k2 and beyond: a synthetic cannabinoid primer

K2 AND BEYOND: A SYNTHETIC CANNABINOID PRIMER.

INTRODUCTION

Barry K Logan

Fredric Rieders Family Renaissance Foundation

Marijuana

Most popular recreational drug after alcohol and tobacco.

Main psychoactive component THC

#1 Drug in the DRE program

Some 25 million Americans have smoked marijuana in the past year, and more than 14 million do so regularly.

Possession and use illegal under federal law, but states have variable policies on enforcement and prosecution.

6.8% of Friday and Saturday evening drivers test positive for use.

http://www.erowid.org/plants/salvia/salvia.shtml

http://www.erowid.org/plants/salvia/salvia.shtml

Synthetic Cannabinoids

Chemicals designed to have CB1/CB2 binding properties.

Chemically diverse structure classes.

First synthesized as investigational drugs in the 1980’s.

Adopted by the “Research Chemical” movement in 2000’s.

Sold as “Legal highs”, “Incense blends”, “Potpourri”

Very dynamic market with patchwork scheduling and regulation.


Learning ObjectivesAfter attending this workshop, attendees will be able to:Explain cannabinoid pharmacology.Understand the history and chemistry of synthetic cannabinoids (SCS) and their role in therapeutics.Develop and implement analytical methods for the identification of SCS in biological and non-biological matrices.Describe the current knowledge on the metabolism of SCS and the results of controlled drug administration studies.Explain the current legal status of SCS.


Mechanism of Action and Pharmacology.History of Synthetic CannabinoidsIdentification of Synthetic Cannabinoids in Botanical materialIdentification of Synthetic Cannabinoids in Biological MatricesHuman pharmacodynamics and Pharmacokinetics. Legal Status

SYNTHETIC CANNABINOIDS IN HERBAL INCENSE PRODUCTSLindsay E. Reinhold, M.F.S., F-ABC

AAFS Annual Conference

February 2011

Topics

IntroductionSamples TestedReference MaterialsSample PreparationMethods of IdentificationSample ResultsFuture Challenges

Synthetic Cannabinoid Basics

What are synthetic cannabinoids? Chemicals that mimic the actions or have a

similar structure to THC Classical – structurally related to THC Non-Classical – alternative structures, but

produces the same desired effects as THC

Molecular Structures

THCClassical

HU-210/HU-211JWH-133

JWH-018Naphthoylindoles

JWH-073JWH-081JWH-122JWH-200JWH-210JWH-398WIN55,212-2

CP47,497(C8)Non-Classical

CP47,497(C7)CP55,940HU-308

Molecular Structures

AM-694Benzoylindoles

AM-630AM-1241

JWH-250Phenylacetylindoles

JWH-203JWH-251

History Initially chemicals were synthesized for medicinal

research. JWH prefaced compounds are named for John W.

Huffman at Clemson University for research on the relationship between the structure of drugs and brain receptor activity.

HU prefaced compounds are named for Hebrew University where these compounds were first synthesized and investigated.

CP 47,497 was initially developed by Pfizer for its analgesic effects.

AM prefaced compounds are fluorinated and named for Northeastern University professor Alexandros Makriyannis

WIN compounds were developed by Sterling Winthrop

Legal StatusDEA Schedule I – pending approval for Temporary Scheduling

CP47,497 (C7 analog)(non-classical)

CP 47,497 (C8 analog) – Cannabicyclohexanol(non-classical)

HU-210(classical)

JWH-018(non-classical)



Samples Tested

175+ samples Botanical Botanical Residue Charred Residue (Paraphernalia) Liquids Solids (Rock-like) Solids (Powder) Capsules

Reference Materials

Manufacturers/Suppliers Cayman Chemicals Toronto Research Chemicals Cerilliant Sigma BOC Sci Tocris

Clan Labs Chinese Labs European Labs “Bathtub” Labs Mountain Industry

Current Analytical Scope

AM-694 AM-2201 CB-25 CB-52 CP47,497 (C7) CP47,497 (C8) CP55,940 HU-210 HU-211 HU-308 HU-331 JWH-015 JWH-018 JWH-019

JWH-073 JWH-081 JWH-122 JWH-133 JWH-200 JWH-203 JWH-210 JWH-250 JWH-251 JWH-398 RCS-4 RCS-8 WIN 55,212-2 WIN 55,212-3

Sample Preparation

Qualitative Identification Approximately 30mg botanical samples Can ID on residues Acid/Base extraction Multiple aliquots for different analyses

