Cannabinoids in humans. ii. the influence of three methods of hydrolysis on the conentration of thc and two metabolites in urine

Abstract

Glucuronide conjugates of cannabinoids were previously identified in humans. For gas chromatographic-mass spectrometric (GC-MS) analysis of the unconjugated compounds in human urine, it is necessary to cleave the glucuronide moiety. Base hydrolysis and two forms of enzymatic hydrolysis were compared in this study to examine any quantitative differences between the hydrolysis methods. Human volunteers (n = 8) each smoked one marijuana cigarette containing 3.58% 9-tetrahydrocannabinol (THC) and submitted urine samples prior to smoking, 5 rain after smoking, and hourly for 8 h thereafter. Urine (1 mL) was buffered to the optimum pH for each form of enzyme tested. β-Glucuronidase from Escherichia coli (bacteria) or Helix pomatia (mollusk) was added to the specimens, followed by overnight incubation at 37°C. Following hydrolysis, the samples were extracted using hexane-ethyl acetate (7:1) and derivatized with N, O-bis(trimethylsilyl)-trifluoroacetamide plus 1% trimethylchlorosilane, which converted the cannabinoids to their trimethylsilyl derivatives. GC-MS analysis revealed striking differences between the hydrolysis methods. Concentrations of unconjugated THC and 11-hydroxy-THC (11-OH-THC) using E. coli were significantly increased over all other methods tested (p