest time constraint that labs face

� ~75% of the work activity and operating cost in an analytical lab is spent processing and preparing samples for injection� Choice of format can significantly improve workflow and decrease processing time


processing time

� Typically the least amount of effort is spent developing a rugged sample preparation method

Magical Method

Sample Preparation OptionsSample Preparation Options

Technique Advantages Disadvantages Appropriate Matrices

Dilution � Simple� Cheap� Easy to automate

� No cleanup� No enrichment� Non-selective

� Urine

Protein Precipitation

� Simple� Quick� Minimal method development

� Minimal selectivity; does not remove most matrix interferences

� No enrichment� Substantial solvent evaporation may be needed

� Whole blood, plasma, serum

Liquid-LiquidExtraction

� Offers better clean up than protein precipitation

� Less selective than SPE; does not remove endogenous phospholipids

� Urine, plasma,serum, oral fluid

Non-

selective


Extraction precipitation� Can be optimized for different compound classes

remove endogenous phospholipids� Cumbersome; requires user intervention

� Difficult to automate� Not ideal for highly polar drugs and metabolites

� Solvent evaporation needed

serum, oral fluid

Lipid/protein removal plates

� Simple, universal method� Quick� Minimal method development

� Minimal selectivity� No enrichment� Substantial solvent evaporation may be needed

� Whole blood, plasma, serum

Solid-Phase Extraction (SPE)

� Best cleanup option� Fast; easy to automate� Achieves the highest recovery and reproducibility

� Can be manipulated for optimum recovery and cleanup

� Variety of device formats and sorbent chemistries

� May require method development to optimize the protocol

� Perceived to be difficult and costly

� Urine, whole blood, plasma,serum, oral fluid

Highly

selective

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

� Cleanup of phospholipids and proteins in plasma and serum (also blood)– Fast, easy in-well protein precipitation; precipitated proteins and phospholipids are left behind in the wells

– Significant time savings; protocol eliminates extract transfer and evaporation steps (also in plate format)

– Generic protocol; no method development


– Generic protocol; no method development

� Extracts can be directly injected and analyzed

� Suitable for a wide variety of acidic, basic, and neutral compounds

Pass-through method

Phospholipids Remaining in the Extract:Phospholipids Remaining in the Extract:Ostro vs. LLE and PPTOstro vs. LLE and PPT

MRM of m/z 184-184

%

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

100 184.4 > 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

1.90

2.622.56 2.68

LLE 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 2.800

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™

� Ostro is superior to LLE and PPT for phospholipid removal

Phospholipid BuildPhospholipid Build--up Over Time: up Over Time: Ostro vs. PPTOstro vs. PPT


� After 200 injections, no phospholipid build up in the system when injecting samples extracted with Ostro plate

SolidSolid--Phase Extraction (SPE)Phase Extraction (SPE)

� SPE is gaining acceptance in clinical and forensic labs – Cleans up, concentrates, and produces a final sample that is in an appropriate solvent for further analysis

� Uses polymeric or silica-based chromatographic particles packed into a variety of formats– 96-well plates, cartridges, etc.


– 96-well plates, cartridges, etc.

� SPE is considered to be a very versatile sample preparation technique for various analytes in complex matrices– Blood, serum, plasma, oral fluid, tears, nasal fluid, CSF, urine, feces, meconium, postmortem samples, and many more!

� It’s the best technique for minimizing matrix interferences including proteins, phospholipids, salts, and other endogenous compounds

OasisOasis®® Family of Sorbents:Family of Sorbents:ReversedReversed--Phase and MixedPhase and Mixed--ModeMode

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

Oasis 2x4 MethodologyOasis 2x4 Methodology

� A simple, logical approach to the selection of an SPE sorbent and protocol

� Two protocols and four sorbents – For extraction of acids, bases, and neutrals


– Optimized to achieve high SPE recoveries while removing matrix components that may interfere with analysis

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.

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–Eluates can be directly injected


–Eluates can be directly injected

–Saves time

–No evaporative loss

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

� Compatible with most liquid handling robotic systems for automated high throughput SPE

Narrow and Tall bed

OverviewOverview



� Application Examples– Opioids and Metabolites in Urine

� Summary


OOpioids and Metabolites in Urinepioids and Metabolites in Urine

Assay Use

Quantification of opioids and metabolites in urine

Analytes

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


Goals

� Increase sample cleanliness, sensitivity and accuracy compared to sample dilution

� Accurate quantification of a comprehensive panel of opioid drugs and metabolites

� Direct analysis of glucuronide metabolites without hydrolysis

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

Comprehensive opioid panelComprehensive opioid panel

Natural opiates and metabolites

Semi-synthetic opioids

Synthetic narcotic analgaesics


10 Codeine

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

All bases→Oasis MCX

Extraction Methodologies for UrineExtraction Methodologies for Urine

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(Mixed-mode strong cation exchange SPE)

Sample Dilution Protocol

Inject 10 µL


300 µL pretreated sample

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

• A short evaporation step (<5 min) implemented to evaporate and reconstitute in the mobile phase

• Prevents solvent effects for early eluting compounds

Opioid ChromatographyOpioid Chromatography

Separation of isobaric compounds

(highlighted compounds)

13

17

Compound


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

ACQUITY BEH C18

1.7 µm, 2.1 x 100mm


1,

3

4,

97

6

12

15,16

21

18,1920

22

23

24

26

25

14

11

2

5

10

8

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

Time1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

% Recovery on % Recovery on Oasis MCX SPE Oasis MCX SPE 6 Lots of Urine6 Lots of Urine

