Insights into the human metabolism of hexahydrocannabinol by non-targeted liquid chromatography-high-resolution tandem mass spectrometry

Abstract

Hexahydrocannabinol (HHC), 6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10a-hexahydrobenzo[c]chromen-1-ol, is a semi-synthetic cannabinoid that has presented challenges to analytical laboratories due to its emergence and spread in the drug market. The lack of information on human pharmacokinetics hinders the development and application of presumptive and confirmatory tests for reliably detecting HHC consumption. To address this knowledge gap, we report the analytical results obtained from systematic forensic toxicological analysis of body-fluid samples collected from three individuals suspected of drug-impaired driving after HHC consumption. Urine and plasma samples were analyzed using non-targeted liquid chromatography-high-resolution tandem mass spectrometry. The results provided evidence that HHC undergoes biotransformation reactions similar to other well-characterized cannabinoids, such as ∆9-tetrahydrocannabinol or cannabidiol. Notably, HHC itself was only detectable in plasma samples, not in urine samples. The observed Phase I reactions involved oxidation of C11 and the pentyl side chain, leading to corresponding hydroxylated and carboxylic acid species. Additionally, extensive glucuronidation of HHC and its Phase I metabolites was evident.

Figure 1.

Molecular structure of HHC. (a) Possible sites of metabolic transformation and (b) likely occurring fragmentation reactions in MS-MS experiments are highlighted.

Figure 2.

Fragmentation spectra of (a) an unknown compound detected in one of the analyzed plasma samples, (b) cannabigerol in a reference sample and (c) HHC in a reference sample.

Figure 3.

Representative XICs of HHC and proposed human transformation products: (a) HHC, (b) HHC glucuronide, (c) hydroxylated HHC, (d) glucuronide of hydroxylated HHC, (e) HHC oxidized at C11 to the carboxylic acid, (f) glucuronide of HHC oxidized at C11 to the carboxylic acid, (g) HHC oxidized at C3′ to the carboxylic acid, and (h) glucuronide of HHC oxidized at C3′ to the carboxylic acid. The color code refers to the possible sites of metabolic transformation of HHC as depicted in Figure 1.

Figure 4.

Representative fragmentation spectra of the proposed HHC Phase II metabolites. (a) M1.1: HHC glucuronide 1, (b) M3.2: HHC-OH (6–9) glucuronide 2, (c) M4.1: HHC-OH (1′–5′) glucuronide 1, (d) M6: HHC-COOH glucuronide and (e) M8: a putative metabolite obtained by oxidation of C3′ to the carboxylic acid and subsequent glucuronidation (HHC-COOH (3′) glucuronide).

Figure 5.

Proposed metabolic transformation of HHC in humans based on the screening of plasma and urine samples for HHC metabolites.