Jacob Bell
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Urinary Cannabinoid Detection Times after Controlled Oral Administration of {Delta}9-Tetrahydrocannabinol to Humans
Richard A. Gustafson1, Barry Levine2, Peter R. Stout3, Kevin L. Klette4, M.P. George5, Eric T. Moolchan1 and Marilyn A. Huestis1,a
1 Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Dr., Baltimore, MD 21224.
2 Office of the Chief Medical Examiner, 111 Penn St., Baltimore, MD 21201.
3 Aegis Sciences Corp., 345 Hill Ave., Nashville, TN 37210.
4 Navy Drug Screening Laboratory, PO Box 113, Bldg. H-2033, Naval Air Station, Jacksonville, FL 32212.
5 Quest Diagnostics, Inc., 506 East State Pkwy., Schaumburg, IL 60173.
aAuthor for correspondence. Fax 410-550-2971; e-mail mhuestis@intra.nida.nih.gov.
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Background: Urinary cannabinoid excretion and immunoassay performance were evaluated by semiquantitative immunoassay and gas chromatography—mass spectrometry (GC/MS) analysis of metabolite concentrations in 4381 urine specimens collected before, during, and after controlled oral administration of tetrahydrocannabinol (THC).
Methods: Seven individuals received 0, 0.39, 0.47, 7.5, and 14.8 mg THC/day in this double-blind, placebo-controlled, randomized, clinical study conducted on a closed research ward. THC doses (hemp oils with various THC concentrations and the therapeutic drug Marinol®) were administered three times daily for 5 days. All urine voids were collected over the 10-week study and later tested by Emit II®, DRI®, and CEDIA® immunoassays and by GC/MS. Detection rates, detection times, and sensitivities, specificities, and efficiencies of the immunoassays were determined.
Results: At the federally mandated immunoassay cutoff (50 µg/L), mean detection rates were <0.2% during ingestion of the two low doses typical of current hemp oil THC concentrations. The two high doses produced mean detection rates of 23—46% with intermittent positive tests up to 118 h. Maximum metabolite concentrations were 5.4—38.2 µg/L for the low doses and 19.0—436 µg/L for the high doses. Emit II, DRI, and CEDIA immunoassays had similar performance efficiencies of 92.8%, 95.2%, and 93.9%, respectively, but differed in sensitivity and specificity.
Conclusions: The use of cannabinoid-containing foodstuffs and cannabinoid-based therapeutics, and continued abuse of oral cannabis require scientific data for accurate interpretation of cannabinoid tests and for making reliable administrative drug-testing policy. At the federally mandated cannabinoid cutoffs, it is possible but unlikely for a urine specimen to test positive after ingestion of manufacturer-recommended doses of low-THC hemp oils. Urine tests have a high likelihood of being positive after Marinol therapy. The Emit II and DRI assays had adequate sensitivity and specificity, but the CEDIA assay failed to detect many true-positive specimens.
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Introduction
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
{Delta}9-Tetrahydrocannabinol (THC)1 is the major psychoactive cannabinoid in the marijuana plant Cannabis sativa. Clinical marijuana smoking studies have characterized the urinary pharmacokinetics of THC after inhalation (1)(2)(3)(4)(5)(6); ingestion of cannabis has been investigated to a lesser extent (7)(8)(9)(10)(11). Since the mid-1990s, food products derived from cannabis plants and advertised as nutritional supplements have been available in the US; one such item is hemp seed oil. Manufacturers suggest that hemp oil is of value as a nutritional supplement because of its high concentration of the essential fatty acids {omega}-linolenic and linoleic acid. Although the hemp seed kernel does not have a substantial THC concentration, hemp oils have been found to contain a wide range of THC concentrations (12)(13)(14). The THC content of hemp oil is dependent on the effectiveness of seed cleaning and drying procedures and oil filtration processes. THC contamination originates from plant resin covering the surrounding seed hull. Ingestion of these products may produce positive urine drug tests (13)(15). This has led to questions about the validity of urine drug tests intended to deter drug use in treatment, workplace, criminal justice, and military programs.
Several studies have reported that ingestion of hemp oil causes positive urine tests for cannabinoids (12)(16)(17)(18). Lehmann et al.(12) reported THC concentrations of 3—1500 µg/g in 25 hemp oil samples. Six individuals ingested one or two tablespoons of hemp oil containing 1500 µg/g THC (11 and 22 g of hemp oil, or ~16.5—33 mg THC). Positive urine specimens were observed with a 50 µg/L cannabinoid immunoassay cutoff and a 15 µg/L 11-nor-9-carboxy-{Delta}9-tetrahydrocannabinol (THCCOOH) gas chromatography—mass spectrometry (GC/MS) cutoff for up to 6 days. Costantino et al. (18) reported that seven individuals ingesting 15 mL of hemp oil of an unknown THC concentration had positive urine drug tests by immunoassay at a cutoff of 20 µg/L for up to 48 h after ingestion. GC/MS analysis of urine specimens for THCCOOH, the primary urinary metabolite of THC, identified concentrations up to 78.6 µg/L. This is substantially above the federally mandated urine THCCOOH confirmation cutoff concentration of 15 µg/L. It is of concern that legitimate consumption of hemp oil may be interpreted as illicit drug exposure and that hemp oil ingestion may be used to conceal illicit cannabis use.
During the past few years, there has been a reduction in the THC concentration of hemp food products (19). The decrease is attributable to improved quality-control measures, such as more thorough seed cleaning. The Drug Enforcement Agency and Justice Department added an interpretive rule to 21 CFR Part 1308 in the Federal Register in October 2001 (20). This addition to the Controlled Substances Act declared that any product containing any THC is a Schedule I controlled substance. However, hemp oils with considerable THC concentrations continue to be available in other countries. Therefore, the potential for hemp oil ingestion to explain a positive urine drug test remains.
Within the last decade, cannabinoid receptors and endogenous ligands have been identified, and research into the medicinal uses of cannabinoids has expanded (21)(22). Synthetic THC medications, such as dronabinol (Marinol®), may be prescribed for the treatment of nausea and vomiting from cytotoxic chemotherapy unresponsive to conventional anti-emetics and for AIDS wasting syndrome (22). In addition, clinical studies are underway with drugs extracted from the cannabis plant and administered via a bronchial nebulizer or sublingual liquid for the treatment of neurologic disorders and pain relief (23).
Marijuana smoking is the most prevalent route of administering cannabis, but oral consumption of cannabis-containing foodstuffs, such as brownies or tea, is common. Cone et al. (11) reported urine cannabinoid results after consumption of brownies laced with marijuana plant material. Participants ate one or two brownies, each containing 22.4 mg of THC. After ingestion of two brownies, an individual's urine was positive by immunoassay (20 µg/L cutoff) for up to 11 days and by GC/MS (5 µg/L cutoff) up to 14 days.