Quantitative Analysis Must grind entire sample 15-30mg botanical material in 1mL MeOH 4 calibrators and 2 controls (2 stock solutions) 3 replicates + spike

Methods of Identification

Presumptive TestsMacro/Microscopic AnalysisDuquenois –LevineThin Layer Chromatography (TLC)

High Performance Liquid Chromatography (HPLC)Diode-Array Detection (DAD)

Macro/Microscopic Analysis

Clearly different macroscopically from Marijuana

No identifiable microscopic hairs similar to Marijuana

Variable matrix

Duquenois-Levine Color Test

- Solvent System: 9:1 Toluene:Diethylamine

-Visualization Spray:1.5g Dianiside Tetrazotized in 50:50 MeOH:DI Water(Fast Blue B Spray)

- Distinct red or red/orange bands for the CB, CP and most of the HU compounds evaluated

- Obvious UV absorbance (254nm) for the AM, JWH and WIN compounds evaluated

TLC

TLC - Solvent System: 18.5:18:3:1 EtOAc:CH2Cl2:MeOH:NH4OH

-Visualization Series:1: UV (254 nm)2: UV (366 nm)3: Fluorescamine Spray + UV 366 nm4: Ninhydrin Spray + heat5: 10% H2SO4 Spray + UV 366 nm6: Acidified Iodoplatinate Spray7: 50% Nitric Acid Spray + heat8: Mercuric Sulfate Spray9: D-MAB Spray + heat

HPLC/DAD

HP 1100 Series HPLC with a UV DAD 5um Aquasil C18 4.6 x 100 mm Column 70:30 ACN:Water + 0.1% TFA 1.0 mL / min flow 40°C 316 nm

Methods of Identification

Confirmatory TestsGas Chromatography / Mass

Spectrometry (GC/MS) BSTFA derivatization

High Performance Liquid Chromatography (HPLC)

Tandem Quadrupole Mass Spectrometry (MS/MS)

Accurate-Mass Time-of-Flight Liquid Chromatography / Mass Spectrometry (LC-TOF)

CP47,497 (C8 analogue)(a.k.a.Cannabicyclohexa

nol)

CAS# 70434-92-3Formal Name: 5-(1,1-dimethyloctyl)-2-[(1R,3S)-3- hydroxycyclohexyl]-phenolMol.Form. C22H36O2

Mol.Wt. 332.27153

1 Internal Standard2 JWH-1333 CP47,497(C7) Di-

TMS4 CP47,497(C8) Di-

TMS5 HU-308 Mono-TMS6 CP55,940 Tri-TMS7 JWH-2518 HU-210/211 Di-TMS9 JWH-25010 RCS-411 JWH-01512 JWH-07313 AM-69414 JWH-01815 CB-25 Di-TMS16 JWH-01917 AM-220118 CB-52 Di-TMS19 JWH-12220 RCS-821 JWH-39822 JWH-21023 CB-52 Mono-TMS24 JWH-08125 JWH-20026 WIN55,212-2/3

CP47,497 (C8 analogue)(a.k.a.Cannabicyclohexa

nol)

Di-TMS DerivativeMol.Form. C28H52O2Si2Mol.Wt. 476.88

0.02 µg/mL CP47,497 (C8)

Underivatized

Derivatized

LC/MS/MS Extremely sensitive for most compounds

of interest Identifying approximately 0.1% JWH-018 in samples

Trace levels Bi-products/contaminants from synthesis

GC/MS identifying 2-10 ng/g JWH-018 (sample dependent)

LC/MS/MS

Strong Response Good Response Weak Response

JWH-015 AM-2201 CP47,497(C7)

JWH-018 JWH-081 CP47,497(C8)

JWH-019 JWH-200 CP55,940

JWH-073 JWH-251 HU-210

JWH-122 JWH-398 HU-211

JWH-210 JWH-133

JWH-250 WIN55,212-2

RCS-4 WIN55,212-3

RCS-8~ 0

.01

ng/m

cL ~ 0.1

ng/

mcL

~ 1 n

g/m

cL

LC/MS/MS JWH-200 2.57

WIN55,212-2/3 3.50AM-2201 4.56CP55,940 4.79JWH-015 4.84JWH-250 5.00RCS-4 5.12JWH-073 5.16JWH-251 5.31JWH-018 6.49CP47,497(C7) 6.85JWH-081 6.94JWH-019 7.23JWH-122 7.23RCS-8 7.30HU-210/211 7.58CP47,497(C8) 7.68JWH-398 7.79JWH-210 7.79JWH-133 11.04

