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.