The availability of cannabinoid-containing food products, increased interest in cannabinoid-based therapeutics, and the continued abuse of oral cannabis have emphasized the need for a controlled clinical investigation of the pharmacokinetics and pharmacodynamics of oral THC. Scientific data are required for the accurate interpretation of cannabinoid tests and for making reliable administrative drug-testing policy. The objectives of the present study were to characterize urinary cannabinoid excretion after THC ingestion and to determine whether measurable physiologic and behavioral effects accompany positive urine cannabinoid tests. In addition, the sensitivities, specificities, and efficiencies of three commercially available cannabinoid immunoassays, CEDIA®, DRI®, and Emit® II, were assessed. These data are needed for deterrence, drug treatment, parole, and probation programs to develop more effective and reliable urine cannabinoid-monitoring procedures.
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Abstract
Introduction
Materials and Methods
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participants and study design
Seven healthy individuals with a history of marijuana use resided in the secure clinical research unit of the Intramural Research Program (IRP), National Institute on Drug Abuse (NIDA), National Institutes of Health, while participating in a protocol designed to characterize the pharmacokinetics and pharmacodynamics of oral THC. The NIDA Institutional Review Board approved the study. All participants provided written informed consent, were under continuous medical supervision, and were financially compensated for their participation. The characteristics and drug use histories of the participants are summarized in Table 1 .
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Table 1. Participant demographics.
Before admission, each participant underwent thorough medical (physical exam, electrocardiography, and blood and urine chemistries) and psychologic evaluations, including past and recent drug use history. Participants did not receive the first drug administration until urine cannabinoid concentrations were <10 µg/L by fluorescence polarization immunoassay (Abbott© Laboratories). Twenty-four-hour medical surveillance and a closed, secure ward prevented access to unauthorized licit or illicit drugs. In addition, frequent urine drug tests were performed.
This protocol was a randomized, double-blind, double-dummy, placebo-controlled clinical study. Each participant participated in five dosing conditions, each of which entailed supervised administration of 15 mL (~1 tablespoon) of hemp oil and two capsules three times per day with meals for 5 consecutive days. Participants freely selected food choices, without restriction, from the clinical research unit menus. After 5 days of dosing, there was a 10-day washout period. Individuals resided in the secure clinical unit for 10—13 weeks.
Hemp oils were assayed by GC/MS to accurately determine the concentration of THC. Flax oil was administered as the placebo. The liquid low-dose hemp oil contained 9 µg/g THC for a total daily dose of 0.39 mg. The low-dose hemp oil administered in capsules contained 92 µg/g THC for a total daily dose of 0.47 mg. The liquid high-dose hemp oil contained 347 µg/g THC for a total daily dose of 14.8 mg. Dronabinol (synthetic THC in sesame oil; 2.5 mg of THC per capsule) was administered as a positive control with a daily total dose of 7.5 mg.
Dosing conditions were randomized and assigned by the IRP pharmacy to ensure that research staff and participants were blind to the administered dose. The five dosing conditions were as follows: (a), placebo oil and placebo capsules; (b), low-dose hemp oil and placebo capsules; (c), high-dose hemp oil and placebo capsules; (d), placebo oil and low-dose capsules; and (e), placebo oil and dronabinol capsules. The hemp oil capsules and dronabinol capsules were contained within larger capsules to maintain the double-blind condition.
specimen collection and analysis
Every urine void was collected from admittance to discharge from the clinical unit. Specimens (n = 4381) were collected in polypropylene containers, refrigerated immediately after urination, and measured for total volume. Aliquots of the urine specimens were stored in 5-mL polypropylene screw-cap tubes and 30-mL bottles at -30 °C within 48 h of collection. After the protocol was complete, frozen specimens were assembled and coded. Specimens were randomized within a large batch to eliminate potential analytical bias. Each specimen was analyzed for THCCOOH by GC/MS with a 15 µg/L cutoff according to published procedures with a limit of quantification of 2.5 µg/L (21)(24). Aliquots from each specimen also were screened with the following cannabinoid reagents: CEDIA (Microgenics), DRI (Diagnostic Reagents, Inc.) and Emit II (Dade Behring). The CEDIA assays were performed on a Hitachi 704 automated clinical analyzer (Roche Diagnostics) with a 50 µg/L cutoff. The DRI assays were conducted on the AU 800 analyzer (Olympus) with a 50 µg/L cutoff. The Emit II assays also were performed on an AU 800 analyzer with cutoffs of 20, 50, and 100 µg/L. Each batch of samples contained assayed negative and positive controls.
data analysis
Detection rates and times were calculated for each participant at each dose. The detection rate is the number of urine samples that screen positive from the first dose to the last specimen of the 2-week session, divided by the total number of samples collected during that session. The first positive is the time from the first dose to the first positive urine sample, and the last positive is the time from the last dose to the last positive sample. The first negative is the time from the last dose to the first negative sample. Urinalysis data were evaluated further by calculating the number of true positive (TP; positive by immunoassay at 50 µg/L and by GC/MS at 15 µg/L THCCOOH), false positive (FP; positive by immunoassay but negative by GC/MS), false negative (FN; negative by immunoassay but positive by GC/MS), and true negative (TN; negative by immunoassay and by GC/MS) results for each immunoassay at each dose. In addition, immunoassay performance was evaluated by calculating mean sensitivity, specificity, and efficiency over the five doses. Sensitivity was calculated by dividing the number of TP results by the sum of FN and TP results. Specificity was determined by dividing the number of TN results by the sum of FP and TN results. The efficiency of an assay is the sum of TP and TN results divided by the total number of samples. Sensitivities, specificities, and efficiencies were multiplied by 100 to yield percentage values.
