K2 and Beyond: A Synthetic Cannabinoid Primer
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K2 and Beyond: A Synthetic Cannabinoid Primer

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Presented by NMS Labs at the AAFS Annual Conference, February 2011, by Dr. Barry Logan, Lindsay Reinhold, and Dr. Sherri Kacinko

Presented by NMS Labs at the AAFS Annual Conference, February 2011, by Dr. Barry Logan, Lindsay Reinhold, and Dr. Sherri Kacinko

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  • Synthetic drugs that mimic the effects of cannabis through binding at the same receptors, principally the CB1 receptor, more of which about momentarily. The drugs were developed in the 1980’s and 1990’s as potential cannabinoid agonists that might possess some of the advantageous effects of cannabis, appetite stimulation, and anti- nausea properties, blood pressure, and much flaunted as adjuncts in cancer pain therapy, without the euphoric intoxicating effects for which the drug was popular recreationally. Now the drugs are sold widely on the internet and convenience stores and gas stations, smoke shops and head shops. The compounds that we will discuss are largely unregulated in most o the US but there is a rapid trend towards state or even local regulation as public concern grows. The DEA is looking to develop controls for these drugs and recently appealed for information through the SOFT newsletter.
  • So what do we know about this phenomenon, and what can be done to prepare for investigations of its possession and use as it is outlawed, and to answer questions about the toxicity associated with its use? Unfortunately, the K2/spice phenomenon appears to be more than just a fad, but rather represents the emergence of an industry designed to continually change its products based on attempts to regulate each new compound that comes along. We will briefly review the origins of the synthetic cannabinoids Consider what products are on sale in the K2 online and retail markets and what drugs they contain Consider their mechanisms of action and pharmacology Analytical challenges Consider what is known about their effects from published self-experimentation and some recent human administration studies And finally consider some impaired driving cases with associated toxicology results.
  • So what do we know about this phenomenon, and what can be done to prepare for investigations of its possession and use as it is outlawed, and to answer questions about the toxicity associated with its use? Unfortunately, the K2/spice phenomenon appears to be more than just a fad, but rather represents the emergence of an industry designed to continually change its products based on attempts to regulate each new compound that comes along. We will briefly review the origins of the synthetic cannabinoids Consider what products are on sale in the K2 online and retail markets and what drugs they contain Consider their mechanisms of action and pharmacology Analytical challenges Consider what is known about their effects from published self-experimentation and some recent human administration studies And finally consider some impaired driving cases with associated toxicology results.
  • Just some introductory historical perspective. Slides are self explanatory.
  • To complicate the matter further, these chemicals are already in circulation. As certain compounds are banned, there are plenty of replacement chemicals that quickly show up in new formulations. This list is getting bigger as more analogous materials are synthesized and distributed. We have also been seeing compounds added to these mixtures that are not synthetic cannabinoids such as 5-methoxy-dallyltryptamine (5-MeO-DALT).
