Normalization of Cannabinoid Urine Concentrations to Urine Creatinine Concentrations

Normalization of the cannabinoid drug concentration to the urine creatinine concentration aids in the differentiation of new vs prior cannabis use and reduces the variability of drug measurement due to urine dilution. Due to the long half-life of drug in the body, especially in chronic cannabis users, toxicologists and practitioners are frequently asked to determine if a positive urine test represents a new episode of drug use or represents continued excretion of residual drug. Random urine specimens contain varying amounts of creatinine depending on the degree of concentration of the urine. Hawks first suggested creatinine normalization of urine test results to account for variations in urine volume in the bladder (Hawks 1983). Whereas urine volume is highly variable due to changes in liquid, salt, and protein intake, exercise, and age, creatinine excretion is much more stable. Manno et al. recommended that an increase of 150% in the creatinine normalized cannabinoid concentration above the previous specimen be considered indicative of a new episode of drug exposure (Manno et al. 1984). If the increase is greater than or equal to the threshold selected, then new use is predicted. This approach has received wide attention for potential use in treatment and employee assistance programs, but there has been limited evaluation of the usefulness of this ratio under controlled dosing conditions. Huestis et al. conducted a controlled clinical study of the excretion profile of creatinine and cannabinoid metabolites in a group of six cannabis users who smoked two different doses of cannabis separated by weekly intervals (Huestis and Cone 1998b). As seen in Fig. 6, normalization of urinary THCCOOH concentration to the urinary creatinine concentration produces a smoother excretion pattern and facilitates interpretation of consecutive urine drug test results. A relative operating characteristic (ROC) curve was constructed from sensitivity and specificity data for 26 different cutoffs ranging from 10% to

ng/mL 75

75 ng/mg

0 30 60 90 120 150 180 Hours

Fig. 6. Urine concentrations of 11-nor-9-carboxy-49-tetrahydrocannabinol (THCCOOH; ng/ml, and ng/mg creatinine) for one subject after smoking a single 3.55% THC cigarette. (Reproduced from the Journal of Analytic Toxicology by permission of Preston Publications, a division of Preston Industries; Huestis and Cone 1998b, Fig. 3 therein)

ng/mL 75

75 ng/mg

0 30 60 90 120 150 180 Hours

Fig. 6. Urine concentrations of 11-nor-9-carboxy-49-tetrahydrocannabinol (THCCOOH; ng/ml, and ng/mg creatinine) for one subject after smoking a single 3.55% THC cigarette. (Reproduced from the Journal of Analytic Toxicology by permission of Preston Publications, a division of Preston Industries; Huestis and Cone 1998b, Fig. 3 therein)

200%. The most accurate ratio (85.4%) was 50%, with a sensitivity of 80.1% and a specificity of 90.2%, with 5.6% false-positive and 7.4% false-negative predictions. If the previously recommended increase of 150% was used as the threshold for new use, sensitivity of detecting new use was only 33.4%, specificity was high at 99.8%, and there was an overall accuracy prediction of 74.2%. To further substantiate the validity of the derived ROC curve, urine cannabinoid metabolite and creati-nine data from another controlled clinical trial that specifically addressed water dilution as a means of specimen adulteration were evaluated (Cone et al. 1998). Sensitivity, specificity, accuracy, percentage false positives, and percentage false negatives were 71.9%, 91.6%, 83.9%, 5.4%, and 10.7%, respectively, when the 50% criterion was applied. These data indicate selection of a threshold to evaluate sequential creatinine-normalized urine drug concentrations can improve the ability to distinguish residual excretion from new drug usage.

Oral Fluid Testing

Oral fluid is also a suitable specimen for monitoring cannabinoid exposure and is being evaluated for driving under the influence of drugs, drug treatment, workplace drug testing, and for clinical trials (Cairns et al. 1990; Gross and Soares 1978; Gross et al. 1985; Mura et al. 1999; Soares et al. 1976,1982). Adequate sensitivity is best achieved with an assay directed toward detection of THC, rather than 11-OH-THC or THCCOOH. The oral mucosa is exposed to high concentrations of THC during smoking and serves as the source of THC found in oral fluid. Only minor amounts of drug and metabolites diffuse from the plasma into oral fluid (Hawks 1983). Following intravenous administration of radiolabeled THC, no radioactivity could be demonstrated in oral fluid (Hawks 1982). No measurable 11-OH-THC or THCCOOH was found in oral fluid collected immediately following and up to

