Human diagnostic markers for intake of semi-synthetic cannabinoids (9R)- and (9S)-hexahydrocannabinol

Background: Hexahydrocannabinol (HHC) emerged on the illicit drug market in 2022, especially in cannabinoid consumables. HHC has a chiral center and is present as 9(R) and 9(S) epimers, exhibiting different potencies. This study explores the enantioselective metabolism of 9(R)- and 9(S)-HHC to identify and differentiate intake of these two toxicants. Methods: 9(R)- and 9(S)-HHC were incubated separately with human hepatocytes for 3 h and analyzed by liquid chromatography-high resolution mass spectrometry. In addition, authentic urine samples collected 0.5, 3 and 24 h after HHC intake were also analyzed to identify biomarkers. In silico predictions were performed to complement in vitro and in vivo experiments with data mining analysis. Results: In silico metabolites predicted were transformed via hydroxylation, carboxylation, and alcohol oxidation to ketones and further conjugated through phase II glucuronidation and sulfation reactions. Thirteen metabolites were identified following 9(R)-HHC in vitro incubation and nine for 9(S)-HHC incubate. In human urine, nineteen metabolites were identified, ten and six specific 9R- and 9S-HHC metabolites, respectively, and three undefined for a specific isomer. However, 9(R)- and 9(S)-HHC were not detected in the urine under these analytical conditions. Conclusion: The stereoselective metabolism of 9(R)- and 9(S)-HHC was observed in in vitro incubation and in vivo authentic urine. Phase I metabolites were substantially conjugated as glucuronides. Without hydrolysis, MU1 and MU10 are recommended markers for 9(R)-HHC and MU7 for 9(S)-HHC consumption. MU3, MU4, and MU16 are proposed as metabolite biomarkers for HHC intake, after hydrolysis. Additionally, difference in HHC and Δ9-THC metabolism was observed.