LC/TOF

Some limitations (isomers) Useful tool for unknowns Not currently used in casework,

but may be required to validate standards with questionable provenance

K2 Blonde

Analytical Technique

JWH-018 JWH-073 JWH-200

TLC Positive Positive None Detected

GC/MS Positive (approx. 12 mg/g)

Positive (approx. 13 mg/g)

None Detected

LC/TOF Positive Positive Positive

K2 Standard


JWH-018 JWH-073

TLC Positive Positive



LC/TOF Positive Positive

K2 Citron


JWH-018 JWH-073





K2 (unknown variety)


CP47,497 (C8) JWH-018 JWH-073

TLC Positive None Detected None Detected


None Detected None Detected

LC/TOF None Detected Positive Positive

K2 Summit


JWH-018 JWH-073





K2 Blue


JWH-018

TLC Positive


LC/TOF Positive

K2 Pink


JWH-018

TLC Positive


LC/TOF Positive

K2 Latte


JWH-018 JWH-073 JWH-250

TLC Positive Positive Positive


Positive (approx. 0.03 mg/g)


LC/TOF Positive Positive Positive

K2 Pineapple Express


JWH-018

TLC Positive

GC/MS Positive (approx. 5.4 mg/g)

K2 Blueberry


JWH-018

TLC Positive

GC/MS Positive (approx. 5.5 mg/g)

Sample Summary

As of 2/14/11: 175 samples tested 28 samples contained no cannabinoids,

but contained some other drug 3 samples were negative 49 contained 5-MeO-DALT in addition to

multiple cannabinoids 31 contained only a single cannabinoid

(either JWH-018, JWH-250 or CP47,497(C8))

Samples Tested

Controlled Non-Controlled Non-Cannabinoid

JWH-018 AM-694 5-MeO-DALT

JWH-073 AM-2201 Mitragynine

JWH-200 JWH-081 MDPV

CP47,497(C8 analog) JWH-122 Methylone

JWH-210 Mephedrone

JWH-250 Butylone

RCS-4 4-MEC

RCS-8 ???

Future Challenges

Constantly changing market Black market changes more quickly

than testing/standards can be developed legitimately

Distributors are more aware of the market and holding on to their edge

Clan labs are constantly developing new products

Reference materials are not available quickly

Future Challenges

Legal Status The USA is 1-2 years behind the majority of the

rest of the world in controlling these substances The DEA is the agency that initiates controlling

the compounds and they are significantly behind

State legislation being used Haven’t figured out how to ban them all and

then enforce the ban How do we test for EVERYTHING? One analyte becomes controlled, three more

appear on the market

Future Challenges

Criminalistics can try to keep up Toxicology is more difficult (urine analysis) Metabolites are unknown and standards

don’t exist Large dosing studies haven’t been done Most products contain multiple analytes extremely complicated to identify metabolites, if even possible How do we develop testing?

Questions?

Acknowledgements:Barry LoganFran DiamondAllan XuSherri Kacinko

SYNTHETIC CANNABINOIDS: IDENTIFICATION OF METABOLITES AND ANALYTICAL METHODS FOR BIOLOGICAL MATRICES

Sherri L. Kacinko

Overview

Methods for the identification & quantification of synthetic cannabinoids in plasma & whole blood

Metabolism of synthetic cannabinoids

Methods for the identification & quantification of synthetic cannabinoid metabolites in urine

Whole Blood & Serum

Published Methods

CP47497-C8: Auwärter et al. JMS Letters, 44:832-7, 2009.

JWH-018: Teske et al. J Chrom B, 878(27): 2659-63, 2010.

Auwärter et al.

Self-administration of ‘Spice Diamond’

Modified method used for routine cannabinoid analysis in whole blood

C18 SPE, TMS derivatization & GC-EI-MS

Auwärter et al.

Contents of ‘Spice Diamond’ identified by GCMS, TLC, UV, NMR

OH

OH

CP 47,497-C8

Auwärter et al.

Auwärter et al, Fig. 3

Auwärter et al.

Auwärter et al, Fig. 6

Teske et al.