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immunoassay detection rates and times for 50 µg/L cutoff
Urinary detection rates and times varied across assays and doses (Table 2 ) and demonstrated considerable interindividual variability. Only one participant of seven had any positive cannabinoid urine results at the low dose of 0.39 mg/day; he had two positive samples with the Emit II assay and one with the DRI, for detection rates of 0.2% and 0.1%, respectively. Three participants produced one, two, and two positive specimens with the Emit II assay after the daily ingestion of 0.47 mg THC. All participants produced positive specimens after administration of the high doses. The mean detection rates were 30.7—45.7% for the 7.5 mg/day dose and 23.5—41.2% for the 14.8 mg/day dose of liquid hemp oil. The Emit II assay consistently had the shortest times to the first positive specimen, and the CEDIA assay had the longest; times ranged from 1.9 to 52.8 h for the 7.5 mg/day dose and 4.0 to 32.5 h for the 14.8 mg/day THC dose, respectively. The inverse was true for the last positive and first negative specimens; Emit II had the largest window of detection and CEDIA the smallest. Times to last positive cannabinoid test after the 7.5 and 14.8 mg/day doses were 15.9—91.1 and 19.3—117.5 h, respectively, for the Emit II assay and 15.9—67.0 and 12.6—67.3 h for the CEDIA assay. The first negative specimen, for both doses and in all three assays, generally occurred before the last positive specimen. The shortest time to the first negative specimen was 2.6 h with the CEDIA assay after the 14.8 mg/day dose, and the longest time was 83.9 h with the Emit II assay after the daily dose of 7.5 mg of THC. DRI results for the two high doses were consistently intermediate between those of the Emit II and CEDIA assays.
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Table 2. Cannabinoid immunoassay data for 50 µg/L cutoff.
immunoassay detection rates and times for emit ii at 20 and 100 µg/L cutoffs
The results for the Emit II immunoassay at cutoffs of 20 and 100 µg/L are shown in Table 3 . Lowering the cutoff to 20 µg/L increased detection rates considerably and widened the detection time window. This increased the number of participants with positive samples at the lower doses: three of seven participants had positive tests at the 0.39 mg/day dose and six with the 0.47 mg/day dose. Mean detection rates for participants with positive specimens with the two lower doses were 5.1% and 12.8%, respectively. The mean detection rates for the 7.5 and 14.8 mg/day doses also increased to 55.9% and 53.6%, respectively. Mean times to first positive for the higher doses were just under 6 h; this decreased the times by approximately one-fourth from those observed with the higher 50 µg/L cutoff value. The mean times of the last positive and first negative tests were increased 26—52% compared with those of the 50 µg/L cutoff. The longest last positive time was 132 h (5.5 days) after the 14.8 mg/day dose, and the longest first negative time was 94.9 h (4 days) after the 7.5 mg/day dose.
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Table 3. Emit II cannabinoid immunoassay data for 20 and 100 µg/L cutoffs.
No specimens tested positive at the 100 µg/L cutoff after the 0.39 and 0.47 mg/day doses. As expected, the two high doses had fewer positive specimens at this cutoff compared with the 50 µg/L cutoff. The mean detection rates for the 7.5 and 14.8 mg/day doses were 25.8% and 16.7%, respectively. Mean times to first positive specimen increased, and the times to last positive and first negative decreased.
gc/ms detection rates and times at a 15 µg/L cutoff
THCCOOH concentrations in all urine specimens were quantified by GC/MS; the detection rates and times are presented in Table 4 . As observed with the immunoassay data, THCCOOH concentrations and detection times reflected considerable interindividual variability. Four of the seven participants produced urine samples at or above the 15 µg/L cutoff during the lowest dosing session of 0.39 mg/day. For the higher 0.47 mg/day dosing session, two of seven participants had positive test results. The mean numbers of specimens with THCCOOH at or above the 15 µg/L cutoff were 3.1 and 2.4 for the 0.39 and 0.47 mg/day doses, respectively. Approximately one-third of the specimens were positive after the 7.5- and 14.8-mg daily doses. With the exception of the lowest dose, mean times for first positive urine specimens were <24 h. Generally there were intermediate negative specimens before the last positive specimens for all four doses. Three participants had a positive specimen after the last 0.39 mg/day dose, and one after the last 0.47 mg/day dose. The longest times for a last positive test were 110.5 h after the 7.5 mg/day dose and 84.2 h for the 14.8 mg/day dose. First negative times for the high doses ranged from 0.3 to 91.6 h.
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Table 4. GC/MS THCCOOH quantification data for 15 µg/L cutoff.
The 0.39 and 0.47 mg/day doses produced maximum urine THCCOOH concentrations (cmax) of 7.3—38.2 and 5.4—31 µg/L, respectively, with mean times to the highest THCCOOH concentration (tmax) of 99.9 and 85.9 h, respectively. The mean cmax for the 7.5 mg/day dose (145.7 µg/L) was larger than that of the 14.8 mg/day dose (116.0 µg/L). The highest THCCOOH concentration was 436 µg/L, after a daily dose of 7.5 mg, and the lowest cmax at the 14.8 mg/day dose was 19.0 µg/L. The mean tmax values were 52.1—119 h for the 7.5 mg/day dose and 46.0—157.4 h for the 14.8 mg/day dose.
immunoassay performance characteristics
Immunoassay semiquantitative and GC/MS quantitative results (n = 4381) were used to calculate TP, FP, FN, and TN values. These data determined the sensitivity, specificity, and efficiency of each immunoassay (Table 5 ). The two high doses produced the majority of TP specimens. There were 528 TP specimens with the Emit II assay and slightly fewer (n = 517) with the DRI assay. The CEDIA assay identified far fewer TP specimens (n = 397), but had a higher number of TN results (n = 3716). The Emit II and DRI assays had 3526 and 3658 TN specimens, respectively.
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Table 5. Cannabinoid immunoassay performance at 50 µg/L cutoff.1
The Emit II assay provided the highest mean sensitivity of 84%, with the DRI and CEDIA assays having values of 82.5% and 62%, respectively. Mean specificity values were all >90%; the CEDIA assay was the most specific at 99.3%, followed by the DRI assay at 97.8% and the Emit II at 94.7%. The three assays also produced mean efficiency values >90%, in part because of the high number of TN values. The DRI assay had the highest overall efficiency at 95.2%, with the CEDIA and Emit II assays having efficiencies of 93.9% and 92.8%, respectively.
tp detection rates
The detection rate data for TP specimens, those specimens positive at a 50 µg/L immunoassay cutoff and a THCCOOH concentration at or above 15 µg/L by GC/MS, are shown in Table 6 . The mean TP detection rates for the Emit II and DRI assays at the lowest dose, 0.39 mg/day, were 0.2% and 0.1%, respectively. One participant had 1.6% and 0.8% TP urine specimens with the Emit II and DRI assays, respectively. Three of the remaining six participants had urine samples with THCCOOH concentrations at or above the GC/MS cutoff, but none was positive by immunoassay. The 0.47 mg/day dose produced TP in two participants, but only with the Emit II assay. TP detection rates for these two participants were 1.9% and 1.0%, for a mean TP detection rate of 0.4%. Administration of the two low doses produced no TP tests with the CEDIA assay. Mean detection rates for the 7.5 mg/day dose were 35.9%, 34.8%, and 29.0%, respectively, for the Emit II, DRI, and CEDIA assays. The 14.8 mg/day dose had lower mean detection rates than the 7.5 mg/day dose: rates for the Emit II, DRI, and CEDIA immunoassays were 28.1%, 27.9%, and 22.5%, respectively.
