High-Resolution Mass Spectrometry for Characterizing the Metabolism of Synthetic Cannabinoid THJ-018 and Its 5-Fluoro Analog THJ-2201 after Incubation in Human Hepatocytes Xingxing Diao, 1 Ariane Wohlfarth, 1 Shaokun Pang, 2 Karl B. Scheidweiler, 1 and Marilyn A. Huestis 1* BACKGROUND: Despite increasing prevalence of novel psychoactive substances, no human metabolism data are currently available, complicating laboratory documenta- tion of intake in urine samples and assessment of the drugs’ pharmacodynamic, pharmacokinetic, and toxico- logical properties. In 2014, THJ-018 and THJ-2201, synthetic cannabinoid indazole analogs of JWH-018 and AM-2201, were identified, with the National Forensic Laboratory Information System containing 220 THJ- 2201 reports. Because of numerous adverse events, the Drug Enforcement Administration listed THJ-2201 as Schedule I in January 2015. METHODS: We used high-resolution mass spectrometry (HR-MS) (TripleTOF 5600 + ) to identify optimal me- tabolite markers after incubating 10 mol/L THJ-018 and THJ-2201 in human hepatocytes for 3 h. Data were acquired via full scan and information-dependent acqui- sition triggered product ion scans with mass defect filter. In silico metabolite predictions were performed with MetaSite and compared with metabolites identified in human hepatocytes. RESULTS: Thirteen THJ-018 metabolites were detected, with the major metabolic pathways being hydroxylation on the N-pentyl chain and further oxidation or glucu- ronidation. For THJ-2201, 27 metabolites were ob- served, predominantly oxidative defluorination plus subsequent carboxylation or glucuronidation, and glucu- ronidation of hydroxylated metabolites. Dihydrodiol for- mation on the naphthalene moiety was observed for both compounds. MetaSite prediction matched well with THJ-018 hepatocyte metabolites but underestimated THJ-2201 oxidative defluorination. CONCLUSIONS: With HR-MS for data acquisition and processing, we characterized THJ-018 and THJ-2201 metabolism in human hepatocytes and suggest appropri- ate markers for laboratories to identify THJ-018 and THJ-2201 intake and link observed adverse events to these new synthetic cannabinoids. © 2015 American Association for Clinical Chemistry Synthetic cannabinoids (SCs) interact with endogenous cannabinoid receptors and elicit cannabimimetic effects similar to those of  9 -tetrahydrocannabinol (1, 2 ). SC 3 abuse causes serious toxicities, including acute kidney injury, myocardial infarction, and death (3, 4 ). Despite these health hazards, SC intake persists. Many SCs (e.g., JWH018, JWH-073, AKB48, and XLR-11) have been scheduled in the US, Japan, and most European coun- tries; however, more structurally diverse compounds con- tinuously emerge. Two new SCs, THJ-018 [1-naphthalenyl(1-pentyl- 1H-indazol-3-yl)-methanone] and its 5-fluoro analog, THJ-2201 [(1-(5-fluoropentyl)-1H-indazol-3-yl)(naph- thalen-1-yl)methanone] (Fig. 1), were found in the US, Japan, and Russia in 2014 (5–8). The indole core of JWH-018 and AM2201 is replaced by indazole in THJ- 018 and THJ-2201. The National Forensic Laboratory Information System has detailed 220 THJ-2201 reports since January 2014 (7). Although no THJ-018 preva- lence data are available, drug-user forums suggested in- creasing popularity for both analogs (9). In January 2015, THJ-2201 became a Schedule I compound in the US (7); THJ-018 and THJ-2201 were scheduled in Ja- pan in August 2014 (10 ). Currently, THJ-018 and THJ- 2201 pharmacological data are unavailable; however, 1 Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, NIH, Baltimore, MD; 2 SCIEX, Redwood City, CA. * Address correspondence to this author at: Chemistry and Drug Metabolism, IRP, Na- tional Institute on Drug Abuse, NIH, 251 Bayview Blvd, Suite 200 Room 05A721, Balti- more, MD 21224. Fax 443-740-2823; e-mail mhuestis@intra.nida.nih.gov. Received May 17, 2015; accepted July 21, 2015. Previously published online at DOI: 10.1373/clinchem.2015.243535 © 2015 American Association for Clinical Chemistry 3 Nonstandard abbreviations: SC, synthetic cannabinoid; THJ-018, 1-naphthalenyl(1- pentyl-1H-indazol-3-yl)-methanone; THJ-2201, (1-(5-fluoropentyl)-1H-indazol-3-yl) (naphthalen-1-yl)methanone; HR-MS, high-resolution mass spectrometry; HLM, human liver microsome; NPS, novel psychoactive substance; KHB, Krebs-Henseleit buffer; IDA, information-dependent acquisition; MDF, mass defect filter; CYP450, cytochrome P450; CL int, micr , microsomal intrinsic clearance; CL int , intrinsic clearance; CL H , hepatic clear- ance; ER, extraction ratio. Clinical Chemistry 62:1 157–169 (2016) Drug Monitoring and Toxicology 157 Downloaded from https://academic.oup.com/clinchem/article/62/1/157/5611966 by guest on 19 June 2022