60%

80%

100%

120%

%Recovery


0%

20%

40%

%Recovery

0.80

1.00

1.20

1.40

Matr

ix F

acto

rs ** ** *

*

*

**

**

*

� Mixed-mode SPE� Dilution

Improved Matrix Factors and %CV with Improved Matrix Factors and %CV with Oasis MCX SPE vs. dilution Oasis MCX SPE vs. dilution -- 6 Lots of Urine6 Lots of Urine


0.00

0.20

0.40

0.60

Matr

ix F

acto

rs

*

* Statistically significant difference in Matrix Factors (12/26 compounds) - reduced %CV in 23/26 compounds

QC Results QC Results –– Diluted UrineDiluted Urine

QC Concentration (ng/mL)

7.5 75 250 400

%CV Bias %CV Bias %CV Bias %CV Bias

Morphine-3-gluc 10.3% -5.7% 6.1% -2.3% 2.3% -4.2% 6.2% -5.0%

Oxymorphone-3-gluc 18.1% -8.7% 6.8% -2.8% 4.0% -8.1% 7.0% -8.5%

Hydromorphone-3-gluc 14.5% 3.3% 4.5% 4.1% 6.9% -5.4% 5.8% -9.3%

Morphine-6-gluc 23.1% 4.7% 17.5% -1.4% 9.3% -0.4% 3.5% -10.4%

Morphine 26.9% -29.7% 7.9% 1.3% 9.4% 7.0% 16.6% 2.7%

Oxymorphone 23.3% 19.7% 9.7% 9.9% 5.8% 0.7% 5.4% -10.0%

Hydromorphone 14.1% 8.3% 5.0% 5.7% 5.1% 0.7% 3.4% -4.7%

Codeine-6-β-d-gluc 11.5% -14.0% 7.0% -4.5% 8.0% -9.3% 4.4% -10.4%

Mean %CV @ 7.5 ng/mL = 10.2%11 compounds >10%


Codeine-6-β-d-gluc 11.5% -14.0% 7.0% -4.5% 8.0% -9.3% 4.4% -10.4%

Dihydrocodeine 9.4% 10.0% 8.0% 14.8% 5.6% -2.1% 5.3% -3.2%

Codeine 10.5% 5.3% 4.7% 2.0% 8.0% -5.5% 3.9% -8.5%

Oxycodone 20.4% 0.3% 6.8% 5.6% 3.4% -2.8% 3.4% -4.9%

6-Acetylmorphone 7.7% -13.3% 9.5% -8.9% 2.8% -13.8% 5.2% -7.1%

O-desmethyl Tramadol 3.6% -0.7% 4.9% 5.9% 3.3% -3.9% 2.5% -7.8%

Hydrocodone 8.2% -10.0% 3.6% -4.2% 6.4% -9.1% 5.8% -14.7%

Norbuprenorphine-gluc 5.3% -3.3% 2.7% 2.8% 5.0% -6.2% 3.0% -12.4%

Norfentanyl 11.2% -18.7% 3.7% 6.9% 3.7% -3.6% 0.6% -7.9%

Tramadol 1.5% -13.0% 3.6% -6.9% 1.3% -12.8% 0.8% -16.1%

Normeperedine 4.6% -0.7% 5.1% 5.7% 3.1% -6.2% 0.7% -10.8%

Meperidine 1.7% -2.3% 7.0% 3.2% 2.1% -5.5% 2.7% -8.2%

Buprenorphine-gluc 4.5% -36.0% 3.6% -12.3% 4.9% -15.6% 2.1% -18.2%

Norbuprenorphine 9.2% -4.7% 2.8% 6.2% 5.6% -3.0% 1.7% -9.0%

Fentanyl 3.3% -6.7% 2.9% 3.9% 3.9% -4.1% 1.4% -8.2%

Buprenorphine 6.4% -9.3% 3.8% 0.6% 3.7% -7.6% 2.3% -10.9%

EDDP+ 1.7% -0.7% 3.3% 4.4% 1.0% -4.3% 2.1% -8.7%

Propoxyphene 8.2% -6.7% 2.2% 1.2% 2.8% -8.1% 4.5% -12.5%

Methadone 6.0% -7.0% 2.5% 0.7% 3.4% -6.9% 4.4% -12.6%

QC Results QC Results –– Oasis MCX SPE Oasis MCX SPE


7.5 75 250 400

%CV Bias %CV Bias %CV Bias %CV Bias

Morphine-3-gluc 8.3% -5.3% 5.2% -0.7% 2.2% 0.0% 3.6% -3.4%

Oxymorphone-3-gluc 9.7% -1.0% 3.0% 2.5% 4.9% -4.0% 3.7% -7.0%

Hydromorphone-3-gluc 7.8% 6.3% 5.8% 1.9% 2.9% 0.9% 3.7% -0.5%

Morphine-6-gluc 8.7% 10.7% 6.7% -0.1% 5.1% -3.7% 4.0% -5.8%

Morphine 10.1% 8.7% 7.7% 0.8% 5.1% -13.2% 4.3% -2.2%

Oxymorphone 5.1% 4.7% 4.2% -2.3% 4.7% -2.6% 4.5% -3.6%

Hydromorphone 1.6% 5.7% 3.0% 0.9% 3.7% -0.9% 1.2% -2.8%

Codeine-6-β-d-gluc 4.0% 3.7% 3.8% -1.9% 5.0% 2.9% 2.6% 5.4%

Mean %CV @ 7.5 ng/mL= 3.5%1 point >10%


Codeine-6-β-d-gluc 4.0% 3.7% 3.8% -1.9% 5.0% 2.9% 2.6% 5.4%

Dihydrocodeine 0.8% 2.0% 1.1% 1.1% 0.6% -2.5% 2.8% -5.5%

Codeine 4.7% 2.3% 0.6% 1.1% 1.9% -1.9% 0.9% -3.7%

Oxycodone 5.2% 1.0% 2.3% 0.7% 3.4% -2.2% 2.8% -5.5%

6-Acetylmorphone 5.3% 2.7% 4.3% 1.6% 2.3% -1.7% 0.7% -2.1%

O-desmethyl Tramadol 1.9% 4.3% 1.3% 0.0% 0.7% -1.2% 0.7% -3.8%

Hydrocodone 1.9% 1.3% 1.3% -0.7% 1.6% -2.3% 0.9% -4.7%

Norbuprenorphine-gluc 3.6% 4.0% 3.1% 1.8% 3.9% 2.0% 1.3% 0.5%

Norfentanyl 0.0% 1.3% 2.3% 3.3% 1.2% 0.4% 2.2% 1.7%

Tramadol 0.0% 1.3% 0.3% 2.4% 0.8% -3.8% 0.5% -7.7%

