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

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.

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.

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.

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, M_U1 and M_U10 are recommended markers for 9(R)-HHC and M_U7 for 9(S)-HHC consumption. M_U3, M_U4, and M_U16 are proposed as metabolite biomarkers for HHC intake, after hydrolysis. Additionally, difference in HHC and Δ⁹-THC metabolism was observed.