  • No distinctive color test Duquenois-Levine did not give anything specific
  • Good response for HU and CP compounds While UV absorbance was observed for JWH and WIN compounds, no band was observed upon use with visualization spray
  • Overspraying with iodoplatinate could result in a false negative for all but JWH-200 and WIN55,212 Observable results for all compounds with UV (254nm) JWH018, 019, 073, 200 and WIN 55 also gave responses for UV (366nm) and 10% H2SO4 HU210, HU211, JWH015, 018, 019, 073, 200 and WIN 55 also gave responses with 50% Nitric No responses were observed for any compounds with Fluorescamine, Ninhydrin, Mercuric Sulfate or D-MAB visualization sprays
  • 125x greater response from the derivatized standard
  • 1 st picture is from the Missouri sample 2 nd picture was K2 Blonde purchased from www.k2-incense-online.com 2 separate samples tested (Missouri and www.k2-incense-online.com); both contained the same compounds
  • From Missouri
  • 1 st picture is from Missouri 2 nd picture is from www.k2-incense-online.com 2 separate samples analyzed (Missouri and www.k2-incense-online.com); both contained the same compounds
  • From Missouri
  • 1 st picture is from Missouri 2 nd picture is from www.k2-incense-online.com 2 separate samples analyzed (Missouri and www.k2-incense-online.com); both contained the same compounds
  • 2 separate samples analyzed (www.topk2.com and North Carolina); both contained the same compound 1 st picture is from www.topk2.com 2 nd picture is from North Carolina
  • 2 separate samples analyzed (www.topk2.com and www.k2-incense-online.com); both contained the same compound 1 st picture is from www.topk2.com 2 nd picture is from www.k2-incense-online.com
  • From www.k2herbal.com
  • From www.k2-incense-online.com
  • From www.k2-incense-online.com
  • N,N -diallyl-5-methoxytryptamine (Psychedelic Tryptamine)
  • TMS derivative of CP47497
  • Selected ion monitoring mode
  • *Multiple dihydroxy metabolites identified
  • None Detected 774 JWH-073 Only 304 JWH-018 Only 108 JWH-018 & JWH-073 88 May not have been positive for one of the compounds because only the mono-hydroxy metabolite was present
  • The terminal OH is present with a fairly high intensity but the unknown is most intense. So what is this peak? It is most likely hydroxylation up the chain. This explains the intensity and the poor peak shape.
  • Every week we are seeing anecdotal reports in the news and additional reports in the scientific media
  • Every week we are seeing anecdotal reports in the news and additional reports in the scientific media
  • Every week we are seeing anecdotal reports in the news and additional reports in the scientific media
  • Every week we are seeing anecdotal reports in the news and additional reports in the scientific media
  • Every week we are seeing anecdotal reports in the news and additional reports in the scientific media
  • Every week we are seeing anecdotal reports in the news and additional reports in the scientific media
  • Every week we are seeing anecdotal reports in the news and additional reports in the scientific media
  • Every week we are seeing anecdotal reports in the news and additional reports in the scientific media
  • makes some of these reports circumstantial. However
  • Including acute intoxications, with anxiety, paranoia, gross impairment, and marked tachycardia.
  • more credible reports are now appearing in the medical literature also.
  • Including acute intoxications, with anxiety, paranoia, gross impairment, and marked tachycardia.
  • Sobolevsky and coworkers reported urine profiles of three subjects in Russia who had smoked a product containing JWH-018. The effetc proifile was again similar to that expected of marijuana.
  • They made tentative identifications of more than 13 possible metabolites in these subject’s urine. These include monohydroxylated metabolites on the indole ring, M1, M3 but could not identify the position of hydroxylation. There are actually 4 possible positional isomers of this one monohydroxylated metabolite, so considering that poly hydroxylated compounds are known, the positional isomers have not been determined. Our investigations suggest that the ring hydroxylated metabolites are very minor constituents in human urine, although standards have now been synthesized. They also report terminal hydroxylation on the N-alkyl chain, M2 which we have confirmed as a major metabolite in human urine. The major metabolite is also a mono-hydroxy, but not on any of the positions shown in the Sobolevsky paper. M4, carboxy is also present in small amounts in human urine. M6 and M7 are variants of some of the polyhydroxylated compounds. M8, through M11 are again polyhydroxylated but also reduced, now on the naphthyl ring. And M 12, 13 and other variants are N- delakylated and hydroxylated. So as we will see a complex soup of hydroxylated, reduced and oxidised compounds
  • Detected CP 47,497 C7 and C8 analogs and JWH-018 in the blood. No data on urine. No cross reactivity with THC-COOH immunoassay.
  • Teske and coworkers conducted self administration of a product containing JWH-018. They reported sickness, sedation and dry mouth, hot flushes, burning eyes, and thought disruption. No change in pupil size. But pulse and blood pressure were noticeably elevated.