Quantification of JWH-018 in serum Liquid-Liquid extraction followed by LC-

MS/MS analysis using d5-Diazepam as an internal standard

Validation parameters: linearity, LOD, LOQ, selectivity, accuracy, precision, matrix effect, extraction efficiency, & process efficiency

Method applied to serum collected from 2 individuals who smoked ‘Smoke’

Teske et al.

Linear range: 0.5 – 20 ng/mL (Calculated LOD = 0.07 ng/mL)

Intra-day imprecision = 2.4-4.8% RSD

Inter-day imprecision = 13.5-14.8 % RSD

% Bias ≤ ± 10.5%

Significant ion suppression noted

Teske et al.

Kacinko et al.

Submitted to JAT

Identification & Quantification JWH-018, JWH-019, JWH-073 & JWH-250 in whole blood

Kacinko et al.

Single step liquid-liquid extraction

Internal standards: d9-JWH-018 & d9-JWH-073

LC-positive ion ESI – MS/MS

Kacinko et al.

Validation parameters: sensitivity, linearity, within- & between-run imprecision, accuracy, specificity, extraction efficiency, matrix effect, process efficiency, extract & sample stability, dilution integrity, carryover, & matrix-matching.

Kacinko et al.

0.2 mL sample + saturated sodium bicarb + saturated NaCl.

1% ethyl acetate in hexane, evaporate, recon

Acquity HHS T3 column (100x2.1mm 1.8 micron) on Waters Premier. Formic acid/methanol gradient

Linear Range: 0.1 – 20 ng/mL

Kacinko et al.

Analyte Retention Time

Quantifier Transition

Qualifier Transition

d9-JWH-018

3.12 351 155 351 127

JWH-018 3.13 342 155 342 127JWH-019 3.30 356 155 356 127d9-JWH-

0732.92 337 155 337 127

JWH-073 2.94 328 155 328 127JWH-250 2.93 336 121 none

Kacinko et al.

100

0

126.92

155.18

214.26

342.17

127.05227.96

356.39

0

155.12100

143.89

N

O

JWH-019

N

O

JWH-018

100

0

100

100 200 300 400

0 m/z

126.86

155.12

200.10

328.18

121.11

200.16144.34

Kacinko et al.

NOO

JWH-250

N

O

JWH-073

Kacinko et al.

Validation Parameter

Conc.(ng/mL)

JWH-018

JWH-019

JWH-073

JWH-250

Average within-run

imprecision (%RSD)

0.10.315

4.35.42.6

19.3*11.69.5

7.94.31.9

7.04.53.6

Between-run imprecision

(%RSD)

0.10.315

7.95.93.2

35.7*20.6*23.0*

10.24.32.1

8.64.76.9

Accuracy (%)0.10.315

99.293.793.7

112.070.3*67.3*

99.192.293.0

104.793.691.8

*Outside method validation acceptance criteria

Kacinko et al.

JWH-250

JWH-073

JWH-018

JWH-019

Kacinko et al.

Validation

Parameter

Conc.(ng/mL)

JWH-018

JWH-019

JWH-073 JWH-250

Extraction

Efficiency (%)

0.315

75.563.2

63.660.6

80.871.0

92.879.4

Matrix Effect (%)

0.315

36.749.4

42.164.7

31.132.2

22.018.6

Process Efficienc

y (%)

0.315

47.732.0

36.821.4

55.648.1

72.464.6

Kacinko et al.

Whole Blood Additive

Conc.(ng/mL)

JWH-018 JWH-073 JWH-250

Sodium Fluoride (n=2)

0.510

103.198.7

111.8106.0

88.586.6

Potassium Oxalate (n=3)

0.510

96.094.6

102.3107.0

87.587.6

Sodium EDTA(n=3)

0.510

105.398.1

113.5107.7

88.587.5

Potassium EDTA (n=2)

0.510

109.693.2

104.0103.5

94.087.4

Sodium Heparin (n=3)

0.510

99.992.0

102.997.4

88.284.3

Lithium Heparin (n=2)

0.510

103.191.5

98.496.7

94.582.8

NMS Labs

All blood analyzed from 9/27/2010 – 2/15/2011

69 quantitative analyses (JWH-018, JWH-073, JWH-205)

24 qualitative analyses (JWH-018, JWH-073, JWH-019, JWH-205)

NMS Labs – Qualitative

Qualitative: 51 negative 42 positive 27 positive for JWH-018 only 7 positive for JWH-018 & JWH-073 5 positive for JWH-018 & JWH-250 2 positive for all three compounds No specimens positive for JWH-019

