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Powerful Sample Prep and LC Column Solutions for Forensic Toxicology Applications

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Solutions for Forensic Toxicology Applications

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About Today’s Presenter…About Today’s Presenter…

� Jonathan Danaceau, Ph.D., Senior Applications Chemist, Waters Corporation

� Dr. Jonathan Danaceau is a Senior Applications Chemist within Waters’ Consumable Business Unit. Jon received his B.S. in Biology from Allegheny College (Meadville, PA) and his Ph.D. in Neuroscience from the University of Utah (Salt


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 where he has been focusing on sample preparation and chromatography solutions for various applications, including forensic toxicology and clinical research.

Sample Preparation and LC Column Solutions

for Forensic Toxicology Applications


Jonathan Danaceau, Ph.D.

Senior Applications Chemist

Waters Corporation

OverviewOverview

� Goal of Sample Preparation

� Sample Preparation Options

� Application Examples– Opioids and metabolites in urine and oral fluid

– Synthetic cannabinoids in urine and whole blood

– THC and metabolites in whole blood



– Expanded Toxicology Panel from Urine

� Summary

OverviewOverview



� 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


– Synthetic cathinones “Bath Salts” in urine


� 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 OptionsSample Preparation Options

Technique Advantages Disadvantages Appropriate Matrices

Dilution � Simple� Cheap� Easy to automate

� No cleanup� No enrichment� Non-selective

� Urine

Non-

selective


Highly

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

Non-

selective


Highly

selective





� Urine






Lipid/protein removal

� Simple, universal method� Quick

� Minimal selectivity� No enrichment


Non-

selective


removal plates

� Quick� Minimal method development


plasma, serum

Highly

selective





� Urine










Non-

selective


removal plates



plasma, serum

Liquid-LiquidExtraction

� Offers better clean up than protein precipitation

� Can be optimized for different compound classes

� Less selective than SPE; does not remove endogenous phospholipids

� Cumbersome; requires user intervention

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

� Solvent evaporation needed

� Urine, plasma,serum, oral fluid

Highly

selective





� Urine










Non-

selective


removal plates



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

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

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

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 often 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

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 analgesics


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

UPLC Conditions

Column ACQUITY BEH C18 1.7 µm; 2.1 x 100

LC ACQUITY UPLC

Flow Rate 0.4 mL/min

MPA Water with 0.1% FA

MPB ACN with 0.1% FA

Column Temp 30 ˚C

MS Conditions

XEVO® TQD ESI+

Capillary: 1.0 kV

Source Temp: 150 ˚C

Desolvation Temp: 350 ˚C

Cone Gas Flow: 50 L/Hr

Desolvation Gas Flow: 900 L/Hr

UPLC and MS conditionsUPLC and MS conditions


Column Temp 30 ˚C

Inj. Vol 10 µL


MRM Transitions optimized individually

Time Flow %A %B Curve

0 0.4 98 2 6

6 0.4 47.2 52.8 6

6.5 0.4 98 2 11

8 0.4 98 2 11

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)


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

Matrix Factors

**

*

� 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

Matrix Factors

**

* **

*

*

*

*

* 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

QC Concentration (ng/mL)

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 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

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)


Load 100 µL pretreated sample

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

60%

80%

100%

120%

Recovery of Opioids from Oral Fluid

%Recovery Using Oasis MCX µElution Plate


0%

20%

40%

Average Recovery = 91%

Linearity and calibration curve results

Compound R2 Max. %CV Max. %Bias LOQ (ng/mL)

Morphine 0.989 16.4% 9.7% 5

Oxymorphone 0.997 9.4% 1.9% 5

Hydromorphone 0.997 11.8% 1.6% 5

Dihydrocodeine 0.996 6.5% 5.9% 5

Codeine 0.994 6.7% 5.9% 5

Oxycodone 0.996 5.7% 6.8% 5

6-Acetylmorphone (6-AM) 0.996 7.6% 4.2% 5

O-desmethyl Tramadol 0.999 4.3% 1.7% 5

Hydrocodone 0.998 5.0% 2.3% 5

5-500 ng/mL Calib. Curve

(1.25-125 for Fentanyl andnorfentanyl

All R2 Values ≥ 0.99


Hydrocodone 0.998 5.0% 2.3% 5

Norfentanyl 0.998 4.2% 3.2% 1.25

Tramadol 0.999 3.3% 3.6% 5

Normeperedine 0.999 3.9% 1.8% 5

Meperidine 0.999 2.6% 2.0% 5

Norbuprenorphine 0.996 5.5% 6.5% 5

Fentanyl 0.999 2.9% 1.5% 1.25

Buprenorphine 0.999 3.3% 2.0% 5

EDDP+ 0.999 2.5% 1.2% 5

Propoxyphene 0.999 3.6% 2.3% 5

Methadone 0.999 2.9% 2.2% 5

QC Results - Oral fluid

QC Concentration (ng/mL) (N=4)

