Mass Spectrometry-based Detection of Newly Emerging Psychoactive Drugs Kenichi (Ken) Tamama, M.D., Ph.D. Associate Professor of Pathology University of Pittsburgh School of Medicine Medical Director, Clinical Toxicology Laboratory University of Pittsburgh Medical Center Learning objectives • Describe the current drug-of-abuse situation of emerging psychoactive drugs • Summarize laboratory detection of psychoactive drugs • Explain the challenges in detecting the emerging psychoactive drugs DEA: “Drugs of abuse as a national threat in the US” http://www.post-gazette.com/ • Drug overdose deaths have become the leading cause of injury death in the US, surpassing the number of deaths by motor vehicles and by firearms every year since 2008. • In 2015, approximately 140 people died every day from drug poisoning. (DEA NDTA 2017) Heroin • Diacetyl morphine (→ 6-MAM → Morphine) • Detectable by opiate EIA • Schedule I Controlled substance (high potential for abuse, no medical use) • Heroin overdose deaths are increasing in many cities and counties across the United States • Heroin availability is increasing in areas throughout the nation. • Increased demand for, and use of, heroin is being driven by both increasing availability of heroin in the US market and by some CPD abusers using heroin. (DEA NDTA 2017) • Pittsburgh is in the western PA, facing OH and WV (Tri-State area) • PA: 5.6 death rate per 100,000 • OH: 13.3 death rate per 100,000 (Worst in the nation) • WV: 11.8 death rate per 100,000 (2nd worst in the nation) (DEA NDTA 2017) Fentanyl • Synthetic opioid • 80 to 100 times stronger than morphine • 25 to 40 times more potent than heroin • Not detected by opiate EIA (fentanyl EIA is needed) • Schedule II (high potential for abuse, acceptable medical use) • Clandestinely-produced fentanyl is sometimes added to heroin to increase its effects, or mixed with adulterants and diluents and sold as heroin • In March 2015, DEA issued a nationwide alert about the dangers of fentanyl and fentanyl analogues/ compounds, stating “Fentanyl is commonly laced in heroin, causing significant problems across the country, particularly as heroin use has increased.” • 79%↑ in synthetic opioid deaths between 2014 and 2015 (CDC) Fentanyl analogs (Fentalogs) identified at the UPMC lab (Li, Am J Clin Pathol. 2017) • Carfentanyl • The most potent opioid (10,000 times more potent than morphine) • Used as a tranquilizing agent for elephants • Between July 5 and July 26, 2016, paramedics in Akron, OH registered at least 236 drug overdoses with at least 14 being fatal, linked to suspected carfentanyl. (DEA NDTA 2017) Other opioids (Mitragynine, AH-7921 and U-47700) • Mitragynine • Major psychoactive alkaloid of the plant kratom indigenous to South East Asia. • Emerging in the US as a legal psychoactive product available on-line. • In the U.S., mitragynine has been used recreationally as well as a means to treat symptoms of opioid withdrawal outside of established medical programs . • 7-hydroxymitragynine, a minor constituent of kratom, has 46 times higher potency than mitragynine . • AH-7921 and U-47700 • Synthetic opioids developed in the mid-70s • AH-7921 is as potent as morphine • U-47700 is 7.5 times more potent than morphine • In 2016, DEA first encountered U-47700 responsible for at least 80 deaths in the United States for the year. (Li, Am J Clin Pathol. 2017; DEA NDTA 2017) Synthetic Designer Drugs • Synthetic designer drugs refer to man-made substances created to mimic the effects of controlled substances, and are oftentimes unscheduled and unregulated. • The two most commonly used types of synthetic designer drugs in the United States are • Synthetic cannabinoids • Amphetamine-like stimulants including cathinones • Synthetic designer drugs might be attractive to drug users subjected to drug screening (e.g., inmates, parolees, and probationers), as drug screens often do not have the ability to identify all of these compounds. (DEA NDTA 2015) Rapid spread of designer drugs (DEA NDTA 2013) Synthetic cannabinoids • Synthetic cannabinoids, also commonly known as “Spice” and “K2,” are chemicals synthesized in laboratories and simulate the biological effects of THC. • Synthetic cannabinoids are the 4th most popular drug used among 8th graders, the 3rd most popular drug used among 10th graders and the 4th most popular drug used among 12th graders. (DEA NDTA 2015) Synthetic cannabinoids are rapidly evolving 2010 2012 (Designer_Drug_Trends_February_2014, from www.nmslab.com) (DEA NDTA 2017) Synthetic cannabinoids F • 4 major structural groups AM2201 • Alkylindoles XLR-11 • Naphthoylindoles (JWH-018, JWH-073, AM2201) • Phenylacetylindoles (JWH-250) AM-694 • Benzolindoles (AM-694) • Cyclopropylindoles (XLR-11) APICA AKB-48 • Adamantyllindoles (APICA) • Cyclohexylphenols (CP47,497) AB-PINACA • Indazole carboxamides (AKB-48 (APINACA), AB-PINACA, AB-CHMINACA) • Classical cannabinoids (HU-210) AB- CHMINACA Amphetamine-type stimulants • Cathinone is an active compound in Catha edulis (khat), a flowering plant indigenous to the Horn of Africa and the Arabian Peninsula • Cathinones are also commonly known as “bath salts,” can produce pharmacological effects similar to amphetamine and MDMA. • Other types of stimulants (“2Cs or dimethoxyphenethylamines”, “Dimethoxyamphetamines”) are also emerging as well. • Synthetic cathinones are “rebranded” and sold as MDMA, or “Molly” • Methylone has been seized in all forms of supposed MDMA. Amphetamine-type stimulants • β-keto amphetamines Butylone Methylone Ethylone Methcathinone Mephedrone • Pyrrolidinophenones MDPV Amphetamine Pyrovalerone αPVP • 2C (dimethoxyphenethylamines) Due to their moderate structural similarity to amphetamine, β-keto amphetamines and 2C-B 2C-I dimethoxyamphetamine appear to cross- • Dimethoxyamphetamines react weakly with some of amphetamine immunoassays. DOET DOM Arylcyclohexylamines • Dissociative and hallucinogenic agents • Ketamine, Phencyclidine (PCP), and their analogs [methoxetamine (MXE), 2- oxo-PCE, etc.] • Recreationally abuse • Super K (Ketamine), Wet (PCP + THC), Mexxy, m-ket, k-max(MXE) • 2-oxo-PCE cases in Hong Kong • The analogs as novel psychoactive substances (NPS) • No statistics provided in the DEA reports • 3% of the seized drug in European Drug Report 2016 • Limited scientific literature • Several cases at the UPMC (EMCDDA, European Drug Report 2018) GABA-mimetics • GHB (γ-hydroxybutyrate​) • Originally synthesized as an active analog of GABA in 1960​ • Ingested for euphoria, relaxation, sedation during rave parties • Drug rape substance as it is "color- and odorless“ • Schedule I in CSA • The total number of GHB abuse seems to be comparatively low • Pathophysiology • GHB itself is present in the brain and serves as both a precursor and a degradation product of GABA • γ-butyrolactone (GBL) and 1,4-butanediol (BD) are precursors of GHB • Therapeutic usage for nacrolepsy (Sodium Oxybate or Xyrem®) • Phenybut (β-phenyl-γ-aminobutyric acid) • Developed in Russia in 60s and has been used for anxiolysis, preoperational sedation and alcohol withdrawal • Structurally similar to baclofen • Recreational abuse cases have been reported recently • Available through the Internet (O'Connell, Am J Med. 2014; Schep Clin Toxicol. 2012; Drasbek, Acta Neurol Scand. 2006 ) GABA-mimetics (Modified from Drasbek, Acta Neurol Scand 2006, Lapin, CNS Drug Rev. 2001) Laboratory detection of emerging drugs of abuse • Immunoassays • Mostly not FDA-approved • Cross-reactivity of these compounds with the existing kits is often unknown • 2D molecular similarity calculation to predict cross-reactivity of these compounds • Mass Spectometry • GC-MS • Traditionally regarded as a gold standard platform • LC-MSMS or LC-high resolution MS GC-MS • GC and MS combined • Originally developed in 1950’s • Older than LC-MS • Samples have to be delivered as a gas • Analytes have to be volatile • Output from GC • Sample derivatization often needed for GC • Hard ionization J Am Soc Mass Spectrom 1993, 4, 367-371 • EI (Electron ionization) The expanding role of mass spectrometry in biotechnology Henry’s Clinical Diagnosis and Laboratory Management. 2011 Comprehensive drug screening at UPMC • Immune-based screening of the major drugs of abuse • Amphetamine-EIA, Barbiturate-EIA, Benzodiazepine-EIA, Buprenorphine-EIA, Cocaine metabolite-EIA, Methadone-EIA, Opiate-EIA, Oxycodone/oxymorphone-EIA, Phencyclidine-EIA, THC metabolite-EIA • GC-MS-based screening • Untargeted comprehensive screening • For screening of the drugs not covered by immunoassays • For confirmation of immunoassays • Full scan mode and selected ion monitoring for major drugs • Laborious procedures, but offered 24/7 • Preliminary report for stat orders • Final report after the pathologist’s review in the next business day Sample preparation Comprehensive drug screening by GC-MS Opiate confirmation by GC-MS X1 • Sample preparation is laborious for both comprehensive drug screening and drug confirmation. • CDS: >60 min for sample preparation • Opiate confirmation: >60 min for acid hydrolysis, >20 min for SPE, >20 min for derivatization • GC-MS run time: 45 min • The time for the reagent preparation is NOT included! A case of comprehensive drug screening by GC-MS Drugs of abuse (parental and metabolites) and impurities • Cocaine and metabolites (ecgonine methyl ester, trimethoxycocaine, cocaethylene, ethylecgonine) • 6MAM (heroin metabolite), acetylcodeine, codeine • Fentanyl, parafluoroisobutyryl fentanyl, butanoyl-4-fluorofentanyl Cutting agent • Levamisole • Quinine