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.

Development of a rapid column-switching LC-MS/MS method for the quantification of THCCOOH and THCCOOH-glucuronide in whole blood for assessing cannabis consumption frequency

“…The amount of THCCOOH-glucuronide is calculated as the difference between total and free THCCOOH [12]. Since hydrolysis can be variable and/or incomplete this procedure can lead to ambiguous results [27,28]. The use of LC-MS/MS eliminates the need of costly and time-consuming alkaline and/ or enzymatic glucuronide hydrolysis, enabling direct measurement of free and glucuronidated THCCOOH.…”

Urinary Cannabinoid Disposition in Occasional and Frequent Smokers: Is THC-Glucuronide in Sequential Urine Samples a Marker of Recent Use in Frequent Smokers?

“…Studies documenting urinary THCCOOH and THCCOOH-glucuronide disposition used workplace (18 ), random cannabis abstainers ' (19 ) or stability samples (20 ), or they compared THCCOOH before and after hydrolysis (13,(21)(22). Unconjugated THC and 11-OH-THC are not detected or detected in low concentrations (11-OH-THC only) in urine and require enzymatic or tandem enzymatic-alkaline hydrolysis to cleave glucuronides (19,(23)(24). However, given the inability to definitively ensure complete hydrolysis, direct glucuronide analysis provides more accurate information regarding urinary metabolite disposition.…”

Validated Gas Chromatographic–Negative Ion Chemical Ionization Mass Spectrometric Method for Δ9-Tetrahydrocannabinol in Sweat Patches

“…9 -Tetrahydrocannabinol (THC) 4 is the major psychoactive constituent of cannabis (e.g., marijuana, hashish, and hashish oil). THC is lipophilic and distributes widely throughout the body.…”

“…Toxicologic analysis to determine cannabis use includes measurement of THC, the active metabolite 11-hydroxy-tetrahydrocannabinol, and the inactive metabolite 11-nor-9-carboxy-tetrahydrocannabinol. Numerous methods for the analysis of cannabinoids in blood and urine have been developed, primarily using gas chromatography-mass spectrometry (GC/MS) in electron impact ionization (EI) mode (1)(2)(3)(4)(5)(6).…”