NMS Labs – Blood Distribution

NMS Labs – Concentrations

JWH-018: 0.12 – 20 ng/mL

JWH-073: 0.11 – 1.6 ng/mL

JWH-250: 0.23 – 8.8

NMS Labs – Concentrations

Summary

CP 47,497 – C8, JWH-018, JWH-019, JWH-073, & JWH-250 detectable in blood as parent compounds

Lack of deuterated internal standards may complicate quantification of these compounds

Methods should be developed keeping in mind ability to add new compounds

Metabolism

The Challenge

Identify markers for the use of herbal incense products containing synthetic cannabinoids

Develop a method for the routine identification of these markers

Validate a qualitative method in the absence of certified reference material

Identify Markers

Literature Review

Blood & urine from known administrations

Identify Markers

Literature Review

WIN 55212-2 - Zhang, et al. Drug Metab & Disp 30(10): 1077-86, 2002.

AM-694 – Zhang, et al. J Mass Spec 39(6): 672-81, 2004.

JWH-015 - Zhang, et al. Analytical Bioanalytical Chem, 386(5): 1345-55, 2006.

Zhang, et al.

Incubated with rat liver microsomes

Used LC/MS, LC-MS/MS & NMR to elucidate structures of metabolites

Similarities in major metabolic pathway -Dihydroxylation via epoxide intermediary

WIN 55212-2 Metabolic Pathway

Zhang et al. 2002 Fig. 8

AM-630 Metabolic Pathway

Zhang et al. 2004 Fig. 11

JWH-015 Metabolic Pathway

Zhang et al. Fig. 9

JWH-018 Studies

Sobolevsky et al, Forensic Science International, 200(1-3):141-47, 2010 Urine from 3 persons known to have

smoked JWH-018 Analysis of hydrolyzed urine by

GC-MS/MS & LC-MS/MS Wintermeyer et al, Analytical

Bioanalytical Chem, 398(5): 1241-53, 2010. Incubation with human liver microsomes Analysis by LC-MS/MS

JWH-018 Metabolic Pathway

Wintermeyer et al, Fig. 2

JWH-018 Metabolites

Sobolevsky et al, Fig. 6

NMS Labs Studies

In vitro – human liver microsomes

In vivo – rats

Urine from Missouri administration study

Human Liver Microsomes

NH

O

N

O

N

O

+Om/z 358

RT 10.4, 10.7, 9.8

K-2m/z 342

N

O

N

OOH

OH

+2O+2Hm/z 376

RT 10.0, 10.2

HO OH

N-Dealkylationm/z 272RT 9.4

+2Om/z 374RT 10.5

or

NH

O

N

O

N

O

+Om/z 358

RT 10.4, 10.7, 9.8

K-2m/z 342

N

O

N

OOH

OH

+2O+2Hm/z 376

RT 10.0, 10.2

HO OH

N-Dealkylationm/z 272RT 9.4

+2Om/z 374RT 10.5

or

RMI Laboratories, North Wales, PA

Rat Studies

Intraperitoneal injection (10 mg/kg)

Blood: 1 hour= Parent only 3 hour= mono-hydroxy desalkyl

(prominent) Other metabolites= mono-, di-, & tri-

hydroxy metabolites; carboxy-, reduced di-hydroxy

Rat Studies

5 hour urine: Mono-hydroxy-desalkyl (3) is

prominent, tri-hydroxy (5), mono-hydroxy (>5), dihydroxy (3), carboxy trace.

Human Blood

Blood from controlled administration of JWH-018 & JWH-073

LC-MS/MS identified Parent Mono-hydroxy Di-hydroxy Tri-hydroxy Mono-hydroxy desalkyl Di-hydroxy desalkyl Carboxylic acid

Human Blood

Urine Methods

Urine Test Development

Worked with authentic positive urine: LC-TOF work identified exact mass of

predicted metabolites. LC-MS/MS for MRM studies to verify

general structure.

Liu et al, 2010

Hybrid triple quad/linear ion trap system

MRM triggered enhanced product ion scan with library confirmation

“Dilute & shoot” Urine metabolites: mono-hydroxy, di-

hydroxy, hydroxylated N-dealkyl, carboxy, reduced di-hydroxy with corresponding glucuronidated moieties


Worked with authentic positive urine: LC-APCI-MS/MS for intact

glucuronide of mono-hydroxy as a direct injection screening test

LC-ESI-MS/MS following enzymatic hydrolysis. Monitored for all mono-, di- & tri-hydroxy metabolites.