Normeperedine 2.0% -0.3% 1.6% 0.4% 1.2% -4.5% 1.4% -7.2%

Meperidine 0.7% -1.0% 0.5% -2.5% 2.4% -3.1% 1.7% -3.0%

Buprenorphine-gluc 2.7% 7.7% 1.8% 3.7% 1.6% 6.8% 1.3% 10.3%

Norbuprenorphine 1.2% 3.0% 3.8% 3.6% 1.5% -1.6% 1.0% -5.7%

Fentanyl 0.0% 1.3% 1.1% 2.4% 1.0% -2.7% 1.0% -3.2%

Buprenorphine 2.3% 0.7% 1.9% 2.9% 1.9% -1.1% 1.3% -0.7%

EDDP+ 1.3% 2.0% 1.1% 0.0% 0.9% -2.7% 1.1% -3.1%

Propoxyphene 0.8% 0.7% 0.5% 4.5% 0.9% -2.6% 1.9% -5.3%

Methadone 0.7% 1.0% 1.5% 4.3% 1.0% -1.4% 1.2% -3.4%

Reduced Total AnalyticalError vs. Dilute and Shoot

OverviewOverview



� Application Examples– Opioids and Metabolites in Whole Blood

� Summary


OOpioids and Metabolites in Whole Bloodpioids and Metabolites in Whole Blood

Assay Use

� Quantitative screening of opioids and metabolites in whole blood

– Post-mortem analysis, OUI

Analytes

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


analgesic compounds

Goals

� Clean up complex matrix– Proteins, cellular debris, phospholipids, etc.

� Simple, in-well procedure

� High throughput compatibility– In-well lysis and PPT, 96-well format

Whole Blood Whole Blood Extraction Methodology Extraction Methodology with Ostro Plate*with Ostro Plate*

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

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

Add 600 µL of ACN containing IS to all wells

Vortex for 3 minutes


* Similar protocol to the generic Ostro protocol for plasma and serum but with some modifications to account for the use of whole blood.


Evaporate to dryness under N2

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

Inject 10 µL

Elute into a 96-well collection plate

� Simple, in-well lysis and PPT- No need to transfer supernatant

� 96-well format- High throughput

� Phospholipid and protein removal- Cleaner than PPT alone

% Recovery on % Recovery on Ostro Plate from Ostro Plate from Whole BloodWhole Blood

60%

80%

100%

120%

Opioid Recovery in WB


0%

20%

40%

60%

Opioid Linearity, Accuracy, and Opioid Linearity, Accuracy, and Precision in Whole BloodPrecision in Whole Blood

% Accuracy %CV

R2 Mean S.D. Mean S.D.

Morphine-3-β-d-glucuronide 0.985 99.7% 4.9% 9.6% 4.6%

Oxymorphone-3-b-d-glucuronide 0.983 100.7% 4.7% 8.4% 6.6%

Hydromorphone-3-b-d-glucuronide 0.986 100.8% 8.3% 8.2% 3.8%

Morphine 0.986 101.0% 7.4% 10.0% 2.9%

Oxymorphone 0.989 98.9% 6.5% 5.7% 3.3%

Hydromorphone 0.988 99.4% 8.1% 4.6% 2.1%

Codeine-6-β-d-glucuronide 0.973 99.8% 13.5% 6.5% 4.2%

Dihydrocodeine 0.984 99.8% 11.4% 7.0% 2.2%

5-500 ng/mL; N=3 replicates/curve point

93% of curve points within 15% of nominal value


Codeine 0.979 101.1% 14.1% 4.3% 2.2%

Oxycodone 0.986 99.0% 12.4% 4.4% 3.6%

6-Acetylmorphone (6-AM) 0.984 100.4% 7.1% 11.7% 3.4%

O-desmethyl Tramadol 0.990 100.3% 6.7% 5.2% 2.6%

Hydrocodone 0.990 100.5% 6.8% 5.5% 4.8%

Norbuprenorphine-glucuronide 0.989 101.0% 7.4% 11.1% 6.9%

Tramadol 0.988 100.5% 10.2% 3.4% 1.9%

Normeperedine 0.995 100.4% 4.9% 4.2% 2.6%

Meperidine 0.994 100.3% 6.4% 4.2% 2.3%

Norbuprenorphine 0.989 100.5% 7.6% 6.6% 4.4%

Fentanyl 0.992 99.6% 5.8% 5.2% 2.3%

Buprenorphine 0.994 100.3% 5.5% 5.5% 2.3%

Propoxyphene 0.990 100.5% 7.5% 4.2% 1.7%

Methadone 0.994 100.3% 6.3% 2.8% 0.4%

Mean accuracy and precision (%CV) of opioid drugs and metabolites extracted from whole blood samples N=9 curve points from 5-500 ng/mL. R2 values are listed to the right of compound names.

