Investigation of phase II metabolism of 11-hydroxy-Δ-9-tetrahydrocannabinol and metabolite verification by chemical synthesis of 11-hydroxy-Δ-9-tetrahydrocannabinol-glucuronide

(−)-Δ-9-tetrahydrocannabinol ((−)-Δ-9-THC) is the main psychoactive constituent in cannabis. During phase I metabolism, it is metabolized to (−)-11-hydroxy-Δ-9-tetrahydrocannabinol ((−)-11-OH-Δ-9-THC), which is psychoactive, and to (−)-11-nor-9-carboxy-Δ-9-tetrahydrocannabinol ((−)-Δ-9-THC-COOH), which is psychoinactive. It is glucuronidated during phase II metabolism. The biotransformation of (−)-Δ-9-tetrahydrocannabinol-glucuronide ((−)-Δ-9-THC-Glc) and (−)-11-nor-9-carboxy-Δ-9-tetrahydrocannabinol-glucuronide ((−)-Δ-9-THC-COOH-Glc) is well understood, which is mainly due to the availability of commercial reference standards. Since such a standardized reference is not yet available for (−)-11-hydroxy-Δ-9-tetrahydrocannabinol-glucuronide ((−)-11-OH-Δ-9-THC-Glc), its biotransformation is harder to study and the nature of the glucuronide bonding—alcoholic and/or phenolic—remains unclear. Consequently, the aim of this study was to investigate the biotransformation of (−)-11-OH-Δ-9-THC-Glc in vitro as well as in vivo and to identify the glucuronide by chemically synthesis of a reference standard. For in vitro analysis, pooled human S9 liver fraction was incubated with (−)-Δ-9-THC. Resulting metabolites were detected by high-performance liquid chromatography system coupled to a high-resolution mass spectrometer (HPLC-HRMS) with heated electrospray ionization (HESI) in positive and negative full scan mode. Five different chromatographic peaks of OH-Δ-9-THC-Glc have been detected in HESI positive and negative mode, respectively. The experiment set up according to Wen et al. indicates the two main metabolites being an alcoholic and a phenolic glucuronide metabolite. In vivo analysis of urine (n = 10) and serum (n = 10) samples from cannabis users confirmed these two main metabolites. Thus, OH-Δ-9-THC is glucuronidated at either the phenolic or the alcoholic hydroxy group. A double glucuronidation was not observed. The alcoholic (−)-11-OH-Δ-9-THC-Glc was successfully chemically synthesized and identified the main alcoholic glucuronide in vitro and in vivo. (−)-11-OH-Δ-9-THC-Glc is the first reference standard for direct identification and quantification. This enables future research to answer the question whether phenolic or alcoholic glucuronidation forms the predominant way of metabolism.