  • They reported quantitative concentrations in in serum, with peak concentrations occurring in the range of 8-10 ng/mL within a few minutes of smoking, and concentrations falling below 1ng/mL within 3 hours. Trace concentrations were reported at 24 hours (<0.1ng/mL)
  • In July 2010 the University of Missouri sponsored a K2 administration study. Following approval by the University’s IRB, six subjects were administered low doses of smokeable products containing JWH-018, JWH-073 or CP-47,497. Subjects completed SFST’s and a DRE exam, some cognitive tests, and underwent medical assessments. Blood urine and oral fluid were collected, refrigerated or frozen and shipped to NMS labs for analysis. The K2 was analyzed for SC content and the presence of any other drugs before administration.
  • In July 2010 the University of Missouri sponsored a K2 administration study. Following approval by the University’s IRB, six subjects were administered low doses of smokeable products containing JWH-018, JWH-073 or CP-47,497. Subjects completed SFST’s and a DRE exam, some cognitive tests, and underwent medical assessments. Blood urine and oral fluid were collected, refrigerated or frozen and shipped to NMS labs for analysis. The K2 was analyzed for SC content and the presence of any other drugs before administration.
  • In July 2010 the University of Missouri sponsored a K2 administration study. Following approval by the University’s IRB, six subjects were administered low doses of smokeable products containing JWH-018, JWH-073 or CP-47,497. Subjects completed SFST’s and a DRE exam, some cognitive tests, and underwent medical assessments. Blood urine and oral fluid were collected, refrigerated or frozen and shipped to NMS labs for analysis. The K2 was analyzed for SC content and the presence of any other drugs before administration.
  • In July 2010 the University of Missouri sponsored a K2 administration study. Following approval by the University’s IRB, six subjects were administered low doses of smokeable products containing JWH-018, JWH-073 or CP-47,497. Subjects completed SFST’s and a DRE exam, some cognitive tests, and underwent medical assessments. Blood urine and oral fluid were collected, refrigerated or frozen and shipped to NMS labs for analysis. The K2 was analyzed for SC content and the presence of any other drugs before administration.
  • In July 2010 the University of Missouri sponsored a K2 administration study. Following approval by the University’s IRB, six subjects were administered low doses of smokeable products containing JWH-018, JWH-073 or CP-47,497. Subjects completed SFST’s and a DRE exam, some cognitive tests, and underwent medical assessments. Blood urine and oral fluid were collected, refrigerated or frozen and shipped to NMS labs for analysis. The K2 was analyzed for SC content and the presence of any other drugs before administration.
  • In July 2010 the University of Missouri sponsored a K2 administration study. Following approval by the University’s IRB, six subjects were administered low doses of smokeable products containing JWH-018, JWH-073 or CP-47,497. Subjects completed SFST’s and a DRE exam, some cognitive tests, and underwent medical assessments. Blood urine and oral fluid were collected, refrigerated or frozen and shipped to NMS labs for analysis. The K2 was analyzed for SC content and the presence of any other drugs before administration.
  • In July 2010 the University of Missouri sponsored a K2 administration study. Following approval by the University’s IRB, six subjects were administered low doses of smokeable products containing JWH-018, JWH-073 or CP-47,497. Subjects completed SFST’s and a DRE exam, some cognitive tests, and underwent medical assessments. Blood urine and oral fluid were collected, refrigerated or frozen and shipped to NMS labs for analysis. The K2 was analyzed for SC content and the presence of any other drugs before administration.
  • The data show the rapid rise, distribution and decline of the parent compounds in blood, combined with the rapid rise in blood pressure, systolic shown here. Importantly within one hour of this pharmacologically significant dose, the concentrations had dropped to less than 1ng.
  • Urine samples from the MO subjects were collected for between 2-5 hours. Describe One subject provided a 24 hour urine which was positive close to the method threshold. Oral fluid was collected, but had very weak positives, one hour after use. This may be a function of the collection devices and is being investigated further. Our general experience is that the SC compounds do tend to stick tightly to plastic, and glass should be used whenever possible.
  • The effects were no surprisingly similar to those associated with marijuana. (Read from list)

K2 and Beyond: A Synthetic Cannabinoid Primer K2 and Beyond: A Synthetic Cannabinoid Primer Presentation Transcript

  • 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
  • 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.
