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.

Current technology establishes marijuana use based upon detection of the pharmacologically inactive cannabinoid metabolite (11-nor-delta9-carboxy-tetrahydrocannabinol-9-carboxylic acid, THC-COOH) in urine. No accurate prediction of time of use is possible because THC-COOH has a half-life of 6 days. To determine if a temporal relationship between marijuana use and metabolite excretion patterns could be established, eight healthy user-volunteers (18-35 years old) smoked marijuana cigarettes containing 0% (placebo), 1.77%, and 3.58% delta9-tetrahydrocannabinol (THC). Plasma and urine were collected prior to smoking, 5 min after smoking, and hourly thereafter for 8 h for measurement of cannabinoid concentrations by gas chromatography-mass spectrometry. Mathematical models proposed for determination of recent marijuana use were applied to data from this study and verified the temporal use of marijuana. One subject, who later admitted chronic marijuana use (urine baseline THCCOOH, 529.2 ng/mL; plasma, 75.5 ng/mL), excreted 8beta-dihydroxy-THC, peaking 2 h postsmoking (92.3 ng/mL). Urinary THC, the psychoactive component of marijuana, concentrations peaked 2 h after smoking and declined to assay limit of detection (LOD) (1.5 ng/mL) by 6 h. 11-Hydroxy-delta9-tetrahydrocannabinol (11-OH-THC) and THCCOOH were detectable for the entire 8-h testing period but continued to decrease. Urinary concentrations of THC greater than 1.5 ng/mL suggests marijuana use during the previous 8-h time period.

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In previous work we demonstrated and replicated a significant association between increased absolute and relative power and interhemispheric coherence of EEG alpha activity over the bilateral frontal-central cortex ("alpha hyperfrontality") in daily marihuana users as contrasted with nonusers. In this report we focused our analyses on subjects who reported smoking marihuana on a daily basis for 15 to 24 consecutive years. Compared to nonuser controls and subjects who had used marihuana on a daily basis for shorter periods of time, subjects with excessively long cumulative exposures to THC were found to have significantly elevated absolute power of theta activity over bilateral frontal-central cortex, as well as significantly increased interhemispheric coherence of theta activity across central and posterior regions. Concurrent reaction time studies conducted in our laboratory suggest that very long duration cumulative marihuana exposure might be associated with slowed cognitive processing.

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