10000 1000 E 100

0 1 10 100 Hours

Fig. 7. Excretion patterns of ^-tetrahydrocannabinol (THC) concentrations (ng/ml) in oral fluid and plasma, and urinary 11-nor-9-carboxy-49-tetrahydrocannabinol (ng THCCOOH/mg creatinine) in one human subject following smoking of a single cannabis cigarette (3.55%). The ng THCCOOH/mg creatinine ratio is illustrated for all urine specimens collected through the last positive specimen. Analyses were performed by GC-MS at cutoff concentrations of 0.5 ng/ml for oral fluid and plasma and 15 ng/ml for urine. (Reproduced from the Journal of Analytic Toxicology by permission of Preston Publications, a division of Preston Industries; Huestis and Cone 2004, Fig. 2 therein)

0 1 10 100 Hours

Fig. 7. Excretion patterns of ^-tetrahydrocannabinol (THC) concentrations (ng/ml) in oral fluid and plasma, and urinary 11-nor-9-carboxy-49-tetrahydrocannabinol (ng THCCOOH/mg creatinine) in one human subject following smoking of a single cannabis cigarette (3.55%). The ng THCCOOH/mg creatinine ratio is illustrated for all urine specimens collected through the last positive specimen. Analyses were performed by GC-MS at cutoff concentrations of 0.5 ng/ml for oral fluid and plasma and 15 ng/ml for urine. (Reproduced from the Journal of Analytic Toxicology by permission of Preston Publications, a division of Preston Industries; Huestis and Cone 2004, Fig. 2 therein)

7 days after cannabis smoking with a GC/MS LOQ of 0.5 ng/ml (Huestis and Cone 1998a). Similarly, 11-OH-THC and THCCOOH were not detected in the oral fluid of 22 subjects who were documented cannabis users (Kintz et al. 2000). Oral fluid collected with the Salivette collection device was positive for THC in 14 of these 22 participants. Although no 11-OH-THC or THCCOOH was identified by GC/MS, cannabinol and cannabidiol were found in addition to THC. Hours after smoking, the oral mucosa serves as a depot for release of THC into the oral fluid. In addition, as detection limits continue to decrease with the development of new analytical instrumentation, it maybe possible to measure low concentrations of THCCOOH in oral fluid.

Detection times of cannabinoids in oral fluid are shorter than in urine, and more indicative of recent cannabis use (Cairns et al. 1990; Gross et al. 1985). Oral fluid THC concentrations temporally correlate with plasma cannabinoid concentrations and behavioral and physiological effects, but wide intra- and inter-individual variation precludes the use of oral fluid concentrations as indicators of drug impairment (Huestis and Cone 1998a; Huestis et al. 1992a). THC may be detected at low concentrations by radioimmunoassay for up to 24 h after use. Figure 7 depicts excretion of THC in oral fluid and plasma and creatinine-normalized THCCOOH excretion in urine in one subject after smoking a single 3.55% cannabis cigarette (Huestis and Cone 2004). After smoking cannabis, oral fluid cannabinoid tests were positive for THC by GC/MS/MS with a cutoff of 0.5 ng/ml for 13±3 h (range 1-24) (Niedbala et al. 2001). After these times, occasional positive oral fluid results were interspersed with negative tests for up to 34 h. Peel et al. tested oral fluid samples from 56 drivers suspected of being under the influence of cannabis with the enzyme-multiplied immunoassay test (EMIT) screening test and GC/MS confirmation (Peel et al. 1984). They suggested that the ease and non-invasiveness of sample collection made oral fluid a useful alternative matrix for detection of recent cannabis use. Oral fluid samples also are being evaluated in the European Union's Roadside Testing Assessment (ROSITA) Project to reduce the number of individuals driving under the influence of drugs and to improve road safety. The ease and non-invasiveness of oral fluid collection, reduced hazards in specimen handling and testing, and shorter detection window are attractive attributes of this method for identifying the presence of potential performance-impairing drugs.

In a recent study of smoked and oral cannabis use, the Intercept DOA Oral Specimen Collection Device and GC/MS/MS (cutoff 0.5 ng/ml) were paired to monitor oral fluid cannabinoids in ten participants (Niedbala et al. 2001). Oral fluid specimens tested positive following smoked cannabis for an average of 13±3 h (range 1-24). After these times, occasional positive oral fluid results were interspersed with negative tests for up to 34 h. A different oral fluid collection device, the Cozart RapiScan device, utilizes a 10 ng/ml cannabinoid cutoff to screen for cannabis use (Jehanli et al. 2001). Positive oral fluid cannabinoid tests were not obtained more than 2 h after last use, suggesting that much lower cutoff concentrations were needed to improve sensitivity. A procedure for direct analysis of cannabi-noids in oral fluid with solid-phase microextraction and ion trap GC/MS has been developed with a limit of detection of 1.0 ng/ml (Hall et al. 1998). Detection of cannabinoids in oral fluid is a rapidly developing field; however, there are many scientific issues to resolve. One of the most important is the degree of absorption of the drug to oral fluid collection devices.

