title: Cannabinoids in humans. II. The influence of three methods of hydrolysis on the concentration 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% delta 9-tetrahydrocannabinol (THC) and submitted urine samples prior to smoking, 5 min after smoking, and hourly for 8 h thereafter. Urine (1 mL) was buffered to the optimum pH for each form of enzyme tested. beta-Glucuronidase from Escherichia coli (bacteria) or Helix pomatia (mollusk) was added to the specimens, followed by overnight incubation at 37 degrees 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 < .05). These results demonstrate the species-dependent nature of glucuronidase activity in hydrolyzing THC and 11-OH-THC glucuronides and the ineffectiveness of base hydrolysis on these hydroxylated compounds. The need for further study to find the optimum conditions necessary for the complete hydrolysis of cannabinoid conjugates is suggested.
authors.affiliation: Louisiana State University, Department of Pharmacology, USA.
authors.forename: P M
authors.initials: PM
authors.lastname: Kemp
authors.forename: I K
authors.initials: IK
authors.lastname: Abukhalaf
authors.forename: J E
authors.initials: JE
authors.lastname: Manno
authors.forename: B R
authors.initials: BR
authors.lastname: Manno
authors.forename: D D
authors.initials: DD
authors.lastname: Alford
authors.forename: M E
authors.initials: ME
authors.lastname: McWilliams
authors.forename: F E
authors.initials: FE
authors.lastname: Nixon
authors.forename: M J
authors.initials: MJ
authors.lastname: Fitzgerald
authors.forename: R R
authors.initials: RR
authors.lastname: Reeves
authors.forename: M J
authors.initials: MJ
authors.lastname: Wood
mesh_terms: D013759:Dronabinol / Q000032:analysis / Q000378:metabolism / Q000652:urine*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D005966:Glucuronidase; D006801:Humans; D006868:Hydrolysis; D008297:Male
chemical_list: D013759:Dronabinol; D005966:Glucuronidase