XIC of +MRM (14 pairs): 534.2/358.2 amu from Sample 13 (# 13) of ED01_0719K2_AX.wiff (Heated Nebulizer) Max. 9600.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5Time, min

0

2000

4000

6000

8000

9600

In

te

ns

it

y,

c

ps

5.27

4.874.203.733.38

Workstation: NMS815Printing Time: 3:04:32 PMPrinting Date: Wednesday, January 26, 2011

Electronic Signature: noAnalyst Version: 1.4.2Page 1 of 1

Acq. Time: 12:07Acq. Date: Monday, July 19, 2010Acq. File: ED01_0719K2_AX.wiff

Sample Name: # 13Sample Number: N/ABatch Name: K2-2.dab

Sample Comment: Collected by: NMS815\labmanSample ID: 13

Project: Production_042309Method Name: N/AResults Name: N/A


0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5Time, min

0

1000

2000

3000

In

te

ns

it

y,

c

ps

3.07 5.34

4.89

7.602.11








0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5Time, min

0

500

1000

1500

In

te

ns

it

y,

c

ps

5.30

4.89

1.98







Intact glucuronide of mono-hydroxy JWH-018

Mono-hydroxy JWH-018

Mono-hydroxy JWH-018


XIC of +MRM (35 pairs): 358.2/155.0 amu from Sample 2 (Hydrolyzed 19-2... Max. 4.2e6 cps.

2 4 6 8 10 12Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e64.2e6

Inte

ns

ity, c

ps

6.80

7.33

6.003.47

JWH-073 Human Urinary Metabolites

IS

HO

COOH

HO-N-dealkylation2HO

3HO

Workstation: NMS815Printing Time: 3:10:57 PMPrinting Date: Wednesday, September 15, 2010


Acq. Time: 10:07Acq. Date: Friday, September 03, 2010Acq. File: ED01_090310K2_AX.wiff

Sample Name: Hydrolyzed 19-20-21 mix 1Sample Number: N/ABatch Name: K2 090310.dab


Project: ProductionMethod Name: N/AResults Name: N/A


Screening MethodSource

Fragmentation

Collision Cell

Fragmentation

JWH-018-OH-GLUC

534358 358155 358127

JWH-073OH-GLUC

520344 344155 344127


Confirmation MethodPrecursor

IonProduct Ions

OH-JWH-018 358 155 127 284 186

2OH-JWH-018*

376374

214 171155 127

3OH-JWH-018 374 189 171OH-JWH-073 344 155 127

2OJ-JWH-073* 362360

200 171155 127

3OH-JWH-073 378 189 171

Final Monitored Transitions

N

O

OH

N

O

OH

OH

N

O

OHN

O

OH

OH

358

344

374

362

358

344

374

362

155

155

155

155

127

127

127

127

JWH-018

JWH-073

Analysis Summary

1 mL urine + JWH-018 (d9) Enzymatic hydrolysis for 1 hour at

60oC 10.4 Borax Buffer, extract MTBE Evaporate & reconstitute XBridge C18 3.5 um, 2.1 x 100mm Formic acid / ACN gradient Waters TQD MS with an ACQUITY UPLC Positive & Negative control pools

Validation

Pedigreed positive urine controls Pooled urine from administration study Diluted until criteria not met for

approx 50% of replicates Positive control = 10x less dilution

Pedigreed negative urine control Blind positive (50) & negative (50)

urine samples Stability

Acceptance Criteria

Negative control must be negative Positive control must be positive Retention Time – ±2% of the positive

control Signal to noise ratio of each transition

must be at least 10:1 Transition ratios +/- 30% of positive

control Detection of both mono-hydroxy & di-

hydroxy metabolites

JWH-018 Metabolites

Positive Control

Positive Case

JWH-073 Metabolites

Positive Control

Positive Case

Urine Distribution (n=1404)

Summary

Validated LC-MS/MS method for the identification of mono- & di-hydroxy metabolites of JWH-018 & JWH-073 All known positive specimens tested

positive All known negative specimens tested

negative Stable for at least 30 days

Reportable when both the mono- & di-hydroxy metabolites are present

Urine Metabolites - Continued

Major Mono-hydroxy metabolite

Certified reference material available for the 2-, 4-, 5-, 6- & 7- mono-hydroxy metabolites

N

O

JWH-073

45

6

7

2

Future Challenges

Identification of metabolites of other compounds

Continuing to produce scientifically sound methods before certified reference materials are available

Just trying to keep up!

