Identification of Tetrahydrocannabidiol Metabolites in Human Urine

Tetrahydrocannabidiol (H4CBD) is an emerging semisynthetic cannabinoid, which has been known since 1940. Like hexahydrocannabinol (HHC), it is easily obtained by hydrogenation of available phytocannabinoids, in the case of H4CBD by hydrogenation of cannabidiol (CBD). H4CBD shows a weak affinity for the CB₁ receptor, but it is unclear if H4CBD shows psychoactive properties, as reports from users are divided. Only a few countries have placed H4CBD under their narcotic substance law, for example, France and Switzerland. The aim of this study was to identify human Phase I and II metabolites in urine as potential forensic targets. The H4CBD used for this study was bought from an online store and analyzed beforehand using GC-MS. The Phase I and II metabolites were identified using LC-HR-MS/MS and GC-MS after trimethylsilylation. The found H4CBD metabolites were carboxylated, hydroxylated, and bishydroxylated species and their glucuronides with hydroxylation and carboxylation positions on the alicyclic moiety and on the side chain. The tentatively identified metabolites were the carboxylic acids 5″-COOH-H4CBD and 7-COOH-H4CBD, the hydroxylated metabolites (1R,6R)-OH-H4CBD, (1R,6S)-OH-H4CBD, two epimers of 2″-OH-H4CBD, and both epimers of 7-OH-H4CBD. The identified bishydroxylated metabolites were side-chain hydroxylated derivatives of 7-OH-H4CBD. Various other hydroxylated metabolites were found, but their exact hydroxylation positions could not be determined. Some ESI+ spectra of the metabolites showed very unusual fragmentation patterns, like the loss of both oxygens from the resorcinol moiety with subsequent ring contraction and the appearance of radical cations for Phase II metabolites. These unusual patterns were noticed for H4CBD and its side-chain-altered metabolites.