95% of %CVs <15%

OverviewOverview



� Application Examples– Opioids and Metabolites in Oral Fluid

� Summary


OOpioids and Metabolites in Oral Fluidpioids and Metabolites in Oral Fluid

� Oral fluid advantages– Increasingly popular techniques

– Non-invasive

– Easily observed collection

– Reflective of recent use/impairment

– Can be correlated with plasma concentrations


– Can be correlated with plasma concentrationso Bases may be present at higher conc. than plasma

� Oral fluid considerations– Limited sample volume

– Contamination from oral administration

– Collection challenges (devices/inconsistency)

– Protein content of oral fluid (0.3%)

– Salts, matrix components from stabilization buffers

OOpioids and Metabolites in Oral Fluidpioids and Metabolites in Oral Fluid

Assay Use

Quantification of opioids and metabolites in oral fluid

Analytes

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


Goals

� Accurate quantification of a comprehensive panel of opioid drugs and metabolites

� Remove salts, sample stabilization buffer components, proteins, other oral fluid matrix components

� Format appropriate for small sample volume

Extraction Methodology for Oral FluidExtraction Methodology for Oral Fluid


Sample Pretreatment1 mL oral fluid + 3 ml stabilizing buffer*Remove a 400 µL aliquot (100 µL oral fluid)

Add 200 µL of 4% H3PO4 + 20 µL IS (500 ng/mL)

Load

Oasis MCX µElution 96-Well Plate(Mixed-mode strong cation exchange SPE)



Wash200 µL of 2% formic acid; then 200 µL MeOH;

then 200 µL IPA

Elute2 x 50 µL

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

Evaporate under N2 @ 37oC

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

Inject 10 µL* Oral fluid collected using QuantiSal from Immunalysis.