  • Synthetic Cannabinoids
    • Learning Objectives
    • After 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.
  • Synthetic Cannabinoids
    • Mechanism of Action and Pharmacology.
    • History of Synthetic Cannabinoids
    • Identification of Synthetic Cannabinoids in Botanical material
    • Identification of Synthetic Cannabinoids in Biological Matrices
    • Human pharmacodynamics and Pharmacokinetics.
    • Legal Status
  • SYNTHETIC CANNABINOIDS IN HERBAL INCENSE PRODUCTS Lindsay E. Reinhold, M.F.S., F-ABC AAFS Annual Conference February 2011
  • Topics
    • Introduction
    • Samples Tested
    • Reference Materials
    • Sample Preparation
    • Methods of Identification
    • Sample Results
    • Future 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 THC Classical HU-210/HU-211 JWH-133 JWH-018 Naphthoylindoles JWH-073 JWH-081 JWH-122 JWH-200 JWH-210 JWH-398 WIN55,212-2 CP47,497(C8) Non-Classical CP47,497(C7) CP55,940 HU-308
  • Molecular Structures AM-694 Benzoylindoles AM-630 AM-1241 JWH-250 Phenylacetylindoles JWH-203 JWH-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 Status
    • DEA 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) JWH-073 (non-classical) JWH-200 (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 Tests
        • Macro/Microscopic Analysis
        • Duquenois –Levine
        • Thin 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
  • 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 nm
    • 4: Ninhydrin Spray + heat
    • 5: 10% H2SO4 Spray + UV 366 nm
    • 6: Acidified Iodoplatinate Spray
    • 7: 50% Nitric Acid Spray + heat
    • 8: Mercuric Sulfate Spray
    • 9: 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 Tests
        • Gas 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.Cannabicyclohexanol) CAS# 70434-92-3 Formal Name: 5-(1,1-dimethyloctyl)-2-[(1R,3S)-3- hydroxycyclohexyl]-phenol Mol.Form. C 22 H 36 O 2 Mol.Wt. 332.27153
    • Internal Standard
    • JWH-133
    • CP47,497(C7) Di-TMS
    • CP47,497(C8) Di-TMS
    • HU-308 Mono-TMS
    • CP55,940 Tri-TMS
    • JWH-251
    • HU-210/211 Di-TMS
    • JWH-250
    • RCS-4
    • JWH-015
    • JWH-073
    • AM-694
    • JWH-018
    • CB-25 Di-TMS
    • JWH-019
    • AM-2201
    • CB-52 Di-TMS
    • JWH-122
    • RCS-8
    • JWH-398
    • JWH-210
    • CB-52 Mono-TMS
    • JWH-081
    • JWH-200
    • WIN55,212-2/3
  • CP47,497 (C8 analogue) (a.k.a.Cannabicyclohexanol) Di-TMS Derivative Mol.Form. C 28 H 52 O 2 Si 2 Mol.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/mcL ~ 0.1 ng/mcL ~ 1 ng/mcL
  • LC/MS/MS JWH-200 2.57 WIN55,212-2/3 3.50 AM-2201 4.56 CP55,940 4.79 JWH-015 4.84 JWH-250 5.00 RCS-4 5.12 JWH-073 5.16 JWH-251 5.31 JWH-018 6.49 CP47,497(C7) 6.85 JWH-081 6.94 JWH-019 7.23 JWH-122 7.23 RCS-8 7.30 HU-210/211 7.58 CP47,497(C8) 7.68 JWH-398 7.79 JWH-210 7.79 JWH-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 Analytical Technique JWH-018 JWH-073 TLC Positive Positive GC/MS Positive (approx. 9 mg/g) Positive (approx. 9 mg/g) LC/TOF Positive Positive
  • K2 Citron Analytical Technique JWH-018 JWH-073 TLC Positive Positive GC/MS Positive (approx. 10 mg/g) Positive (approx. 10 mg/g) LC/TOF Positive Positive