ACKNOWLEDGEMENTS

Staff of NMS Labs - Research and Development Department

Staff of NMS Labs – LC-MS/MS Department

A special thanks to Matt McMullin

QUESTIONS?

SYNTHETIC CANNABINOIDS: A CONTROLLED ADMINISTRATION STUDY

Barry K Logan

Fredric Rieders Family Renaissance Foundation

K2 Phenomenon

Millard South Shooter Tests Positive for Synthetic Marijuana.Omaha, NE - Tests discover a controversial, yet legal drug, in Robert Butler Juniors' body the day he killed Doctor Vicki Kaspar then himself.

So KMTV Action 3 news talked with a poison control expert.Without wanting to speculate on Butler's use he says the drug can cause suspicious behavior."The confusion, the delirium, people can hurt themselves, they can hurt others, they can hurt other people," says Doctor Ron Kirschner with the Nebraska Regional Poison Center.

K2 Phenomenon

McHenry shop owner had K2 in systemMcHENRY – The owner of House of Glass, who was found unconscious and later died in November, had K2 along with painkillers in his system. The McHenry County Coroner’s Office, which completed its investigation this week, said there was K2 and the painkillers methadone, morphine and Lyrica in Fred Evans’ system when he died.

K2 Phenomenon

Woman Pleads Guilty In Hit, Run That Killed Passenger Devetta Blount Created: 1/12/2011 6:00:45… emergency workers took Watlington to Moses Cone Hospital where she died. Also, Prosecutors said that SBI Lab results revealed Neal had cocaine and … K2 or synthetic marijuana.

K2 Phenomenon

Fake pot sickens two LVC students Lebanon Daily NewsUpdated: 01/31/2011Two Lebanon Valley College students were taken to the hospital last weekend for treatment of an allergic reaction to synthetic marijuana, police said. The students admitted to smoking herbal incense, which caused an allergic reaction, police said. Both were taken to Good Samaritan Hospital and released after treatment.

K2 Phenomenon

Pain pill, not Spice, shows up in DUI-related drug testPublished: Saturday, Jan. 22, 2011SALT LAKE CITY — A man police believed to have been impaired while driving after smoking Spice, pleaded no contest Friday to driving under the influence of drugs and/or alcohol."The question is not whether Spice is legal. The question is whether something is impairing his ability to drive safely," (his attorney) said.He was later subjected to drug testing and Engar said results indicated an opiate drug was present. Beckmann admitted to taking a pain pill in days prior."He has a drug problem," (his attorney) said. "We're looking into the (Corrections Addictions Treatments System) program at the jail. It's just something we'd rather resolve than fight at this point."

K2 Phenomenon

ROCK SPRINGS, WY -- Rock Springs police are reporting several cases of teens apparently overdosing on synthetic marijuana.Memorial Hospital of Sweetwater County says six people have been treated since Jan. 1 for symptoms believed to have been caused by ingesting or smoking fake pot.Three people ages 15, 16 and 17 who went to the hospital Jan. 7 were charged with violating a city ordinance against inhaling toxic vapors. Three Rock Springs High School students, all age 15, were charged Jan. 11 with the same offense after at least one teen suffered adverse symptoms.

K2 Phenomenon

WASHINGTON | Tue Jan 25, 2011 3:20pm EST Seven midshipmen were expelled from the U.S. Naval Academy for using or having a banned marijuana-like substance known as "spice," officials said.It is considered a banned substance by the Departments of Defense and Navy, Carpenter's statement said.

Officials at the Naval Academy in Annapolis, Md. learned about the allegations during the fall semester and consider the investigation to be ongoing, the statement said.

K2 Phenomenon

Synthetic Cannabinoid Effects


The Journal of Emergency Medicine, Epub, 2010


T. Sobolevsky, et al., Detection of JWH-018 metabolites in smoking mixture post-administration urine,Forensic Sci. Int. (2010), doi:10.1016/j.forsciint.2010.04.003

Missouri K2 Administration Lab

Spring 2010, UCMO plans DRE training to include K2 smoking Lab.

NMS Labs contacted regarding ability to test urine.

Protocol designed to include oral fluid, blood and urine.

Study expanded to include additional DRE staff, and six subjects.

Protocol submitted for IRB review.

Approved:

1-3 inhalations per subject, subject to medical staff approval.

Medical screening by staff on-site, EMS on standby.