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Table 6. TP1 detection rate data.
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Discussion
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Abstract
Introduction
Materials and Methods
Results
Discussion
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There continues to be potential for oral cannabis abuse and an increasing likelihood of THC ingestion from medications or food products that may produce positive urine drug tests for cannabinoids. The need for deterring and detecting cannabis abuse continues to be an important component in drug treatment and medical screening, along with judicial, workplace, and military forensic urine drug-testing programs. This study investigated excretion pharmacokinetics after administration of oral THC. This included evaluating the extent to which ingestion of hemp oil and dronabinol could produce a positive urine test for cannabinoids. In the course of the investigation, we evaluated the performances of three commercial cannabinoid immunoassays. This study's data indicate that it is possible but unlikely that a person would test positive for urinary cannabinoids after ingestion of hemp oil. This is valid as long as mandated cannabinoid screening and confirmation cutoffs are used, the THC concentrations in hemp oils remain at their present low values, and individuals take hemp oil according to manufacturers' recommendations.
There are many relevant pharmacologic factors, such as dose, route, and vehicle, and physiologic factors, such as metabolism and excretion, that influence drug concentrations in the circulation. Pharmacologic factors have a significant influence on the amount of drug absorbed into the bloodstream. Perez-Reyes et al. (7) described the efficacy of five different vehicles used in the administration of 35 mg of THC containing 50 µCi of tritium-labeled THC in gelatin capsules. Glycocholate produced the highest THC plasma values, followed by sesame oil. However, there was considerable variability in peak concentration and rate of absorption, even when drug was administered in the same vehicle.
Oral THC bioavailability was estimated to be 10—20% by Wall et al. (8). In their study, participants were dosed with either 15 mg (women) or 20 mg (men) of THC, a percentage of which was radiolabeled, dissolved in sesame oil and contained in gelatin capsules. One drawback of studies with radiolabeled THC is the inability to differentiate labeled THC in plasma from its labeled metabolites, which leads to overestimation of drug concentrations. Possibly a more accurate assessment of oral bioavailability, which used GC/MS to quantify THC in plasma samples, was reported by Ohlsson et al. (25). They estimated a 6% bioavailability after a 20-mg THC dose ingested in a cookie. Slow rates of absorption and low THC concentrations were observed. Low oral THC bioavailability may be attributable to poor absorption, degradation by stomach acid, and/or biotransformation to metabolites during first passage through the liver.
Unlike the inhalation route, THC absorbed from the gastrointestinal system has considerable first-pass metabolism in the liver. Cytochrome P450 enzymes, primarily CYP3A4 and CYP2C9 and -11 (26), oxidize THC into numerous metabolites. The C-11 allylic methyl group may be oxidized to 11-hydroxy-tetrahydrocannabinol (11-OH-THC), a pharmacologically active metabolite. The plasma concentration of 11-OH-THC is ~10% of that of the parent drug after smoking or intravenous infusion of THC, whereas the relative 11-OH-THC plasma concentration increases to 50—100% after oral administration (8). Similar physiologic and pharmacodynamic effects, relative to smoked marijuana, have been observed after ingestion of THC, although THC plasma concentrations were lower after oral ingestion (25). An increase in the equipotent metabolite concentration is a possible explanation for comparable effects. 11-OH-THC may undergo further oxidation to THCCOOH, thereby inactivating the drug before it reaches the heart for systemic distribution. Most of the THCCOOH is rapidly conjugated to a water-soluble glucuronic acid. This glucuronide metabolite is the primary urinary metabolite of THC (27). Immunoassays for the detection of cannabinoids are designed to be specific for THCCOOH with less cross-reactivity to other cannabinoid metabolites (28)(29). After hydrolysis and extraction, THCCOOH is the primary urinary cannabinoid analyte quantified by GC/MS.
The individual TP detection rate data (Table 6Up ) provide useful examples of variability after THC ingestion. The detection rates for participant G were approximately five times those of participant N. Variability in THC concentrations found in smoking studies have been attributed to smoking topography variables such as number of puffs, depth of inhalation, and hold time; therefore, an experienced smoker may attempt to control the degree of pharmacologic effects by titrating the amount of drug absorbed. No such control is available via the oral route, but the variability persists. Other factors that may play a part are age, disease state, body weight, and metabolic differences. One variable that is unique to the oral route and that could affect drug absorption is co-ingestion of food. During this study, participants were allowed unrestricted access to food and drink. In addition, doses were given with meals to simulate common usage conditions. It is possible that controlling dietary intake could have reduced interindividual variability in urinary cannabinoid concentrations.
At present, many commercial cannabinoid immunoassay systems are designed with a 50 µg/L cutoff. In 1994, the US Department of Health and Human Services lowered the required initial immunoassay test cutoff from 100 µg/L to its present value. This brought the mandated cutoff in line with the cutoff established by the Department of Defense in 1992. Administrative cutoffs are established after taking into account many factors, such as goals of the drug-testing program, capabilities of the analytical methods, data from pharmacologic studies, and desired detection time windows. Detection rate data from the present study demonstrate that it is unlikely that a urinary cannabinoid immunoassay will be positive at the 50 µg/L cutoff because of consumption of hemp oils with lower THC concentrations. One participant of seven had two positive samples after the 0.39 mg/day dose by the Emit II assay and one positive by the DRI assay. The time to first positive was >100 h, after the individual had received 15 doses over 5 days. Three participants had positive specimens at the 0.47 mg/day dose, with the first positive samples occurring between 30 and 87 h.
The two higher doses, 7.5 and 14.8 mg/day, produced numerous positive specimens. At the 7.5 mg/day dronabinol dose, one participant produced a specimen positive by all three immunoassays within 2 h of the first dose, which was the first void after dosing. The longest time to first positive at this dose was 52.8 h by the CEDIA assay and 10.6 h by the Emit II assay. The mean time to first negative specimen was shorter for the 14.8 mg daily dose than for the dronabinol dose. Time to first negative for the dronabinol dose was 15.1 (CEDIA) to 83.9 h (Emit II), whereas the range for the higher dose was 2.6 (CEDIA) to 53.9 h (Emit II). As in previous studies (5)(11)(30), the last positive specimen did not necessarily occur before the first negative. In our study, at the 7.5 mg/day dose, all participants had at least one negative sample before the last positive; only one individual had no positive samples after the first negative sample at the 14.8 mg/day dose. Fluctuation of urinary THC concentrations at or around the cutoff may be highly dependent on the individual's state of hydration. These data also demonstrate that an individual may absorb enough drug from hemp oils containing high THC concentrations to produce a positive sample by the first urine void.