  • K2 (unknown variety) Analytical Technique CP47,497 (C8) JWH-018 JWH-073 TLC Positive None Detected None Detected GC/MS Positive (approx. 6 mg/g) None Detected None Detected LC/TOF None Detected Positive Positive
  • K2 Summit Analytical Technique JWH-018 JWH-073 TLC Positive Positive GC/MS Positive (approx. 11 mg/g) Positive (approx. 9 mg/g) LC/TOF Positive Positive
  • K2 Blue Analytical Technique JWH-018 TLC Positive GC/MS Positive (approx. 15 mg/g) LC/TOF Positive
  • K2 Pink Analytical Technique JWH-018 TLC Positive GC/MS Positive (approx. 11 mg/g) LC/TOF Positive
  • K2 Latte Analytical Technique JWH-018 JWH-073 JWH-250 TLC Positive Positive Positive GC/MS Positive (approx. 16 mg/g) Positive (approx. 0.03 mg/g) Positive (approx. 14 mg/g) LC/TOF Positive Positive Positive
  • K2 Pineapple Express Analytical Technique JWH-018 TLC Positive GC/MS Positive (approx. 5.4 mg/g)
  • K2 Blueberry Analytical Technique 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 Logan Fran Diamond Allan Xu Sherri 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
    • C 18 SPE, TMS derivatization & GC-EI-MS
  • Auwärter et al.
    • Contents of ‘Spice Diamond’ identified by GCMS, TLC, UV, NMR
  • 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 d 5 -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.
  • 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: d 9 -JWH-018 & d 9 -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 d 9 -JWH-018 3.12 351  155 351  127 JWH-018 3.13 342  155 342  127 JWH-019 3.30 356  155 356  127 d 9 -JWH-073 2.92 337  155 337  127 JWH-073 2.94 328  155 328  127 JWH-250 2.93 336  121 none
  • Kacinko et al. 100 0 126.92 155.18 214.26 342.17 127.05 227.96 356.39 0 155.12 100 143.89 JWH-019 JWH-018
  • Kacinko et al. 100 0 100 100 200 300 400 0 m/z 126.86 155.12 200.10 328.18 121.11 200.16 144.34 JWH-250 JWH-073
  • Kacinko et al. Validation Parameter Conc. (ng/mL) JWH-018 JWH-019 JWH-073 JWH-250 Average within-run imprecision (%RSD) 0.1 0.3 15 4.3 5.4 2.6 19.3* 11.6 9.5 7.9 4.3 1.9 7.0 4.5 3.6 Between-run imprecision (%RSD) 0.1 0.3 15 7.9 5.9 3.2 35.7* 20.6* 23.0* 10.2 4.3 2.1 8.6 4.7 6.9 Accuracy (%) 0.1 0.3 15 99.2 93.7 93.7 112.0 70.3* 67.3* 99.1 92.2 93.0 104.7 93.6 91.8 *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 (%) 0.3 15 75.5 63.2 63.6 60.6 80.8 71.0 92.8 79.4 Matrix Effect (%) 0.3 15 36.7 49.4 42.1 64.7 31.1 32.2 22.0 18.6 Process Efficiency (%) 0.3 15 47.7 32.0 36.8 21.4 55.6 48.1 72.4 64.6
  • Kacinko et al. Whole Blood Additive Conc. (ng/mL) JWH-018 JWH-073 JWH-250 Sodium Fluoride (n=2) 0.5 10 103.1 98.7 111.8 106.0 88.5 86.6 Potassium Oxalate (n=3) 0.5 10 96.0 94.6 102.3 107.0 87.5 87.6 Sodium EDTA (n=3) 0.5 10 105.3 98.1 113.5 107.7 88.5 87.5 Potassium EDTA (n=2) 0.5 10 109.6 93.2 104.0 103.5 94.0 87.4 Sodium Heparin (n=3) 0.5 10 99.9 92.0 102.9 97.4 88.2 84.3 Lithium Heparin (n=2) 0.5 10 103.1 91.5 98.4 96.7 94.5 82.8
  • 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 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
  • 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
  • Urine Test Development
    • 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.