Pre-administration drug test

Pregnancy test for female subjects

8 hour timeframe.


K2 Standard K2 Citron K2 Summit Herbal Blend

JWH-018 (mg/g) 9 10 11 -

JWH-073 (mg/g) 9 10 9 -

CP47,497 (C7) (mg/g) - - - 6

Free from other known drugs or chemicals


Pre-dosing:

Nurse evaluation, vital signs

Oral Fluid/Urine drug screen

Eye exam

Cognitive tests

DRE evaluation


Dosing:

Pre-dose pulse/blood pressure.

0.3g botanical material placed in water pipe.

Up to three inhalations.


Post-dosing:

Nurse evaluation, vital signs

Blood sampling

Ad lib urine sampling

DRE evaluation

Eye exam

Cognitive tests


Onset of subjective effects within 2-3 minutes.

Subjectively peaking 5-10 minutes.

Taste tobacco/burning garbage/unpleasant

Somatic Effects:Dry mouthLight headednessBuzzedBlurred visionLaughterMotor agitation/restlessnessTime dilationMild anxiety/paranoiaPost intoxication fatigue/exhaustion


Authors Sobolevsky et al, 2010 Teske et al, 2010 Auwater et al, 2009 Elsohly, 2008This Study - subject number

1 2 3 4 5 6

Condition has been reported

Active intoxication, 3 subjects under arrest, positive for JWH-018

Smoking study, 2 subjects smoked Spice

containing JWH-018

Smoking Study, 2 subjects

Cannabicyclohexanol + JWH-018

THC K2 Standard K2 Citron K2 Standard K2 Summit K2 Citron K2 Summit

Red eyes / bloodshot Yes - Yes Yes Yes Yes Yes Yes Yes Yes

Burning of the eyes - Yes - - - Yes - - - -

Xerostomia (dry mouth) - Yes Yes Yes Yes Yes - - Yes Yes

Increased pupil diameter - Yes - Yes Equivocal Equivocal Equivocal Equivocal Equivocal Equivocal

Tachycardia Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Anxiety Yes - - Yes Yes - Yes - Yes Yes

Hallucinations Yes - - Yes - - - - - -

Paranoia Yes - - Yes - - - - Yes Yes

Sickness - Yes - - - - - - - -

Sedation - Yes - Yes Yes - Yes - - Yes

Changes in perception/mood

- - Yes Yes Yes Yes Yes - Yes Yes

Subjective thought disruption/loss of concentration

- Yes - Yes Yes Yes - - Yes Yes

Impaired sense of time Yes - - Yes Yes Yes Yes - - Yes

Tiredness/exhaustion - 6-12 hours 6-24 hours - Yes Yes Yes - - -

Self assessed impairment - - Yes Yes Yes Yes Yes - Yes Yes

Missouri K2 Administration Study

Subject BM

Smoked “K2 Citron”

10mg/g JWH-018/073

0.3g in water pipe

3 inhalations over 30 minutes


Subject BM - Urine

Time JWH-018

Mono-OH Di-OH Tri-OH Glucuronides

Pre-dose

X X X X X

1:15 +/- √ √ X √

2:07 X √ √ √ √

2:40 X √ √ √ √Time JWH-

073Mono-OH Di-OH Tri-OH Glucuronide

s

Pre-dose

X X X X X

1:15 X √ √ X √

2:07 X √ √ √ √

2:40 X √ √ √ √


Psychomotor Effects

Highly variable response

DRE ExamIncreased pulse and blood pressureLack of convergenceNo HGN, or VGNPupils normal, muscle tone normal

SFST’s3-4 inches of sway, leg body tremorsLoss of balanceLoss of motor coordination


Lack of Convergence video

Conclusions

• Subjects were conservatively dosed with typical commercially available K2 products.• Effects were qualitatively similar to marijuana with some additional anxiety/paranoia.• Subjects reported a noticeable hangover effect.• No adverse events to the subjects were reported.• Blood concentrations of the parent drug were typically less than 1ng/mL within 2

hours of smoking.• Urine was positive within 1 hour of administration, for mono-and di-hydroxy

metabolites.

ACKNOWLEDGEMENTS

Staff of NMS Labs, Research and Development and LCMS Departments

Staff of the Missouri Safety Center, University of Central Missouri

QUESTIONS?

k2 and beyond: a synthetic cannabinoid primer

Health & Medicine

analytical

certified

validation

urine test

synthetic

authentic

detected gcms

nms labs