7.5 25 150 300

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

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

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

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

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

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

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

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

5-500 ng/mLCalib. Curve

Mean % Bias = 5.3%

Mean %CV = 4.5%


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

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

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

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

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

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

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

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

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

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

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

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

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

Sample Preparations Strategies for Opioids - Conclusions

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

– Improved accuracy and precision vs. dilution

– µElution format ideal for limited sample volume (OF)

– Excellent linearity, analytical accuracy, and precision.


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 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

� CORTECS Solid Core Columns– 1.6 µm solid-core particles

– Highest efficiency column available

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

UPLC Conditions

Column CORTECS C18 1.6 µm; 2.1 x 100

LC ACQUITY UPLC I-Class

Flow Rate 0.6 mL/min

MPA Water with 0.1% FA

MPB ACN with 0.1% FA

Column Temp 30 ˚C

Inj. Vol 5 µL

MS Conditions

XEVO® TQD ESI+

Capillary: 1.0 kV

Source Temp: 150 ˚C

Desolvation Temp: 600 ˚C

Cone Gas Flow: 0 L/Hr


MRM Transitions optimized individually

UPLC and MS conditionsUPLC and MS conditions


Inj. Vol 5 µL MRM Transitions optimized individually

Time Flow %A %B Curve

0 0.6 70 30 6

2.0 0.6 50 50 6

3.0 0.6 50 50 6

7.0 0.6 10 90 6

7.2 0.6 70 30 6

8.5 0.6 70 30 6

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

%

100

5

6

7

8

9, 10

ACQUITY UPLC BEH C182.1 x 100 mm

Improved Resolution of CORTECSImproved Resolution of CORTECS

5) JWH-073 4-COOH met.6) JWH-073 4-OH met.7) JWH-018 5-COOH met.8) JWH-073 (+/-) 3-OH met.9) JWH-018 5-OH met.10) JWH-018 (+/-) 4-OH met.


Time2.20 2.40 2.60 2.80 3.00 3.20

%

0

100

Time2.40 2.60 2.80 3.00 3.20 3.40

0

5

6

7

89, 10

CORTECS UPLC C182.1 x 100 mm

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 % CVs 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


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.

Vortex for 3 minutes

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

Expanded Toxicology PanelExpanded Toxicology Panel

� Expanded Toxicology Panel– Replace immunoassay screening

o Single analysis vs. multiple methods

o Improved specificity and sensitivity

� HPLC Column Comparison– Attributes of CORTECS 2.7 µm and XBridge BEH Phenyl XP 2.5 µm


– Attributes of CORTECS 2.7 µm and XBridge BEH Phenyl XP 2.5 µm columns

� Simplified Sample Preparation Strategy– Simplified procedure using MCX µElution plates

1) Amphetamine

2) MDA

3) Methamphetamine

4) MDMA

5) Phentermine

6) MDEA

7) BZE

8) PCP

9) Nitrazepam

HPLC Column ComparisonsHPLC Column ComparisonsCompound Key Expanded Compound Key Expanded ToxTox PanelPanel

17) Morphine

18) Oxymorphone

19) Hydromorphone

20) Dihydrocodeine

21) Codeine

22) Oxycodone

23) 6-AM

24) O-desmethyl Tramadol

25) Hydrocodone

amines


9) Nitrazepam

10) Oxazepam

11) Alprazolam

12) Lorazepam

13) Clonazepam

14) Flunitrazepam

15) Temazepam

16) Diazepam

25) Hydrocodone

26) Norfentanyl

27) Tramadol

28) Normeperedine

29) Meperedine

30) Norbuprenorphine

31) Fentanyl

32) Buprenorphine

33) EDDP

34) Propoxyphene

35) Methadone

benzodiazepines

opioids

UPLC I-Class, FL with Column Manager (CMA)

MS Xevo® TQD

MPA 0.1% Formic Acid

MPB 0.1% Formic Acid in ACN

Col Temp 30 ˚C

Inj. Vol 10 µL

Columns CORTECS C18, 2.7 µm; 3.0 x 50 mmXBridge BEH Phenyl XP, 2.5 µm; 3.0 x 50 mm