• Selective cleanup for basic compounds

• µElution format Designed for small volumes• Samples and elution volumes

%Recovery and Matrix EffectsUsing Oasis MCX µElution Plate

60%

80%

100%

120%

140%

% R

ecovery

Opiates in Oral Fluid


Average Recovery = 79% - enables analyte quantification at desired levels

0%

20%

40%

60%

% R

ecovery

Oasis MCX µElution PlateLinearity and QC Results - Oral fluid


7.5 25 150 300

Compound R2 %CV Bias %CV Bias %CV Bias %CV Bias

Morphine-3-β-d-gluc 0.995 10.2% 14.4% 3.5% 9.0% 6.8% 5.3% 3.3% 2.0%

Oxymorphone-3-b-d-gluc 0.994 14.4% 14.9% 5.9% -0.8% 3.8% 11.2% 1.9% 4.2%

Hydromorphone-3-b-d-gluc 0.992 8.2% 8.0% 5.4% 2.2% 7.2% 4.9% 3.9% 2.5%

Morphine-6-gluc 0.993 17.4% 0.8% 6.4% 2.4% 4.6% 3.6% 3.9% 3.3%

Morphine 0.989 15.3% 19.7% 2.7% 18.2% 12.2% 11.6% 6.9% 5.9%

Oxymorphone 0.997 9.2% 2.7% 6.4% 3.3% 2.6% 4.1% 2.7% 5.1%

Hydromorphone 0.997 7.7% 1.1% 3.6% 5.1% 3.2% 5.4% 3.8% 6.4%

Codeine-6-β-d-gluc 0.993 2.6% -7.3% 5.2% 1.9% 3.9% -3.8% 6.0% 5.7%

Dihydrocodeine 0.996 2.3% 6.7% 3.6% 11.4% 2.7% 4.4% 2.2% 1.5%

Codeine 0.994 8.7% 7.2% 3.7% 11.7% 3.8% 4.3% 3.9% 1.4%

5-500 ng/mLCalib. Curve

Mean % Bias = 5.3%

Mean %CV = 4.5%


Codeine 0.994 8.7% 7.2% 3.7% 11.7% 3.8% 4.3% 3.9% 1.4%

Oxycodone 0.996 7.0% 5.3% 5.6% 10.6% 5.1% 7.5% 2.7% 2.2%

6-Acetylmorphone (6-AM) 0.996 5.3% 5.4% 3.6% 8.5% 3.6% 3.3% 7.1% 4.5%

O-desmethyl Tramadol 0.999 5.6% 6.1% 2.5% 7.7% 2.1% 5.8% 1.7% 5.4%

Hydrocodone 0.998 5.6% 6.4% 3.4% 4.6% 2.7% 4.7% 3.0% 6.6%

Norbuprenorphine-gluc 0.992 2.5% -11.4% 2.8% 1.7% 7.1% -4.9% 5.9% 8.8%

Norfentanyl 0.998 7.0% 0.8% 3.9% 8.3% 2.9% 2.6% 3.3% 4.9%

Tramadol 0.999 4.8% 6.4% 3.1% 8.8% 2.6% 6.7% 2.2% 4.8%

Normeperedine 0.999 4.8% -0.7% 3.3% 3.5% 2.2% 3.1% 2.8% 2.4%

Meperidine 0.999 5.5% 5.2% 4.1% 4.9% 2.6% 6.6% 2.5% 6.2%

Buprenorphine-gluc 0.999 4.8% -4.5% 7.0% 2.2% 3.9% 1.1% 3.7% 7.1%

Norbuprenorphine 0.996 5.9% 5.4% 3.6% 8.3% 2.3% 4.8% 1.5% 2.9%

Fentanyl 0.999 4.6% 4.8% 2.5% 7.4% 2.7% 6.8% 1.5% 6.4%

Buprenorphine 0.999 4.5% 6.5% 2.8% 8.1% 3.0% 7.9% 1.5% 7.5%

EDDP+ 0.999 4.7% 4.8% 2.4% 5.8% 2.7% 6.8% 2.5% 7.3%

Propoxyphene 0.999 3.8% 6.8% 3.0% 8.6% 2.4% 7.0% 2.2% 7.0%

Methadone 0.999 5.3% 6.1% 3.2% 8.0% 3.0% 6.8% 2.4% 6.5%

All R2 Values ≥ 0.99

Sample Preparation Strategies for Opioids - Conclusions

� Urine and Oral Fluid– Highly selective clean up – Mixed mode SPE (MCX)

– µElution format ideal for limited sample volume (OF)

– Excellent linearity, analytical accuracy, and precision.

� Whole Blood– Clean up highly complex matrix (Ostro)


– Clean up highly complex matrix (Ostro)

– Removal of proteins, cellular debris, and phospholipids

– Rapid method with minimal method development

OverviewOverview



� Application Examples– Synthetic Cannabinoids in Urine

� Summary


Synthetic CannabinoidsSynthetic Cannabinoids

� Designer drugs that mimic the psychoactive effects of natural cannabinoids– Often referred to or marketed as “Spice” compounds

– Labeled as “not for human consumption” and marketed as a legal alternative to natural cannabis

– Popularity has risen substantially in the last several years


� A growing challenge for law enforcement agencies and forensic laboratories

� Recent legislation has banned some of these compounds, but minor modifications are made to existing structures to circumvent existing laws

� Quantitative analysis in urine and whole blood

Synthetic Cannabinoids Synthetic Cannabinoids in Urinein Urine

Assay Use

Quantification of synthetic cannabinoids in urine

Analytes

22 synthetic cannabinoids and metabolites

Goals


Goals

� Accurate quantification of all compounds

� Universal method to extract neutral, acidic and basic compounds

� Optimize recovery and minimize matrix effects

� Adequate separation of isobaric metabolites

Synthetic Cannabinoids in UrineSynthetic Cannabinoids in Urine

� Oasis HLB µElution– Need to extract neutrals, bases, and acidic metabolites

– Clean up urine matrix

– Concentrate samples

o No need for evaporation

– High recovery and sensitivity


– High recovery and sensitivity

– High throughput

Synthetic Cannabinoids and MetabolitesSynthetic Cannabinoids and Metabolites

No. Compound

1 AM2233

2 RCS-4, M10

3 RCS-4, M11

4 AM 1248

5 JWH-073 4-butanoic acid met.

6 JWH-073 4-hydroxybutyl met.

7 JWH-018 5-pentanoic acid met.

8 JWH-073 (+/-) 3-hydroxybutyl met.

Acids

Bases

Neutrals


9 JWH-018 5-hydroxypentyl met.

10 JWH-018 (+/-) 4-hydroxypentyl met.

11 JWH-015

12 RCS-4

14 JWH-022

13 JWH-073

15 XLR-11

16 JWH-203

17 JWH-018

18 RCS-8

19 UR-144

20 JWH-210

21 AB 001

22 AKB 48

Extraction Methodology with the Extraction Methodology with the Oasis HLB µElution PlateOasis HLB µElution Plate


Sample Pretreatment• Mix 1 mL urine + 0.5 ml of 0.8 potassium

phosphate (pH 7.0)• Add 10 µL of β-glucuronidase and incubate at

40˚C for 1 hr• Add 1.5 mL of 4% H3PO4

Load


Load 600 µL pretreated sample (200 µL urine)

Wash200 µL water, then 200 µL 50:50 H2O:MeOH

Elute2 x 50 µL 60:40 ACN:IPA

Dilute with 75 µL of water

Inject 5 µL

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50

%

0

100

Chromatogram for 22 SyntheticChromatogram for 22 SyntheticCannabinoids and Metabolites Cannabinoids and Metabolites

1) 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

3

21 4

5

6

7

89, 10

Baseline separationof isobaric metabolites


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

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

11) JWH-01512) 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

Column: CORTECS UPLC C18, 2.1 x 100 mm; 1.6 µm

Recovery and Matrix Effects from Urine:Recovery and Matrix Effects from Urine:Oasis HLB µElution PlatesOasis HLB µElution Plates

-20.0%

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

120.0%Recovery

Matrix Effects


-80.0%

-60.0%

-40.0%

-20.0%

� Average recovery was 74%.

� Matrix effects ranged from -49% (ion suppression) to 32% (enhancement), although most were less than 20%.

� Even in instances in which recovery was comparatively low, there was more than adequate sensitivity for the purposes of this assay.