Cannabinoids in Sweat

To date, there are no published data on the excretion of cannabinoids in sweat following controlled THC administration. Sweat testing is being applied to monitor cannabis use in drug treatment, criminal justice, workplace drug testing, and clinical studies (Huestis and Cone 1998a; Kidwell et al. 1998). In 1989, Balabanova and Schneider utilized radioimmunoassay to detect cannabinoids in apocrine sweat (Balabanova and Schneider 1989). Currently, there is a single commercially available sweat collection device, the PharmCheck patch, offered by PharmChem Laboratories in Texas, USA. Generally, the patch is worn for 7 days and exchanged for a new patch once each week during visits to the treatment clinic or parole officer. Theoretically, this permits constant monitoring of drug use throughout the week, extending the window of drug detection and improving test sensitivity. As with oral fluid testing, this is a developing analytical technique with much to be learned about the pharmacokinetics of cannabinoid excretion in sweat, potential reabsorption of THC by the skin, possible degradation of THC on the patch, and adsorption of THC onto the patch collection device. It is known that THC is the primary analyte detected in sweat, with little 11-OH-THC and THCCOOH. Several investigators have evaluated the sensitivity and specificity of different screening assays for detecting cannabinoids in sweat (Mura et al. 1999; Samyn and van Haeren 2000). Kintz et al. identified THC (4-38 ng/patch) in 20 known heroin abusers who wore the PharmChek patch for 5 days while attending a detoxification center (Kintz et al. 1997). Sweat was extracted with methanol and analyzed by GC/MS. The same investigators also evaluated forehead swipes with cosmetic pads for monitoring cannabinoids in sweat from individuals suspected of driving under the influence of drugs (Kintz et al. 2000). THC, but not 11-OH-THC or THCCOOH, was detected (4 to 152 ng/pad) by electron impact GC/MS in the sweat of 16 of 22 individuals who tested positive for cannabinoids in urine. Ion trap tandem mass spectrometry has also been used to measure cannabinoids in sweat collected with the PharmChek sweat patch with a limit of detection of 1 ng/patch (Ehorn et al. 1994).

Cannabinoids in Hair

There are multiple mechanisms for the incorporation of cannabinoids in hair. THC and metabolites may be incorporated into the hair bulb that is surrounded by capillaries. Drug may also diffuse into hair from sebum that is secreted onto the hair shaft and from sweat that is excreted onto the skin surface. Drug may also be incorporated into hair from the environment. Cannabis is primarily smoked, providing an opportunity for environmental contamination of hair with THC in cannabis smoke. Basic drugs such as cocaine and methamphetamine concentrate in hair due to ionic bonding to melanin, the pigment in hair that determines hair color. The more neutral and lipophilic THC is not highly bound to melanin, resulting in much lower concentrations of THC in hair as compared to other drugs of abuse. Usually THC is present in hair at a higher concentration than its THCCOOH metabolite (Cairns et al. 1995; Cirimele 1996; Kintz et al. 1995; Moore et al. 2001). An advantage of measuring THCCOOH in hair is that THCCOOH is not present in cannabis smoke, avoiding the issue of passive exposure from the environment. Analysis of cannabinoids in hair is challenging due to the high analytical sensitivity required. THCCOOH is present in the femtogram to picogram per milligram of hair range. GC/MS/MS is required in most analytical techniques. A novel approach to the screening of hair specimens for the presence of cannabinoids in hair was proposed by Cirimele et al. (1996). They developed a rapid, simple GC/MS screening method for THC, cannabinol, and cannabidiol in hair that did not require derivatization prior to analysis. The method was found to be a sensitive screen for cannabis detection with GC/MS identification of THCCOOH recommended as a confirmatory procedure.

It is difficult to conduct controlled cannabinoid administration studies on the disposition of cannabinoids in hair because of the inability to differentiate administered drug from previously self-administered cannabis. If isotopically labeled drug were administered, it would be possible to identify newly administered drug in hair. There are advantages to monitoring drug use with hair testing including a wide window of drug detection, a less invasive specimen collection procedure, and the ability to collect a second specimen at a later time. However, one of the weakest aspects of testing for cannabinoids in hair is the low sensitivity of drug detection in this alternate matrix. In controlled cannabinoid administration studies conducted by Huestis et al., only about one-third of non-daily users and two-thirds of daily cannabis users had positive cannabinoid hair tests by GC/MS/MS with detection limits of 1 ng/mg for THC and 0.1 ng/mg for THCCOOH. All participants had positive urine cannabinoid tests at the time of hair collection (unpublished data).

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