A sensitive and specific method for extraction and quantification of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THCCOOH) in human urine was developed and fully validated. To ensure complete hydrolysis of conjugates and capture of total analyte content, urine samples were hydrolyzed by two methods in series. Initial hydrolysis was with Escherichia coli beta-glucuronidase (Type IX-A) followed by a second hydrolysis utilizing 10N NaOH. Specimens were adjusted to pH 5-6.5, treated with acetonitrile to precipitate protein, and centrifuged, and the supernatants were subjected to solid-phase extraction. Extracted analytes were derivatized with BSTFA and quantified by gas chromatography-mass spectrometry with electron impact ionization. Standard curves were linear from 2.5 to 300 ng/mL. Extraction efficiencies were 57.0-59.3% for THC, 68.3-75.5% for 11-OH-THC, and 71.5-79.7% for THCCOOH. Intra- and interassay precision across the linear range of the assay ranged from 0.1 to 4.3% and 2.6 to 7.4%, respectively. Accuracy was within 15% of target concentrations. This method was applied to the analysis of urine specimens collected from individuals participating in controlled administration cannabis studies, and it may be a useful analytical procedure for determining recency of cannabis use in forensic toxicology applications.

Background-Generally, urinary 11-nor-9-carboxy-Δ9-Tetrahydrocannabinol (THCCOOH) after alkaline hydrolysis is monitored to detect cannabis exposure, although last use may have been weeks prior in chronic cannabis users. Δ9-Tetrahydrocannabinol (THC) and 11-hydroxy-THC (11-OH-THC) concentrations in urine following E. coli β-glucuronidase hydrolysis were proposed as biomarkers of recent (within 8 h) cannabis use.Objective-To test the validity of THC and 11-OH-THC in urine as indicators of recent cannabis use.Methods-Monitor urinary cannabinoid excretion in 33 chronic cannabis smokers who resided on a secure research unit under 24 h continuous medical surveillance. All urine specimens were collected individually ad libidum for up to 30 days, were hydrolyzed with a tandem E. coli β-glucuronidase/ base procedure, and analyzed for THC, 11-OH-THC and THCCOOH by 1-and 2-dimensionalcryotrap gas chromatography mass spectrometry (2D-GCMS) with limits of quantification of 2.5 ng/ mL.Results-Extended excretion of THC and 11-OH-THC in chronic cannabis users' urine was observed during monitored abstinence; 14 of 33 participants had measurable THC in specimens collected at least 24 h after abstinence initiation. Seven subjects had measurable THC in urine for 3, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of InterestThe authors declare that they have no conflicts of interest. Authors are employed by the Chemistry and Drug Metabolism and Molecular Neuropsychiatry sections of the Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health. 3,4,7,7,12, and 24 days after cannabis cessation. 11-OH-THC and THCCOOH were detectable in urine specimens from one heavy, chronic cannabis user for at least 24 days. NIH Public AccessConclusion-For the first time, extended urinary excretion of THC and 11-OH-THC is documented for at least 24 days, negating their effectiveness as biomarkers of recent cannabis exposure, and substantiating long terminal elimination times for urinary cannabinoids following chronic cannabis smoking.

3,4-Methylenedioxymethamphetamine (MDMA), or ecstasy, is excreted as unchanged drug, 3,4-methylenedioxyamphetamine (MDA), and free and glucuronidated/sulfated 4-hydroxy-3-methoxymethamphetamine (HMMA), and 4-hydroxy-3-methoxyamphetamine (HMA) metabolites. The aim of this paper is to describe the pattern and timeframe of excretion of MDMA and its metabolites in urine. Placebo, 1.0 mg/kg, and 1.6 mg/kg oral MDMA doses were administered double-blind to healthy adult MDMA users on a monitored research unit. All urine was collected, aliquots were hydrolyzed, and analytes quantified by gas chromatography–mass spectrometry. Median Cmax, Tmax, ratios, first and last detection times, and detection rates were determined. Sixteen participants provided 916 urine specimens. After 1.6 mg/kg, median Cmax were 21,470 (MDMA), 2229 (MDA), 20,793 (HMMA), and 876 ng/mL (HMA) at median Tmax of 13.9, 23.0, 9.2 and 23.3 h. In the first 24 h, 30.2–34.3% total urinary excretion occurred. HMMA last detection exceeded MDMA’s by more than 33 h after both doses. Identification of HMMA as well as MDMA increased the ability to identify positive specimens but required hydrolysis. These MDMA, MDA, HMMA, and HMA pharmacokinetic data may be useful for interpreting workplace, drug treatment, criminal justice, and military urine drug tests. Measurement of urinary HMMA provides the longest detection of MDMA exposure yet is not included in routine monitoring procedures.

Recovery of 3,4-methylenedioxymethamphetamine (MDMA) urinary metabolites requires optimization of the hydrolysis of 4-hydroxy-3-methyoxymethamphetamine (HMMA), 4-hydroxy-3-methoxyamphetamine (HMA), and 3,4-methylenedioxyamphetamine (MDA) conjugates prior to chromatographic analysis. Acidic and enzymatic hydrolysis with beta-glucuronidase from Escherichia coli and Helix pomatia were evaluated. Acid hydrolysis yielded 40.0% and 39.3% higher HMA recovery compared to E. coli and H. pomatia hydrolysis, respectively (SE=9.8 and 11.4%). E. coli beta-glucuronidase hydrolysis MDA recovery was 17.1% and 26.5% greater than acid hydrolysis and H. pomatia beta-glucuronidase recovery (SE=3.3 and 6.1%), respectively. HMMA recovery by acid hydrolysis was 336.1% and 159.8% greater than E. coli and H. pomatia beta-glucuronidase (SE=72.8 and 31.6%), respectively. The effects of temperature, time, and acid amount on metabolite recovery were also evaluated. HMA and HMMA acid hydrolysis recoveries were improved at 100 degrees C and above. Effective hydrolysis could be conducted in a dry block heater, GC oven, or autoclave at temperatures from 100 to 140 degrees C. Optimal hydrolysis conditions for the measurement of MDMA metabolite conjugates were addition of 100 microL of hydrochloric acid to 1 mL urine and incubation at 120 degrees C in a GC oven for 40 min. Therefore, based on HMMA, HMA, and MDA recoveries, time efficiency, availability of instrumentation, and cost, acid hydrolysis was preferred to enzyme hydrolysis.