There are commercial cannabinoid immunoassays designed with cutoffs of 20 and 100 µg/L. The 20 µg/L cutoff is used in workplace settings, such as nuclear power plants, where higher sensitivity is desired, yielding a broader detection window. The 100 µg/L cutoff continues to be used in some workplace testing programs, possibly because of contractual obligations. This study found higher detection rates and larger detection time windows with the 20 µg/L cutoff, similar to other published results (30)(31)(32). At this lower cutoff, three participants produced positive specimens with the 0.39 mg/day dose and all but one of seven participants had positive samples at the 0.47 mg/day dose. As expected, increasing the cutoff to 100 µg/L decreased detection rates considerably with no FP specimens at the lower dosing concentrations. These results demonstrate that detection rates and times can vary dramatically depending on choice of immunoassay and cutoff value.
All three immunoassays in this study provided acceptable performance at the 50 µg/L screening cutoff. Overall efficiencies were >90%, with the DRI system having the highest efficiency of 95.2%. The objective of the initial screening test is to identify TP specimens with a minimum number of unconfirmed positive results. To accomplish this task, high immunoassay sensitivity must be balanced with high specificity to reduce the number of specimens that require the expensive and time-consuming GC/MS confirmation. The results from this study indicate a difference in sensitivity between the immunoassays, with the Emit II being the most sensitive (528 TP results) and the CEDIA the least sensitive (397 TP results). The Emit II produced the fewest number of TN results, reflecting its higher sensitivity but lower specificity. Conversely, the CEDIA produced the largest number of TN specimens, providing the highest specificity. The results for the DRI immunoassay were consistently between those of the Emit II and CEDIA systems, indicating a more efficient balance between specificity and sensitivity. Huestis et al. (30) reported similar performance variability among six cannabinoid immunoassays.
To establish a standard for comparing immunoassay performance and to have quantifiable data to compare urinary excretion after oral THC, all urine specimens were analyzed by GC/MS to determine THCCOOH concentration. All doses, with the exception of placebo, produced specimens with THCCOOH >15 µg/L. Interestingly, the 7.5- and 14.8-mg daily doses had nearly equal numbers of specimens above the 15 µg/L GC/MS cutoff despite an almost twofold difference in dose. The most striking disparity between the two doses is in the cmax results. Although statistically insignificant (Student t-test, P >0.05), the mean cmax of 145.7 µg/L for the 7.5 mg/day dose and 116.0 µg/L for the 14.8 mg/day dose yields an inverse dose response. The mean tmax for all doses was consistent, ranging between 85.9 and 103.5 h. These times indicate that the highest urinary concentrations occurred after 12—15 THC doses on days 4—5 of treatment. With the last positive specimen times exceeding 72 h, these data demonstrate that multiple doses of hemp oils with high THC concentrations may produce GC/MS-positive specimens as long as 3 days after last ingestion.
One participant had a quantifiable THCCOOH specimen at the beginning of the placebo dose, immediately after the initial washout phase. The 3.5 µg/L THCCOOH urine concentration was therefore a result of cannabis use before ward admittance. Before starting the first dosing session, a participant's urine needed only to have been negative by immunoassay at a 10 µg/L cutoff.
Previous studies on the occurrence of positive urine samples after hemp oil ingestion examined results after a single dose or a single dose per day for several days (16)(17). The two most recent reports on hemp oil ingestion and urine cannabinoid concentration were not controlled clinical studies (14)(19). In a report by Leson et al. (19), 15 individuals self-administered single daily doses of liquid hemp oil with concentrations ranging from 0.09 to 0.6 mg of THC over a 10-day period. A total of 11 urine specimens were collected from each individual over a 40-day period, with the first morning void specifically not collected. Urine samples were tested by a RIA for cannabinoids (Immunalysis Direct RIA Kit). None of the participants ingesting daily THC doses between 0.09 and 0.45 mg produced positive urine samples at the 50 µg/L cutoff. One specimen, from one of three participants ingesting the 0.6-mg THC daily dose, did screen positive by immunoassay, but GC/MS analysis quantified the THCCOOH concentration at 3.0 µg/L. The authors also reported a cmax of 5.2 µg/L in a specimen after the high dose, which did not screen positive. In the present study, every urine sample was collected to allow for a full evaluation of the potential for positive urine samples. Many of the cmax specimens (15 of 28) occurred in the early morning hours, times not included in the previous study.
Bosy and Cole (14) reported urine cannabinoid results after a daily 15-g hemp oil dose for 7 days. Six hemp oils with THC concentrations ranging from 11.5 to 117.5 µg/g were evaluated, each in a single participant. Urine samples were collected just before dosing and at 6 h post-ingestion. Fluorescence polarization immunoassay and Kinetic Interactions of Microparticles in Solution (KIMS) immunoassay systems with 50 µg/L cutoffs were used to test specimens. Participants ingesting low-dose hemp oils (97.2 and 172.5 µg of THC) had immunoassay results well below the cutoff. Positive specimens occurred by the third and fourth days after medium doses of THC in hemp oil (540 and 546 µg of THC); however, specimens tested negative within 24 h after ingestion ceased. The high-dose hemp oil (1762.5 µg of THC), ingested by one participant, produced a positive urine test on the first day of dosing and tested negative within 72 h of last ingestion. The investigators also reported a peak THCCOOH concentration of 48.7 µg/L after the 1762.5 µg/day dose. The results from the studies by Leson et al. (19) and Bosy and Cole (14) are in general agreement with the data presented here, especially with results from our low doses. The 0.39 and 0.47 mg/day doses produced GC/MS-positive specimens in four and two of seven participants, respectively. The respective cmax means were 19.8 and 12.2 µg/L. These data show that even low doses of THC can produce urine specimens with THCCOOH concentrations >15 µg/L; however, multiple doses appear to be required.
The only previously reported study on oral THC in which all urine specimens were collected for an extended period of time after dosing was conducted by Cone et al. (11). Participants consumed brownies containing marijuana plant material; each brownie contained 22.4 mg of THC, similar to the amount found in a 2.8% THC cigarette. Mean urinary cmax values of 180 and 312 µg/L occurred after single THC doses of 22.4 mg and 44.8 mg, respectively, with individual cmax values of 177—436 µg/L after the higher dose. In a study by Law et al. (9), five individuals ingested meat sandwiches containing cannabis resin with 5 and 20 mg of THC. Urine samples were collected at timed intervals for up to 12 h after consumption and daily thereafter for up to 14 days. After the administration of 20 mg of THC, total urinary cannabinoid concentrations, measured by combined HPLC/RIA analysis, were 185—1063 µg/L 6 h after ingestion. Our study also demonstrate wide ranges in cmax values with the dronabinol dose, yielding a 20-fold difference between the lowest and highest cmax.