  • Urine Test Development Intact glucuronide of mono-hydroxy JWH-018 Mono-hydroxy JWH-018 Mono-hydroxy JWH-018
  • Urine Test Development
  • Urine Test Development Screening Method Source Fragmentation Collision Cell Fragmentation JWH-018-OH-GLUC 534  358 358  155 358  127 JWH-073OH-GLUC 520  344 344  155 344  127
  • Urine Test Development Confirmation Method Precursor Ion Product Ions OH-JWH-018 358 155 127 284 186 2OH-JWH-018* 376 374 214 171 155 127 3OH-JWH-018 374 189 171 OH-JWH-073 344 155 127 2OJ-JWH-073* 362 360 200 171 155 127 3OH-JWH-073 378 189 171
  • Final Monitored Transitions JWH-018 JWH-073 358 344 374 362 358 344 374 362 155 155 155 155 127 127 127 127
  • 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
  •  
  • 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 Drugs part of shooter's troubles By Leslie Reed January 11, 2011. Friends hint at possible drug use — hints that may be substantiated by new drug test results that reveal the presence of K2, a synthetic marijuana substitute, in Butler's body. Authorities have said the designer drug was used by some students at Butler's former school, Lincoln Southwest, and say it's known to cause delusions and paranoia in some users.
  • K2 Phenomenon McHenry shop owner had K2 in system McHENRY – 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 News Updated: 01/31/2011 Two 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 test Published: Saturday, Jan. 22, 2011 SALT 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
  • Synthetic Cannabinoid Effects
  • Synthetic Cannabinoid Effects The Journal of Emergency Medicine, Epub, 2010
  • Synthetic Cannabinoid Effects 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
  • Synthetic Cannabinoid Effects
  • Synthetic Cannabinoid Effects
  • Synthetic Cannabinoid Effects
  • Synthetic Cannabinoid Effects
  • 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.
  • Missouri K2 Administration Lab 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
  • Missouri K2 Administration Lab
    • Pre-dosing:
      • Nurse evaluation, vital signs
      • Oral Fluid/Urine drug screen
      • Eye exam
      • Cognitive tests
      • DRE evaluation
  • Missouri K2 Administration Lab
    • Dosing:
      • Pre-dose pulse/blood pressure.
      • 0.3g botanical material placed in water pipe.
      • Up to three inhalations.
  • Missouri K2 Administration Lab
    • Post-dosing:
      • Nurse evaluation, vital signs
      • Blood sampling
      • Ad lib urine sampling
      • DRE evaluation
      • Eye exam
      • Cognitive tests
  • Missouri K2 Administration Lab
    • Onset of subjective effects within 2-3 minutes.
    • Subjectively peaking 5-10 minutes.
    • Taste tobacco/burning garbage/unpleasant
    • Somatic Effects:
        • Dry mouth
        • Light headedness
        • Buzzed
        • Blurred vision
        • Laughter
        • Motor agitation/restlessness
        • Time dilation
        • Mild anxiety/paranoia
        • Post intoxication fatigue/exhaustion
  • Missouri K2 Administration Lab Authors Sobolevsky et al, 2010 Teske et al, 2010 Auwater et al, 2009 Elsohly, 2008 This 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
  • Missouri K2 Administration Lab 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-073 Mono-OH Di-OH Tri-OH Glucuronides Pre-dose X X X X X 1:15 X √ √ X √ 2:07 X √ √ √ √ 2:40 X √ √ √ √
  • Missouri K2 Administration Lab
    • Psychomotor Effects
    • Highly variable response
    • DRE Exam
      • Increased pulse and blood pressure
      • Lack of convergence
      • No HGN, or VGN
      • Pupils normal, muscle tone normal
    • SFST’s
      • 3-4 inches of sway, leg body tremors
      • Loss of balance
      • Loss of motor coordination
  • Missouri K2 Administration Lab
      • 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?