Human Hepatocyte Incubations and High Resolution Mass Spectrometry to Characterize the Metabolism of Novel Psychoactive Substances

Marilyn Huestis, enior Fellow at the Lambert Center for the Study of Medicinal Cannabis and Hemp Institute of Emerging Medical Professions, Thomas Jefferson University, Philadelphia, USA

Synthetic cannabinoids are highly potent novel psychoactive substances (NPS) that bind to the same receptors as ∆⁹-tetrahydrocannabinol, cannabis’ primary psychoactive component. Initially, synthetic cannabinoids were synthesized as pharmacological probes for investigating the endogenous cannabinoid system and developing potential pharmacotherapies. They are now synthesized in clandestine laboratories and sold on the internet. Because of increasing prevalence and high morbidity and mortality, many synthetic cannabinoids are scheduled as illicit drugs. New synthetic cannabinoids quickly emerge after scheduling, with clandestine manufacturers making minor structure modifications to evade scheduling laws.

Identification of the causative agent in the toxic exposures is critical to tie adverse effects to a specific compound, and to educate the public about the dangers associated with drug intake. A major problem with identifying synthetic cannabinoids intake is the parent drug’s short detection windows in blood and oral fluid. Hence, the urgent need to identify urinary metabolites as markers of intake. Few human metabolism data are available.

Due to the inability to conduct controlled NPS administration studies in humans, we developed a method to identify the most prevalent and the most definitive phase 1 and 2 metabolites for new NPS. A TripleTOF 5600⁺ high-resolution mass spectrometer (HR-MS) was utilized to identify optimal metabolite markers after incubating the NPS in human hepatocytes for 3 h. Data were acquired via full scan and information-dependent acquisition triggered product ion scans with mass defect filter.

For the new THJ-018, 13 metabolites were detected, with the major metabolic pathways being hydroxylation on the N-pentyl chain with further oxidation or glucuronidation. For THJ-2201, 27 metabolites were observed, predominantly oxidative defluorination and subsequent carboxylation or glucuronidation, and glucuronidation of hydroxylated metabolites. Dihydrodiol formation on the naphthalene moiety was observed for both compounds.

With HR-MS for data acquisition and processing, we characterized THJ-018 and THJ-2201 metabolism in human hepatocytes and selected a few appropriate markers for laboratories to identify THJ-018 and THJ-2201 intake, differentiate intake of these two parent synthetic cannabinoids and link observed adverse events to the appropriate compound.