Method ParametersMethod Parameters


18, XBridge BEH Phenyl XP, 2.5 µm; 3.0 x 50 mm

Time (min)

Flow (mL/min)

% MPA % MPB

0.0 0.6 95 5

4.0 0.6 40 60

4.1 0.6 95 5

5.0 0.6 95 5

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50

%

0

100

OpioidOpioid ChromatographyChromatography

CORTECS C18 2.7 µm3.0 x 50 mm

17) Morphine

18) Oxymorphone

19) Hydromorphone

20) Dihydrocodeine

21) Codeine

22) Oxycodone

23) 6-AM

24) O-desmethyl Tramadol

25) Hydrocodone

26) Norfentanyl

17

2021

19 22

24

23

2526

27

30

312928

32

3433

35

18


Time1.00 1.50 2.00 2.50 3.00 3.50

%

0

100

26) Norfentanyl

27) Tramadol

28) Normeperedine

29) Meperedine

30) Norbuprenorphine

31) Fentanyl

32) Buprenorphine

33) EDDP

34) Propoxyphene

35) Methadone

XBridge BEH Phenyl XP2.7 µm, 3.0 x 50 mm

171820,21

19

2223

24

2526

27

30

31

2928

32

3433

35

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80

%

0

100

Enhanced Retention of OpiatesEnhanced Retention of Opiates

17) Morphine

19) Hydromorphone

21) Codeine

25) Hydrocodone

17

25

1921



Time0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80

%

0

100

17

25

1921


Time1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

0

100

Amine ChromatographyAmine Chromatography

1) Amphetamine

2) MDA

3) Methamphetamine

4) MDMA

5) Phentermine

6) MDEA

7) BZE

8) PCP

12

34

5

76

8 CORTECS C18 2.7 µm3.0 x 50 mm


Time1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

0

100

123 4

5 76

8XBridge BEH Phenyl XP2.7 µm, 3.0 x 50 mm

Time2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

%

0

100

Benzodiazepine ChromatographyBenzodiazepine Chromatography

9) Nitrazepam

10) Oxazepam

11) Alprazolam

12) Lorazepam

13) Clonazepam

14) Flunitrazepam

15) Temazepam

9

10 11 1312

1514

16



Time2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00

%

0

100

16) Diazepam

9

10

1113

12

15

1416


Performance Statistics and ConclusionsPerformance Statistics and Conclusions

ColumnParticle Size

(µm)Pressure (Max)

Mean Peak Width(sec)

CORTECS C18 2.7 2206 2.52

XBridge BEH Phenyl XP 2.5 3274 2.94


XBridge BEH Phenyl XP 2.5 3274 2.94

CORTECS – High efficiency, narrow peak widths, and low pressure

XBridge BEH Phenyl XP – Enhanced retention of opiates and other polars

Expanded Toxicology PanelExpanded Toxicology Panel

� Expanded Toxicology Panel– Replace immunoassay screening

o Single analysis vs. multiple methods

o Improved specificity and sensitivity

� HPLC Column Comparison– Attributes of CORTECS 2.7 µm and BEH Phenyl XP 2.5 µm columns


– Attributes of CORTECS 2.7 µm and BEH Phenyl XP 2.5 µm columns

� Simplified Sample Preparation Strategy– Universal simplified procedure using MCX µElution plates

15 Morphine-3-gluc

16 Morphine-6-gluc

17 Morphine

18 Oxymorphone

19 Hydromorphone

20 Naloxone

21 Dihydrocodeine

22 Codeine

23 Noroxycodone

Simplified MCX procedureSimplified MCX procedureCompound Key Expanded Compound Key Expanded ToxTox PanelPanel

1 Amphetamine

2 MDA

3 MDMA

4 MDEA

5 BZE

6 PCP

7 7-Aminoclonazepam

8 alpha-OH Alprazolam


23 Noroxycodone


25 6-MAM

26 Oxycodone

27 Hydrocodone

28 Norfentanyl

29 Tramadol

30 Tapentadol

31 Norbuprenorphine

32 Fentanyl

33 Buprenorphine

34 EDDP

35 Methadone

8 alpha-OH Alprazolam

9 Oxazepam

10 Lorazepam

11 Clonazepam

12 Alprazolam

13 Temazepam

14 Diazepam

UPLC I-Class, FL with Column Manager (CMA)