Dronabinol is synthetic THC dissolved in sesame oil and delivered in a gelatin capsule. It has been cleared by the Food and Drug Administration for the symptomatic treatment of nausea and vomiting for cancer patients receiving chemotherapy and as an appetite stimulant for chronically ill patients with wasting syndrome (22). Natural and synthetic THC are chemically identical, undergo similar metabolism, and produce identical metabolites. Studies have been conducted to discover a biomarker to differentiate the occurrence of THCCOOH in the urine from cannabis abuse and from the ingestion of therapeutic THC (33)(34). To date, no definitive analyte has been identified.
Interest in the therapeutic use of cannabinoids has increased dramatically in the past decade, but little research has focused on the excretion pharmacokinetics of oral THC. ElSohly et al. (33) administered a single 15-mg dronabinol dose to four individuals. Every urine specimen was collected for 24 h, and then specimens were collected once a day for 6 days. The cmax values for three of the participants were 188.7—227 µg/L. These results are within the cmax values found with the 14.8 mg/day dose in the present study. Although there have been THC ingestion studies comparing different THC vehicles, with the exception of our study, there have been no studies on the use of different formulations, such as capsule vs liquid. THC delivered in a capsule as opposed to a liquid may allow greater absorption from the gastrointestinal system. Possibly the capsule provides protection against degradation by stomach acids and delivers a large proportion of THC intact to the intestinal tract for absorption.
Oral abuse of cannabis undoubtedly will continue, and the use of orally administered therapeutic cannabinoids probably will increase. Research is needed to determine how synthetic and naturally occurring cannabinoids, administered via different routes, will affect urine drug-testing programs. We have demonstrated that it is possible but unlikely for a urine specimen to test positive at the federally mandated cutoffs for screening and confirmation after hemp oil ingestion, given the lower THC concentrations currently found in hemp oils and the recommendations of manufacturers for once-a-day dosing. In addition, we have provided cmax and tmax values, detection rates and times, and performance data on the three most commonly used cannabinoid immunoassays. Knowledge of an assay's sensitivity, specificity, and efficiency allows the administrators of drug-testing programs to select the most appropriate immunoassay for their program's goals.
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Acknowledgments
We thank David Darwin, Debbie Price, and the clinical staff of the NIDA IRP Research Unit for technical assistance. We are also grateful to Carole Trojan, Erick Fitzer, Daniel Nichols, and the Navy Drug Screening Laboratory Jacksonville's Screening and Confirmation staff for analytical assistance in this project. We would also like to thank the Research Triangle Institute for GC/MS analyses of the hemp oil and the Department of Defense, Department of Transportation, Substance Abuse and Mental Health Services Administration, and NIDA IRP for funding this project.
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Footnotes
1 Nonstandard abbreviations: THC, {Delta}9-tetrahydrocannabinol; THCCOOH, 11-nor-9-carboxy-{Delta}9-tetrahydrocannabinol; GC/MS, gas chromatography—mass spectrometry; IRP, Intramural Research Program; NIDA, National Institute on Drug Abuse; TP, true positive (number of specimens positive by immunoassay and GC/MS analysis); FP, false positive (number of specimens positive by immunoassay and negative by GC/MS analysis); FN, false negative (number of specimens negative by immunoassay and positive by GC/MS analysis); TN, true negative (number of specimens negative by immunoassay and GC/MS analysis); cmax, highest concentration of drug; tmax, time from first dose to highest concentration of drug; and 11-OH-THC, 11-hydroxy-{Delta}9-tetrahydrocannabinol.
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Source: Urinary Cannabinoid Detection Times after Controlled Oral Administration of {Delta}9-Tetrahydrocannabinol to Humans
Richard A. Gustafson1, Barry Levine2, Peter R. Stout3, Kevin L. Klette4, M.P. George5, Eric T. Moolchan1 and Marilyn A. Huestis1,a
1 Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Dr., Baltimore, MD 21224.
2 Office of the Chief Medical Examiner, 111 Penn St., Baltimore, MD 21201.
3 Aegis Sciences Corp., 345 Hill Ave., Nashville, TN 37210.
4 Navy Drug Screening Laboratory, PO Box 113, Bldg. H-2033, Naval Air Station, Jacksonville, FL 32212.
5 Quest Diagnostics, Inc., 506 East State Pkwy., Schaumburg, IL 60173.
aAuthor for correspondence. Fax 410-550-2971; e-mail mhuestis@intra.nida.nih.gov.
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Background: Urinary cannabinoid excretion and immunoassay performance were evaluated by semiquantitative immunoassay and gas chromatography—mass spectrometry (GC/MS) analysis of metabolite concentrations in 4381 urine specimens collected before, during, and after controlled oral administration of tetrahydrocannabinol (THC).
Methods: Seven individuals received 0, 0.39, 0.47, 7.5, and 14.8 mg THC/day in this double-blind, placebo-controlled, randomized, clinical study conducted on a closed research ward. THC doses (hemp oils with various THC concentrations and the therapeutic drug Marinol®) were administered three times daily for 5 days. All urine voids were collected over the 10-week study and later tested by Emit II®, DRI®, and CEDIA® immunoassays and by GC/MS. Detection rates, detection times, and sensitivities, specificities, and efficiencies of the immunoassays were determined.
Results: At the federally mandated immunoassay cutoff (50 µg/L), mean detection rates were <0.2% during ingestion of the two low doses typical of current hemp oil THC concentrations. The two high doses produced mean detection rates of 23—46% with intermittent positive tests up to 118 h. Maximum metabolite concentrations were 5.4—38.2 µg/L for the low doses and 19.0—436 µg/L for the high doses. Emit II, DRI, and CEDIA immunoassays had similar performance efficiencies of 92.8%, 95.2%, and 93.9%, respectively, but differed in sensitivity and specificity.
Conclusions: The use of cannabinoid-containing foodstuffs and cannabinoid-based therapeutics, and continued abuse of oral cannabis require scientific data for accurate interpretation of cannabinoid tests and for making reliable administrative drug-testing policy. At the federally mandated cannabinoid cutoffs, it is possible but unlikely for a urine specimen to test positive after ingestion of manufacturer-recommended doses of low-THC hemp oils. Urine tests have a high likelihood of being positive after Marinol therapy. The Emit II and DRI assays had adequate sensitivity and specificity, but the CEDIA assay failed to detect many true-positive specimens.