Accuracy, Precision, and SensitivityAccuracy, Precision, and Sensitivity

QC concentration (ng/mL)

2.5 7.5 75

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

AM2233 0.996 95.25 7.59 109.78 8.45 102.48 6.52 102.50

RCS4, M10 0.998 99.00 3.65 103.13 1.97 96.63 4.33 99.58

RCS4, M11 0.999 102.30 2.40 103.53 0.68 96.43 4.11 100.75

AM 1248 0.987 111.43 3.79 110.70 1.73 98.98 2.97 107.03

JWH-073 4-COOH 0.997 104.68 3.29 108.43 1.13 94.58 3.87 102.56

JWH-073 4-OH Butyl 0.999 105.40 2.53 110.30 0.99 93.78 2.59 103.16

JWH-018, 5-COOH 0.998 102.10 4.94 104.53 1.62 97.53 4.61 101.38

JWH-073, 3-OH Butyl 0.999 103.63 3.89 108.00 0.35 98.95 2.47 103.53

Calibration range1-100 ng/mL

• All accuracies within 15% of expected values.


JWH-018, 5-OH Met 0.999 103.40 4.65 107.40 1.88 100.58 3.50 103.79

JWH-018, 4-OH Met 0.999 103.63 2.11 108.60 1.15 100.70 2.75 104.31

JWH-015 0.994 96.65 3.39 99.53 1.81 93.23 3.60 96.47

RCS-4 0.992 98.05 2.27 97.88 2.24 91.85 3.02 95.93

JWH-022 0.993 100.80 3.69 93.50 5.63 93.28 5.68 95.86

JWH-073 0.982 95.48 7.19 88.30 4.51 103.23 6.01 95.67

XLR-11 0.987 105.20 8.37 103.55 1.96 90.85 2.87 99.87

JWH-203 0.990 97.35 5.39 85.65 2.85 93.65 3.00 92.22

JWH-018 0.996 98.48 2.11 86.60 9.38 95.95 6.25 93.68

RCS-8 0.992 98.58 4.09 93.48 10.85 96.23 6.38 96.09

UR-144 0.989 114.30 9.22 94.35 4.15 94.65 2.31 101.10

JWH-210 0.991 89.95 10.86 90.78 14.52 99.80 8.28 93.51

AB 001 0.988 100.28 4.02 86.38 9.66 97.45 5.96 94.70

AKB 48 0.985 104.28 3.58 87.55 5.79 94.35 5.07 95.39

• Most % RSDs less than 10% and none greater than 15%.

• Limits of detection were as low as 0.1 ng/mL ; none greater than 2 ng/mL.

OverviewOverview



� Application Examples– Synthetic Cannabinoids in Whole Blood

� Summary


Synthetic Cannabinoids in Whole BloodSynthetic Cannabinoids in Whole Blood

Assay Use

Quantification of synthetic cannabinoids in Whole Blood

Analytes

22 Synthetic cannabinoids and metabolites


Goals

� A single, robust sample preparation method for synthetic cannabinoid compounds and metabolites (combination of neutral, acidic, and basic compounds)

� High throughput

� High recovery and sensitivity

� Accurate, linear and precise responses

Whole Blood Whole Blood Extraction Methodology Extraction Methodology with Ostro Plate*with Ostro Plate*

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

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

Add 600 µL of ACN containing IS to all wells



* Similar protocol to the generic Ostro protocol for plasma and serum but with some modifications to account for the use of whole blood.


Inject 10 µL

Elute into a 96-well collection plate

% Recovery and matrix effects of % Recovery and matrix effects of Synthetic Cannabinoids from Whole Synthetic Cannabinoids from Whole Blood Using Ostro PlatesBlood Using Ostro Plates

30.0%

50.0%

70.0%

90.0%

110.0%

130.0%

Recovery

Matrix Effect


-30.0%

-10.0%

10.0%

30.0%Matrix Effect

� An average recovery of 92% for all compounds.

� Matrix effects were minimal.

Linearity and Analytical SensitivityLinearity and Analytical Sensitivity

QC concentrations (ng/mL)

7.5 75 300

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

Mean %

Acc.

