In Vitro Metabolic Profiling of 18 Semi-Synthetic Cannabinoids—Hexahydrocannabinol (HHC) and Its Analogs—with Identification in an Authentic Hexahydrocannabiphorol (HHCP) Urine Sample

Abstract

Background Hexahydrocannabinol (HHC) and its analogs are recent additions to semi-synthetic cannabinoids in the recreational drug market. Here, the metabolism of 18 HHC analogs was compared to gain a comprehensive understanding of the structure–metabolism relationship of HHC analogs and to identify urinary biomarkers. Additionally, an authentic urine sample obtained from a suspected hexahydrocannabiphorol (HHCP) user was analyzed. Methods Both 9(R)- and 9(S)-epimers of HHC and 8 analogs were separately incubated with human liver microsomes (HLMs) for 1 h. The resulting products and the urine sample were analyzed by liquid chromatography–high-resolution mass spectrometry in an untargeted approach. Results The metabolites were generated by hydroxylation, dehydrogenation, ketone formation, carboxylation, or hydrolysis, either alone or in combination. Concerning the HHC homologs, for 9(R)-epimers, metabolites with multiple biotransformations, e.g., dihydroxy metabolites, were generally more abundant, and the percentage of hydroxy metabolites tended to increase for the longer side-chain homologs, while the reverse was observed for dihydroxy metabolites. For 9(S)-epimers, a metabolite hydroxylated at the methylcyclohexyl moiety was by far the most abundant metabolite, with similar behaviors among hexahydrocannabivarin, hexahydrocannabutol, and HHC, and between hexahydrocannabihexol and HHCP. Acetylated analogs initially underwent hydrolysis to produce almost identical metabolic profiles as the non-acetylated analogs. Methyl ether analogs did not appear to show any particular metabolic trend. In the clinical urine sample, 3 HHCP (di-)hydroxylated metabolites were detected, matching the HLM results. Conclusions The revealed structure–metabolism relationship could serve as a reference for investigating the metabolism of similar cannabinoids, while the identified biomarkers could facilitate drug testing.