Ongoing Challenges for the Tox Lab in Identifying Receptor Agonists By Sarah Urfer, M.S., D-ABFT-FT Disclosure and Disclaimer

• Disclosure • I work for and own a portion of ChemaTox Laboratory, Inc.

• Disclaimer • I have no affiliation or interest in any of the other companies, brands, groups, or organizations mentioned in this presentation. All information is provided for educational purposes only. Cannabinoid Receptor Agonists AKA SYNTHETIC Just like the Potpourri grandma has Product photos used with permission from Kevin Shanks, M.S., D-ABFT-FT From AIT Laboratories In The Beginning

• Researchers synthesized 100’s, maybe 1000’s of “research chemicals” • Looking for compounds with binding affinity and activity to use as possible therapeutic drugs • John W. Huffman, Organic chemist from Clemson University • JWH-018 one of the first seen on the market 2008 - Germany

• Affinity for the cannabinoid (CB1) receptor 5X greater than THC Today

• Legal Highs are everywhere • “not for human consumption” • Most made in China • New ones made faster than we can: • Regulate • Test for • Characterize • 14 recognizable chemical families • 100+ new compounds a year Effects

• Euphoria • Anxiety • Agitation • Paranoia • Hallucinations • Tachycardia • Hypertension • Excessive sweating • Nausea, and vomiting • Deaths have been reported Popularity

• Growing since 2006 • Population dependent • Monitoring the Future Survey • http://www.drugabuse.gov/trends-statistics/monitoring- future/monitoring-future-study-trends-in-prevalence- various-drugs

Monitoring the Future Study: Trends in Prevalence of Various Drugs for 8th Graders, 10th Graders, and 12th Graders; 2011 - 2014 (in percent)*

Time 8th Graders 10th Graders 12th Graders Drug Period 2011 2012 2013 2014 2011 2012 2013 2014 2011 2012 2013 2014

K2/Spice [5.40 [5.80 (Synthetic Past Year - 4.4 4 3.3 - 8.8 7.4 11.4 11.3 [7.90] ] ] Marijuana)

* Data in brackets indicate statistically significant change from the previous year. The Pattern

• Here today, gone tomorrow • Manufacturer make new drug • Shows up on street • Law Enforcement seizures • Hospital Emergency Departments • Forensic Labs • Regulations put in place • Next day • Manufacturer make new drug A lot of variety STRUCTURES AB-CHMINACA vs THC • N-(1-Amino-3methyl-1- oxoButan-2-yl)- 1CycloHexylMethyl-1H- INdAzole-3-CarboxAmide

• Δ9- Make Your Own Synthetic Cannabinoid http://www.emcdda.europa.eu/topics/pods/synthetic-cannabinoids#panel2

Examples METHODS Moving Target

• Many compounds • 100+ new per year • Takes time to find out what is new • Predicting new compounds? • Development - limited data • Scope • Targeted vs Untargeted • Concentrations are often single digit ng/mL • So Many Possibilities! ELISA

• Fast • Prep and Run time • Low sample volume • Many labs can run them • Lags behind what is current • Need low cutoffs • Metabolites may cross react • Simple data analysis GC/MS

• full scan data acquisition • Patterns can be seen for structures of the • Example: naphthoylindoles fragmentation patterns well studied • Comparison of the spectra with commercial and open spectral databases

• Chromatography–mass spectrometry studies on the metabolism of synthetic cannabinoids JWH-018 and JWH-073, psychoactive components of smoking mixtures, Andrej Grigoryev et al. Journal of Chromatography B Volume 879, Issues 15–16, 1 May 2011, Pages 1126– 1136 LC-MS/MS

• Sensitive – low limits of detection (LOD) • Low sample volume • Targeted • Chromatographic conditions – simple • Limited by available standards

• Development and validation of a liquid chromatography–tandem mass spectrometry method for the quantitation of synthetic cannabinoids of the aminoalkylindole type and in serum and its application to forensic samples, Sebastian Dresen, et al, Journal of Mass Spectrometry Volume 46, Issue 2, pages 163–171, February 2011, DOI: 10.1002/jms.1877 Q-TOF LC/MS

• Detect many possible compounds • Tentative ID: parent drugs & metabolites • Instruments are expensive • Knowledge of how to analyze data • Untargeted or Targeted • Higher LODs

• Comprehensive analytical strategies based on high-resolution time- of-flight mass spectrometry to identify new psychoactive substances María Ibáñez, et al, TrAC Trends in Analytical Chemistry Volume 57, May 2014, Pages 107–117 A to Z WHAT ARE WE LOOKING FOR Reference Lab – 33 compounds • 5F-AB-001 • APINACA (AKB-48) • 5F-ADB-PINACA • BB-22 • 5F-ADBICA • FUB-AKB-48 • 5F-APICA • FUB-PB-22 • 5F-APINACA (5F-AKB-48) • FUBIMINA • 5F-MN-18 • JWH-018 • 5F-PB-22 • JWH-081 • AB-CHMINACA • JWH-122 • AB-FUBINACA • JWH-210 • AB-PINACA • MDMB-CHMINACA • ADB-CHMINACA • MN-18 • ADB-FUBINACA • MN-25 • ADB-PINACA • PB-22 • ADBICA • THJ-018 • AM-2201 • THJ-2201 • APICA • UR-144 • XLR-11 Reality for Most Labs

• Expensive • Time consuming • Instrument time • Instrument types • Is it worth is

• A few reference labs have dedicated programs • Many labs outsource the testing Conclusions

• Data, more data • Standardization • Methods • Cutoffs and LODs • Published methods • Case studies, Research - Interpretation • Faster identification of new compounds • Education and awareness • Public & Professional It’s that time again – LUNCH! References

• Pharmacology, Toxicology, and Adverse Effects of Synthetic Cannabinoid Drugs ss s S. M. R. Gurney, K. S. Scott, S. L. Kacinko, B. C. Presley, B. K. Logan Forensic Science Review 26:53; 01/2014

• http://www.cpdd.org/Pages/Links/Links_PDFs/SyntheticDrugofAbuseUpdateJune2014.pdf %20FINAL.pdf

• Comprehensive review of the detection methods for synthetic cannabinoids and cathinones Akira Namera,corresponding author Maho Kawamura, Akihiro Nakamoto, Takeshi Saito, and Masataka Naga, Forensic Toxicol. 2015; 33(2): 175–194. Published online 2015 Mar 6. doi: 10.1007/s11419-015-0270-0 PMCID: PMC4525208

• Synthetic cannabinoid use among patients in residential substance use disorder treatment: prevalence, motives, and correlates. Bonar EE, Ashrafioun L, Ilgen MA, Drug Alcohol Depend. 2014 Oct 1;143:268-71. doi: 10.1016/j.drugalcdep.2014.07.009. Epub 2014 Jul 17

• Zaitsu K, Katagi M, Nakanishi K, Shima N, Kamata H, Kamata T, Nishioka H, Miki A, Tatsuno M, Iwamura T, Sato T, Tsuchihashi H, Suzuki K. Comprehensive analytical methods of the synthetic cannabinoids appearing in the illicit drug market (in Japanese with English abstract) Jpn J Forensic Sci Tech. 2011;16:73–90.