AM2233 0.997 100.5 2.0% 103.6 3.3% 100.5 2.0% 101.5

RCS4, M10 0.986 97.5 3.9% 106.1 5.7% 101.7 8.4% 101.7

RCS4, M11 0.991 91.3 16.3% 108.8 5.1% 96.8 12.0% 98.9

AM 1248 0.993 83.1 10.0% 106.1 5.7% 105.4 6.4% 98.2

JWH-073 4-COOH 0.991 96.1 9.8% 99.3 7.4% 106.2 9.1% 100.5

JWH-073 4-OH Butyl 0.996 88.7 21.3% 98.1 3.5% 102.2 3.9% 96.3

JWH-018, 5-COOH 0.992 90.7 15.2% 97.8 3.8% 103.7 10.6% 97.4

JWH-073, 3-OH Butyl 0.993 79.0 8.6% 92.9 8.3% 96.6 2.9% 89.5

� R2 values of >0.99 for 21 of the 22 compounds


JWH-073, 3-OH Butyl 0.993 79.0 8.6% 92.9 8.3% 96.6 2.9% 89.5

JWH-018, 5-OH Met 0.995 82.8 10.3% 100.0 10.4% 100.1 3.4% 94.3

JWH-018, 4-OH Met 0.992 82.3 17.9% 103.1 6.3% 96.0 1.9% 93.8

JWH-015 0.993 87.1 4.3% 101.8 3.9% 101.3 2.1% 96.8

RCS-4 0.993 92.5 8.1% 99.6 5.0% 97.3 3.6% 96.4

JWH-022 0.993 85.3 4.9% 100.3 4.8% 97.8 4.2% 94.5

JWH-073 0.994 89.6 6.5% 99.4 6.6% 97.6 4.9% 95.5

XLR-11 0.993 101.4 10.4% 99.6 2.8% 99.7 5.0% 100.2

JWH-203 0.990 82.1 12.2% 96.1 12.2% 94.6 9.3% 91.0

JWH-018 0.994 88.4 2.9% 97.2 3.9% 98.8 3.6% 94.8

RCS-8 0.992 94.3 2.6% 101.9 4.6% 99.4 4.7% 98.5

UR-144 0.994 85.1 5.4% 97.0 6.7% 99.2 3.7% 93.8

JWH-210 0.994 92.7 6.4% 96.3 4.5% 95.6 5.3% 94.8

AB 001 0.992 84.4 8.1% 101.0 4.7% 100.2 10.6% 95.2

AKB 48 0.992 92.8 9.9% 98.5 4.8% 97.7 8.4% 96.4

Mean %

Acc.89.4 100.2 99.5

� Most % CVs less than 10% and none greater than 13%.

� Accurate at all QC levels

Synthetic Cannabinoids Synthetic Cannabinoids -- ConclusionsConclusions

� Urine– Single method for extracting neutral, acidic, and basic compoundso Possibility of using the same technique for related compounds

– Removes salts, enzymes, and buffers– Rapid and simple sample preparation

o 96-well plates utilized– Achieved excellent recovery and sensitivity


– Achieved excellent recovery and sensitivity– No evaporation and reconstitution steps necessary

o µElution format

� Whole Blood– Clean up highly complex matrix (Ostro)– Removal of proteins, cellular debris, and phospholipids– Rapid, universal method with minimal method development

– Excellent recovery with minimal matrix effects– Excellent accuracy and precision

OverviewOverview



� Application Exampleso Bath Salts in Urine

� Conclusion


“Bath Salts”“Bath Salts”

� Synthetic cathinones; variations of the chemical cathinone– central nervous system stimulants

– mimic the effects of drugs such as amphetamine, methamphetamine, cocaine, MDMA, etc.

� Often labeled as “not for human consumption”

� New drugs with modifications to existing cathinone structures


� New drugs with modifications to existing cathinone structures are constantly being developed and marketed – Intended to circumvent drug of abuse legislation aimed at specific compounds

“Bath Salts” “Bath Salts” in Urinein Urine

Assay Use

Quantification of “bath salt” compounds in urine

Analytes

10 synthetic cathinones and metabolites

Goals


Goals

� Accurate quantification of all compounds

� Targeted method to extract basic compounds

� Optimize recovery and minimize matrix effects

� Baseline separation of isobaric metabolites

Extraction Methodology for UrineExtraction Methodology for Urine


Sample Pretreatment100 µL pooled urine + 100 µL 4% H3PO4

Load

Oasis MCX µElution Plate Protocol(Mixed-mode strong cation exchange SPE)



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

Oasis MCX for basic compounds

Bath Salt CompoundsBath Salt Compounds

Drug Alt Name

1 Methylone3,4-methylenedioxy-N-

methylcathinone

2 Ethylone MDEC, bk-MDEA

3 Methedrone 4-methoxymethcathinone

4 α-PPP Alpha-Pyrrolidinopropiophenone


4 α-PPP Alpha-Pyrrolidinopropiophenone

5 MDPPP3',4'-Methylenedioxy-α-

pyrrolidinopropiophenone

6 Butylone Bk-MBDB

7 Mephedrone 4-methylmethcathinone, 4-MMC

8 α-PVP alpha-Pyrrolidinopentiophenone

9 MDPV Methylenedioxypyrovalerone

10 α-PVP Met 1α-Pyrrolidinopentiophenone

metabolite 1

Chromatography of Bath SaltsChromatography of Bath Salts% 4

2,3

5

6

8

9 10

1) Methylone

2) Ethylone

3) Methedrone

4) α-PPP

5) MDPPP

6) Butylone

7) Mephedrone

8) α-PVP

9) MDPV


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 9) MDPV

10) α-PVP Met 1

• Baseline resolution of isobaric compounds (ethylone, butylone)

• 2-min analysis time

LC System: ACQUITY UPLCColumn: ACQUITY BEH C18 1.7 µm,

2.1 x 100 mmMass spectrometer: XEVO® TQD

Recovery and Matrix EffectsRecovery and Matrix EffectsMeOH ElutionMeOH Elution

20.0%

40.0%

60.0%

80.0%

100.0%

120.0%

Recovery


-60.0%

-40.0%

-20.0%

0.0%Matrix Effects

Generic elution solvent (60:40 ACN:MeOH + 5% NH4OH)Can we improve on this?

Recovery and Matrix EffectsRecovery and Matrix EffectsIPA ElutionIPA Elution

40.0%

60.0%

80.0%

100.0%

120.0%

Recovery


� Replacing MeOH with IPA in final elution step improves recovery and eliminates most of the matrix effects

� All matrix effects <12.5%

-20.0%

0.0%

20.0%

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


� 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

Conclusions Conclusions –– “Bath Salts” with MCX“Bath Salts” with MCX

� 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– Simple change in elution co-solvent from MeOH to IPA


– Simple change in elution co-solvent from MeOH to IPA

� No evaporation and reconstitution steps necessary

OverviewOverview



� Application Examples– THC and Metabolites in Blood

� Summary


THC THC and Metabolites in Whole Bloodand Metabolites in Whole Blood

Assay Use

Highly sensitive assay for THC and metabolites in whole blood

Analytes

THC, COOH-THC, OH-THC

Goals


Goals

� Clean up whole blood matrix

� Maximize recovery and minimize matrix effects

� Need excellent linearity and sensitivity

� Selective extraction of acidic compounds (Oasis MAX)

