Identification of human hexahydrocannabinol metabolites in urine

Hexahydrocannabinol (HHC) is a cannabinoid that has been known since 1940 but has only recently found its way into recreational use as a psychoactive drug. HHC has been used as a legal alternative to tetrahydrocannabinol (THC) in many countries, but first countries already placed it under their narcotic substances law. Our aim was to evaluate a reliable analytical method for the proof of HHC consumption by LC-MS/MS and GC-MS. We identified the two epimers of HHC and metabolites after HHC consumption by two volunteers (inhalation by use of a vaporizer and oral intake). LC-HR-MS/MS, LC-MS/MS and GC-MS with literature data (EI-MS spectra of derivatives) and reference compounds - as far as commercially available - were used for metabolite identification. Phase-II-metabolites (glucuronides) of HHC and OH-HHC were found in urine samples with LC-HR-MS/MS and LC-MS/MS. The main metabolite was tentatively identified with GC-MS as 4'OH-HHC (stereochemistry on C9 and C4' unknown). Another major side-chain hydroxylated metabolite found by LC-MS/MS could not be unambiguously identified. Both epimers of 11-OH-HHC were found in considerable amounts in urine. (8R, 9R)-8-OH-HHC was identified as a minor metabolite with GC-MS and LC-MS/MS. While (9S)-HHC was found in urine after oral intake and inhalation of HHC, the more psychoactive epimer (9R)-HHC was only found in urine after inhalation. Several other minor metabolites were detected but not structurally identified. We found that after oral or inhalative consumption the urinary main metabolites of a diastereomeric mixture of HHC are different from the respective, major Δ⁹-THC metabolites (11-OH-Δ⁹-THC and 11-nor-9-carboxy-Δ⁹-THC). Although a sensitive LC-MS/MS and GC-SIM-MS method were set-up for the reference compounds (9R)-11-nor-9-carboxy-HHC and (9S)-11-nor-9-carboxy-HHC, these oxidation products were not detected in urine with these techniques. To further increase sensitivity, a GC-MS/MS method was developed, and the 11-nor-9-carboxy metabolites of HHC were confirmed to be present as minor metabolites.