Extended urinary Δ9-tetrahydrocannabinol excretion in chronic cannabis users precludes use as a biomarker of new drug exposure
Introduction
Cannabinoid detection times in urine depend upon pharmacological factors (e.g., drug dose, route of administration, duration and frequency of use, smoking topography and individual rates of absorption, metabolism and excretion), and methodological issues including analytes evaluated, matrix, type of hydrolysis, cut-off or threshold used, and sensitivity of the method. The primary psychoactive constituent of cannabis, Δ9-tetrahydrocannabinol (THC), is rapidly absorbed during smoking, and due to its high lipophilicity is widely distributed to adipose tissue, liver, lung, and spleen. Body stores of THC increase with increasing frequency and chronicity of cannabis use. THC is rapidly metabolized to the equally psychoactive 11-hydroxy-THC (11-OH-THC), and to the inactive 11-nor-9-carboxy-THC (THCCOOH) metabolite and its glucuronide and sulfate conjugates (Huestis and Smith, 2005). The slow release of THC from fat back into blood was demonstrated to be the rate-limiting step in cannabinoid elimination from the body (Hunt and Jones, 1980). Thus, THC was shown to have a long terminal elimination half-life in blood from chronic cannabis users (Johansson, 1989).
Urine testing remains the most common means of drug monitoring in the United States. The highest numbers of positive urine drug tests in workplace drug testing are for cannabinoids, achieved by immunoassay screening and THCCOOH quantification in urine after alkaline hydrolysis. Urinary THCCOOH excretion patterns have been extensively studied (Smith-Kielland et al., 1999, Huestis et al., 1995, Johansson et al., 1990, Musshoff and Madea, 2006). After occasional use, Huestis et al. reported THCCOOH concentrations above 15 ng/mL by gas chromatography mass spectrometry (GCMS) after alkaline hydrolysis for up to 4 days (Huestis et al., 1996, Huestis and Cone, 1998a). Urinary THCCOOH window of detection ranges from several days in infrequent users (Huestis and Cone, 1998b) to months in frequent users (Ellis et al., 1985, Peat, 1989, Kelly and Jones, 1992, Fraser and Worth, 2003, Johansson and Halldin, 1989). Thus, identification of THCCOOH in urine may not indicate recent cannabis exposure (Dackis et al., 1982, McBurney et al., 1986, Fraser and Worth, 2004).
Huestis and Cone developed and validated a model to differentiate new cannabis use from residual drug excretion in occasional cannabis users based on urine THCCOOH concentrations (Huestis and Cone, 1998b). However, this model may not be accurate in chronic cannabis users during the terminal elimination phase, when THCCOOH concentrations are low, below 20–50 ng/mL. In a search for new biomarkers of recent cannabis use, Kemp et al. showed that THC and 11-OH-THC could be detected in urine after cannabis smoking if Escherichia coli (E. coli) β-glucuronidase hydrolysis was employed to break ether glucuronide bonds (Kemp et al., 1995a). They conducted a controlled cannabis smoking study quantifying THC and 11-OH-THC concentrations in urine specimens collected up to 8 h after cannabis smoking (Kemp et al., 1995b). The presence of THC or 11-OH-THC in urine was stated to indicate cannabis use within 8 h. In attempting to replicate this finding in our cannabinoid smoking studies, we quickly found that 11-OH-THC could be measured for many days after last smoked THC cigarette. Manno et al. later revised their hypothesis to solely rely on the presence of THC in urine (>1.5 ng/mL) to suggest cannabis use in the previous 8 h (Manno et al., 2001). We believed that this hypothesis required evaluation over a much longer timeframe and also, in individuals who were chronic cannabis users.
Cannabis, the most commonly abused drug world wide, is included in workplace, drug treatment, clinical, military and criminal justice drug testing programs. New drug use may have important consequences for employment, child custody, military status, and imprisonment. In treatment programs, urine drug testing is a deterrent to drug use, and is an effective and objective tool in contingency management programs and for evaluating new behavioral and pharmacotherapy treatments. The ability to differentiate new drug use from residual drug excretion would be valuable for clinicians, toxicologists, employee assistance programs, drug treatment providers, clinical trials of cannabis dependence treatment, anti-doping athletic programs, parole officers, attorneys and judges. The interpretation of cannabinoid urine tests would be greatly improved if new cannabis use could be identified in both occasional and chronic cannabis users.