MS Xevo® TQD

MPA 0.1% Formic Acid

MPB 0.1% Formic Acid in ACN

Column BEH Phenyl; 1.7 µm, 2.1 x 100mm

Col Temp 40 ˚C

Inj. Vol 15 µL

Instrument ParametersInstrument Parameters


Time (min)

Flow (mL/min)

% MPA % MPB

0.0 0.6 95 5

5.0 0.6 37.5 62.5

5.1 0.6 95 5

6.0 0.6 95 5

ChromatographyChromatography

%

100

%

100

12

3

45

6

7 9

8

10

1112

13

14Amines, BZE, and PCP Benzodiazepines


Time0.50 1.00 1.50 2.00 2.50 3.00

0 Time2.00 2.50 3.00 3.50 4.00

0

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75

%

0

100

8

151617

18

192021

22

23

24

2526

2728

29

31

30

32

35

34

33

Opioids and Others

Extraction Methodology for UrineExtraction Methodology for Urine

In Well Hydrolysis50 µL urine + 50 µL 0.1 M NH4OAc +

10 µL β-glucuronidase

Add 200 µL 4% H3PO4

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

Load Sample


Wash200 µL 0.02 N HCl in 20% MeOH

Elute2 x 50 µL

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

EvaporateReconsititute with 50 uL 2% ACN/1% FA

Inject 15 µL

Pre-conditioning steps Eliminated

Wash Steps Consolidated

Load Sample

Recovery Recovery –– Simplified ProtocolSimplified Protocol

60%

80%

100%

120%

RecoveryCompound

LOD

(ng/mL)

Morphine-3-gluc 10

Morphine-6-gluc 10

Morphine 10

Oxymorphone 10

Hydromorphone 10

Amphetamine 1

Naloxone 10

Dihydrocodeine 10

Codeine 10

MDA 1

Noroxycodone 10

O-desmethyl Tramadol 1

6-MAM 10


Mean Recovery = 100%

0%

20%

40%Oxycodone 10

MDMA 1

Hydrocodone 10

MDEA 1

7-Aminoclonazepam 1

Norfentanyl 1

BZE 1

Tramadol 1

Tapentadol 1

Norbuprenorphine 10

PCP 1

Fentanyl 0.1

Buprenorphine 1

alpha-OH Alprazolam 10

EDDP 1

Oxazepam 1

Lorazepam 1

Clonazepam 1

Alprazolam 1

Methadone 1

Temazepam 1

Diazepam 1

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 PreparationPPT Followed by Mixed Mode SPEPPT Followed by Mixed Mode SPE

Condition Cartridge1 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)

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

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

OverviewOverview



� Application Examples

� Summary


Summary of Applications andSummary of Applications andSample Preparation StrategiesSample Preparation Strategies

Application Matrix Solution Benefits

Opiates

Urine720004650EN

Oral Fluid720004838EN

Urine Oasis MCX µElution

ACQUITY BEH C18

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

Oral Fluid

Oasis MCX µElution

ACQUITY BEH C18

• Good recovery for all compounds• Linear, accurate and precise data• Limited sample volume

Synthetic Cannabinoids

Urine Oasis HLB µElution • Sorbent appropriate for multiple chemotypes• Linear, accurate and precise


Cannabinoids

Urine720004780EN

Whole Blood720004708EN

CORTECS C18

• Linear, accurate and precise• Effective cleanup• Concentration without evaporation

Whole Blood

Ostro

CORTECS C18

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

Expanded ToxPanel

Urine Oasis MCX µElution

ACQUITY BEH Phenyl

• Excellent recovery for all compound classes• Simplified SPE Method• Rapid Sample Prep and analysis

THC and Metabolites

720004700EN

Whole Blood

PPT and Oasis MAX

ACQUITY BEH C18

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

AcknowledgementsAcknowledgements

Sherri Naughton

Nebila Idris

Erin Chambers

Michelle Wood

Robert Lee


Thank You for Attending!Thank You for Attending!

� Post-Event Landing Page - www.waters.com/Oct16

� 40% Promotional Offer On CORTECS Columns and

Oasis uElution Plates

� 30% Offer on Ostro Plates

� 20% offer on BEH Columns (ACQUITY and Xbridge)

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


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

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

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

– Please eMail - [email protected]

� Visit Us at SOFT: Booth # 101/200, October 21st – 23rd

Thank YouThank You


powerful sample prep and lc column ... - waters corporation€¦ · recorded version of today’s...

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