� Short analytical run times

� Elimination of derivatization step prior to analysis

� Improved sample throughput vs. GC/MS

Sample PreparationSample Preparation

Condition Plate1 mL MeOH then 1 mL 1% NH4OH

Load 1 mL prepared sample

Wash

Oasis MAX Cartridge Protocol(Mixed-mode strong anion exchange SPE)

Sample0.2 mL whole blood

Precipitate 0.4 mL ACN (added dropwise)

Centrifuge

Sample pretreatment(PPT and pretreatment)


Wash0.5 mL 50% ACN

Elute1.5 mL of 49:49:2

hexane:ethyl acetate:acetic acid

Evaporate under N2 at 40 ˚C

Reconstitute in 0.133 mL70% aqueous MeOH

Inject 15 µL

Centrifuge400 x g for 10’

Transfer SupernatentTransfer supernatent (0.4 mL)

To 0.6 mL 1% NH4OH

THC ChromatographyTHC Chromatography0.5 ng/0.5 ng/mLmL in Whole Bloodin Whole Blood

� LC System: ACQUITY UPLC

� Column: ACQUITY BEH C18 1.7 µm, 2.1 x 100 mm

� Flow: 400 µL/min

� MPA: 0.1% formic acid

� MPB: ACN

� Gradient: 60% B to 90% B over 4 minutes

� Mass spectrometer: XEVO® TQ-S

� Ionization: ESI +

Acquisition: MRM

THC-COOH qualifier ion

THC-COOH quantifier ion

THC-OH qualifier ion


� Acquisition: MRM

� Calibration Range: 0.5-50 ng/mL

THC-OH qualifier ion

THC quantifier ion

THC qualifier ion

THC-OH quantifier ion

Mean recovery and matrix effectsMean recovery and matrix effects6 lots of whole blood6 lots of whole blood

60.0

80.0

100.0

Recovery andMatrix Effects


-20.0

0.0

20.0

40.0

THC-OH THC-COOH THC

Recovery

Matrix Effects

Mean % recovery and matrix effects for cannabinoids spiked in whole blood at low (0.5 ng/mL), medium (5.0 ng/mL) , and high (25 ng/mL) concentrations. The error bars are standard deviations.

InterInter--Day Accuracy and PrecisionDay Accuracy and Precision

Inter-day Accuracy% Target

Inter-day Precision%RSD

3.33 ng/mL

16.67ng/mL

33.33ng/mL

3.33 ng/mL

16.67ng/mL

33.33ng/mL

THC-OH 104.1 100.9 97.5 5.0 4.5 6.3

THC-COOH 102.7 99.2 96.5 6.7 4.4 3.8


� Inter-day accuracy and precision assessed by analyzing three quality control (QC) concentrations over 5 different days.

� The mean achieved values for the QC replicates over the 5-day period at the three concentration levels were within 10% of target, and the %RSD was <10%.

THC 106.5 102.5 97.7 5.8 4.7 4.3

UPLC/MS/MS vs. GC/MS/MS Analysis of UPLC/MS/MS vs. GC/MS/MS Analysis of Cannabinoids in Whole BloodCannabinoids in Whole Blood

� Excellent correlation with an alternative GC/MS/MS method for the analysis of cannabinoids in human whole blood samples

� R2 values between the two


� R2 values between the two data sets ranged from 0.9178 for THC-COOH to 0.9961 for THC-OH.

ConclusionsConclusions

� The challenges posed by the matrix (whole blood) and analytical requirements were best met with a two step process– PPT followed by MAX

� Excellent sensitivity, linearity, accuracy and precision

� Minimal matrix effects

� Rapid analytical run time


� Rapid analytical run time

� Excellent correlation with existing GC/MS method without derivatization

OverviewOverview



� Application Examples

� Summary


Summary of Applications andSummary of Applications andSample Preparation StrategiesSample Preparation Strategies

Application Matrix Solution Benefits

Opiates Urine Oasis MCX µElution

• Good recovery for all compounds• Improved linearity, accuracy and precision vs Dilution• Reduced matrix effects

Whole Blood Ostro • Remove phospholipids• Fast and easy – Minimal method development

Oral Fluid Oasis MCX µElution



Best sample prep strategy reduces downtime, reanalysis, false positive/negativesIncreases overall productivity








Synthetic Urine Oasis HLB • Sorbent appropriate for multiple chemotypes


Synthetic Cannabinoids

Urine Oasis HLB µElution

• Sorbent appropriate for multiple chemotypes• Linear, accurate and precise• Effective cleanup• Concentration without evaporation

Whole Blood Ostro • Protein and phospholipid removal• Linear, accurate and precise• Minimal matrix effects• Fast and Easy – Minimal method development















“Bath Salts” Urine Oasis MCX µElution

• Excellent recovery and minimal matrix effects• Linear, accurate and precise• Concentration without evaporation















“Bath Salts” Urine Oasis MCX µElution

• Excellent recovery and minimal matrix effects• Linear, accurate and precise• Concentration without evaporation

THC and Metabolites

Whole Blood PPT and Oasis MAX

• Sensitive, linear, accurate and precise• Good recovery and minimal matrix effects


AcknowledgementsAcknowledgements

Nebila Idris

Erin Chambers

Michelle Wood

Robert Lee

Greg Whitney


Thank You!Thank You!

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