Recent advances in analytical methods have improved sensitivity, specificity and recovery of cannabinoids in biological fluids and tissues. THC and 11-OH-THC are present in urine as glucuronide conjugates that are only effectively recovered following E. coli β-glucuronidase hydrolysis (Kemp et al., 1995a). We showed that to ensure comprehensive hydrolysis of all three cannabinoid conjugates, an initial hydrolysis with E. coli β-glucuronidase followed by a second hydrolysis with strong base (tandem hydrolysis), obtained better recovery of THCCOOH (Abraham et al., 2007). In addition, two-dimensional gas chromatography mass spectrometry (2D-GCMS) is a new technique (Lowe et al., 2007) that reduces background matrix interference allowing superior resolution and specificity, and achieving more reliable cannabinoid quantification at low concentrations. 2D-GCMS was utilized in this study to verify cannabinoid detection times.
The objective of the current research was to examine the time course of THC, 11-OH-THC, and THCCOOH elimination in urine in subjects with a history of chronic, heavy cannabis use, and to test the efficacy of THC and 11-OH-THC as urinary biomarkers of recent cannabis exposure. Participants resided on a secure clinical research unit, under 24 h medical surveillance, while participating in two studies of neurocognitive impairment in abstinent chronic heavy cannabis users. Subjects with the longest histories of cannabis smoking and highest THCCOOH concentrations on admission participated in the present cannabinoid urinary excretion study. Each urine specimen was individually collected throughout an abstinence period of up to 30 days and THC, 11-OH-THC, and THCCOOH concentrations were simultaneously quantified by one-dimensional GCMS. Because of the importance and controversial nature of the data, and availability of new analytical technology, THC, 11-OH-THC, and THCCOOH concentrations and times of last detection were verified by repeat analysis by 2D-GCMS, with low 2.5 ng/mL limits of quantification (LOQ) for each analyte. These are the first data on extended urinary THC, 11-OH-THC and THCCOOH excretion and detection times in chronic, heavy cannabis users, and the first to test the hypothesis that THC and 11-OH-THC in urine are biomarkers of recent cannabis use.
Section snippets
Participants
Two clinical research studies at the Intramural Research Program, National Institute on Drug Abuse investigated neurocognitive impairment and effects of cannabis withdrawal in chronic cannabis users during 30 days of continuously monitored abstinence. Neurocognitive performance, fMRI and cerebral blood flow data from these studies have been previously reported (Bolla et al., 2002, Bolla et al., 2005, Bolla et al., 2008, Eldreth et al., 2004, Herning et al., 2003, Herning et al., 2005, Matochik
Results
A total of 1271 urine specimens were analyzed by 1D-GCMS from 33 participants residing on the closed research unit during monitored abstinence for 3–30 days. All urine specimens from all participants individually collected during the first 72 h were quantified by 1D-GCMS to determine THC, 11-OH-THC, and THCCOOH elimination patterns. Demographic characteristics and self-reported drug use histories of the 33 participants are included in Table 1. Twenty-six subjects’ (13 M, 13 F, 21 African
Discussion
For the first time, the hypothesis that THC and 11-OH-THC in urine indicated new cannabis use was tested with these novel cannabinoid excretion data. Urinary concentrations of THC and 11-OH-THC in chronic, heavy cannabis smokers during extended supervised abstinence and following tandem enzymatic and alkaline hydrolysis are presented. Subjects resided on a secure clinical research unit throughout to prevent access to cannabis and other drugs. Highest analyte concentrations, times of first
Role of funding source
This research was supported by the National Institutes of Health Intramural Research Program of the National Institute on Drug Abuse. This clinical study was approved by the Institutional Review Board (IRB) of the Intramural Research Program of the National Institute on Drug Abuse.
Contributors
Marilyn A. Huestis, Jean Lud Cadet, and Ronald Herning designed the clinical study and developed the protocol. Ross H. Lowe, Tsadik T. Abraham, and William D. Darwin compiled and analyzed the data. Ross H. Lowe, Tsadik T. Abraham and Marilyn A Huestis contributed to the manuscript and all authors have approved the final manuscript.
Conflict of interest
The 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.
Acknowledgement
The authors wish to thank Allan Barnes for assistance with data interpretation and preparation of the manuscript and Jennifer Runkle and Wesen Nebro for technical assistance.
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