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{Delta}9-Tetrahydrocannabinol (THC)1 is the major psychoactive cannabinoid in the marijuana plant Cannabis sativa. Clinical marijuana smoking studies have characterized the urinary pharmacokinetics of THC after inhalation (1)(2)(3)(4)(5)(6); ingestion of cannabis has been investigated to a lesser extent (7)(8)(9)(10)(11). Since the mid-1990s, food products derived from cannabis plants and advertised as nutritional supplements have been available in the US; one such item is hemp seed oil. Manufacturers suggest that hemp oil is of value as a nutritional supplement because of its high concentration of the essential fatty acids {omega}-linolenic and linoleic acid. Although the hemp seed kernel does not have a substantial THC concentration, hemp oils have been found to contain a wide range of THC concentrations (12)(13)(14). The THC content of hemp oil is dependent on the effectiveness of seed cleaning and drying procedures and oil filtration processes. THC contamination originates from plant resin covering the surrounding seed hull. Ingestion of these products may produce positive urine drug tests (13)(15). This has led to questions about the validity of urine drug tests intended to deter drug use in treatment, workplace, criminal justice, and military programs.
Several studies have reported that ingestion of hemp oil causes positive urine tests for cannabinoids (12)(16)(17)(18). Lehmann et al.(12) reported THC concentrations of 3—1500 µg/g in 25 hemp oil samples. Six individuals ingested one or two tablespoons of hemp oil containing 1500 µg/g THC (11 and 22 g of hemp oil, or ~16.5—33 mg THC). Positive urine specimens were observed with a 50 µg/L cannabinoid immunoassay cutoff and a 15 µg/L 11-nor-9-carboxy-{Delta}9-tetrahydrocannabinol (THCCOOH) gas chromatography—mass spectrometry (GC/MS) cutoff for up to 6 days. Costantino et al. (18) reported that seven individuals ingesting 15 mL of hemp oil of an unknown THC concentration had positive urine drug tests by immunoassay at a cutoff of 20 µg/L for up to 48 h after ingestion. GC/MS analysis of urine specimens for THCCOOH, the primary urinary metabolite of THC, identified concentrations up to 78.6 µg/L. This is substantially above the federally mandated urine THCCOOH confirmation cutoff concentration of 15 µg/L. It is of concern that legitimate consumption of hemp oil may be interpreted as illicit drug exposure and that hemp oil ingestion may be used to conceal illicit cannabis use.
During the past few years, there has been a reduction in the THC concentration of hemp food products (19). The decrease is attributable to improved quality-control measures, such as more thorough seed cleaning. The Drug Enforcement Agency and Justice Department added an interpretive rule to 21 CFR Part 1308 in the Federal Register in October 2001 (20). This addition to the Controlled Substances Act declared that any product containing any THC is a Schedule I controlled substance. However, hemp oils with considerable THC concentrations continue to be available in other countries. Therefore, the potential for hemp oil ingestion to explain a positive urine drug test remains.
Within the last decade, cannabinoid receptors and endogenous ligands have been identified, and research into the medicinal uses of cannabinoids has expanded (21)(22). Synthetic THC medications, such as dronabinol (Marinol®), may be prescribed for the treatment of nausea and vomiting from cytotoxic chemotherapy unresponsive to conventional anti-emetics and for AIDS wasting syndrome (22). In addition, clinical studies are underway with drugs extracted from the cannabis plant and administered via a bronchial nebulizer or sublingual liquid for the treatment of neurologic disorders and pain relief (23).
Marijuana smoking is the most prevalent route of administering cannabis, but oral consumption of cannabis-containing foodstuffs, such as brownies or tea, is common. Cone et al. (11) reported urine cannabinoid results after consumption of brownies laced with marijuana plant material. Participants ate one or two brownies, each containing 22.4 mg of THC. After ingestion of two brownies, an individual's urine was positive by immunoassay (20 µg/L cutoff) for up to 11 days and by GC/MS (5 µg/L cutoff) up to 14 days.
The availability of cannabinoid-containing food products, increased interest in cannabinoid-based therapeutics, and the continued abuse of oral cannabis have emphasized the need for a controlled clinical investigation of the pharmacokinetics and pharmacodynamics of oral THC. Scientific data are required for the accurate interpretation of cannabinoid tests and for making reliable administrative drug-testing policy. The objectives of the present study were to characterize urinary cannabinoid excretion after THC ingestion and to determine whether measurable physiologic and behavioral effects accompany positive urine cannabinoid tests. In addition, the sensitivities, specificities, and efficiencies of three commercially available cannabinoid immunoassays, CEDIA®, DRI®, and Emit® II, were assessed. These data are needed for deterrence, drug treatment, parole, and probation programs to develop more effective and reliable urine cannabinoid-monitoring procedures.
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participants and study design
Seven healthy individuals with a history of marijuana use resided in the secure clinical research unit of the Intramural Research Program (IRP), National Institute on Drug Abuse (NIDA), National Institutes of Health, while participating in a protocol designed to characterize the pharmacokinetics and pharmacodynamics of oral THC. The NIDA Institutional Review Board approved the study. All participants provided written informed consent, were under continuous medical supervision, and were financially compensated for their participation. The characteristics and drug use histories of the participants are summarized in Table 1 .
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Table 1. Participant demographics.
Before admission, each participant underwent thorough medical (physical exam, electrocardiography, and blood and urine chemistries) and psychologic evaluations, including past and recent drug use history. Participants did not receive the first drug administration until urine cannabinoid concentrations were <10 µg/L by fluorescence polarization immunoassay (Abbott© Laboratories). Twenty-four-hour medical surveillance and a closed, secure ward prevented access to unauthorized licit or illicit drugs. In addition, frequent urine drug tests were performed.
This protocol was a randomized, double-blind, double-dummy, placebo-controlled clinical study. Each participant participated in five dosing conditions, each of which entailed supervised administration of 15 mL (~1 tablespoon) of hemp oil and two capsules three times per day with meals for 5 consecutive days. Participants freely selected food choices, without restriction, from the clinical research unit menus. After 5 days of dosing, there was a 10-day washout period. Individuals resided in the secure clinical unit for 10—13 weeks.
Hemp oils were assayed by GC/MS to accurately determine the concentration of THC. Flax oil was administered as the placebo. The liquid low-dose hemp oil contained 9 µg/g THC for a total daily dose of 0.39 mg. The low-dose hemp oil administered in capsules contained 92 µg/g THC for a total daily dose of 0.47 mg. The liquid high-dose hemp oil contained 347 µg/g THC for a total daily dose of 14.8 mg. Dronabinol (synthetic THC in sesame oil; 2.5 mg of THC per capsule) was administered as a positive control with a daily total dose of 7.5 mg.
Dosing conditions were randomized and assigned by the IRP pharmacy to ensure that research staff and participants were blind to the administered dose. The five dosing conditions were as follows: (a), placebo oil and placebo capsules; (b), low-dose hemp oil and placebo capsules; (c), high-dose hemp oil and placebo capsules; (d), placebo oil and low-dose capsules; and (e), placebo oil and dronabinol capsules. The hemp oil capsules and dronabinol capsules were contained within larger capsules to maintain the double-blind condition.
specimen collection and analysis
Every urine void was collected from admittance to discharge from the clinical unit. Specimens (n = 4381) were collected in polypropylene containers, refrigerated immediately after urination, and measured for total volume. Aliquots of the urine specimens were stored in 5-mL polypropylene screw-cap tubes and 30-mL bottles at -30 °C within 48 h of collection. After the protocol was complete, frozen specimens were assembled and coded. Specimens were randomized within a large batch to eliminate potential analytical bias. Each specimen was analyzed for THCCOOH by GC/MS with a 15 µg/L cutoff according to published procedures with a limit of quantification of 2.5 µg/L (21)(24). Aliquots from each specimen also were screened with the following cannabinoid reagents: CEDIA (Microgenics), DRI (Diagnostic Reagents, Inc.) and Emit II (Dade Behring). The CEDIA assays were performed on a Hitachi 704 automated clinical analyzer (Roche Diagnostics) with a 50 µg/L cutoff. The DRI assays were conducted on the AU 800 analyzer (Olympus) with a 50 µg/L cutoff. The Emit II assays also were performed on an AU 800 analyzer with cutoffs of 20, 50, and 100 µg/L. Each batch of samples contained assayed negative and positive controls.
data analysis
Detection rates and times were calculated for each participant at each dose. The detection rate is the number of urine samples that screen positive from the first dose to the last specimen of the 2-week session, divided by the total number of samples collected during that session. The first positive is the time from the first dose to the first positive urine sample, and the last positive is the time from the last dose to the last positive sample. The first negative is the time from the last dose to the first negative sample. Urinalysis data were evaluated further by calculating the number of true positive (TP; positive by immunoassay at 50 µg/L and by GC/MS at 15 µg/L THCCOOH), false positive (FP; positive by immunoassay but negative by GC/MS), false negative (FN; negative by immunoassay but positive by GC/MS), and true negative (TN; negative by immunoassay and by GC/MS) results for each immunoassay at each dose. In addition, immunoassay performance was evaluated by calculating mean sensitivity, specificity, and efficiency over the five doses. Sensitivity was calculated by dividing the number of TP results by the sum of FN and TP results. Specificity was determined by dividing the number of TN results by the sum of FP and TN results. The efficiency of an assay is the sum of TP and TN results divided by the total number of samples. Sensitivities, specificities, and efficiencies were multiplied by 100 to yield percentage values.
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immunoassay detection rates and times for 50 µg/L cutoff
Urinary detection rates and times varied across assays and doses (Table 2 ) and demonstrated considerable interindividual variability. Only one participant of seven had any positive cannabinoid urine results at the low dose of 0.39 mg/day; he had two positive samples with the Emit II assay and one with the DRI, for detection rates of 0.2% and 0.1%, respectively. Three participants produced one, two, and two positive specimens with the Emit II assay after the daily ingestion of 0.47 mg THC. All participants produced positive specimens after administration of the high doses. The mean detection rates were 30.7—45.7% for the 7.5 mg/day dose and 23.5—41.2% for the 14.8 mg/day dose of liquid hemp oil. The Emit II assay consistently had the shortest times to the first positive specimen, and the CEDIA assay had the longest; times ranged from 1.9 to 52.8 h for the 7.5 mg/day dose and 4.0 to 32.5 h for the 14.8 mg/day THC dose, respectively. The inverse was true for the last positive and first negative specimens; Emit II had the largest window of detection and CEDIA the smallest. Times to last positive cannabinoid test after the 7.5 and 14.8 mg/day doses were 15.9—91.1 and 19.3—117.5 h, respectively, for the Emit II assay and 15.9—67.0 and 12.6—67.3 h for the CEDIA assay. The first negative specimen, for both doses and in all three assays, generally occurred before the last positive specimen. The shortest time to the first negative specimen was 2.6 h with the CEDIA assay after the 14.8 mg/day dose, and the longest time was 83.9 h with the Emit II assay after the daily dose of 7.5 mg of THC. DRI results for the two high doses were consistently intermediate between those of the Emit II and CEDIA assays.
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Table 2. Cannabinoid immunoassay data for 50 µg/L cutoff.
immunoassay detection rates and times for emit ii at 20 and 100 µg/L cutoffs
The results for the Emit II immunoassay at cutoffs of 20 and 100 µg/L are shown in Table 3 . Lowering the cutoff to 20 µg/L increased detection rates considerably and widened the detection time window. This increased the number of participants with positive samples at the lower doses: three of seven participants had positive tests at the 0.39 mg/day dose and six with the 0.47 mg/day dose. Mean detection rates for participants with positive specimens with the two lower doses were 5.1% and 12.8%, respectively. The mean detection rates for the 7.5 and 14.8 mg/day doses also increased to 55.9% and 53.6%, respectively. Mean times to first positive for the higher doses were just under 6 h; this decreased the times by approximately one-fourth from those observed with the higher 50 µg/L cutoff value. The mean times of the last positive and first negative tests were increased 26—52% compared with those of the 50 µg/L cutoff. The longest last positive time was 132 h (5.5 days) after the 14.8 mg/day dose, and the longest first negative time was 94.9 h (4 days) after the 7.5 mg/day dose.
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Table 3. Emit II cannabinoid immunoassay data for 20 and 100 µg/L cutoffs.
No specimens tested positive at the 100 µg/L cutoff after the 0.39 and 0.47 mg/day doses. As expected, the two high doses had fewer positive specimens at this cutoff compared with the 50 µg/L cutoff. The mean detection rates for the 7.5 and 14.8 mg/day doses were 25.8% and 16.7%, respectively. Mean times to first positive specimen increased, and the times to last positive and first negative decreased.
gc/ms detection rates and times at a 15 µg/L cutoff
THCCOOH concentrations in all urine specimens were quantified by GC/MS; the detection rates and times are presented in Table 4 . As observed with the immunoassay data, THCCOOH concentrations and detection times reflected considerable interindividual variability. Four of the seven participants produced urine samples at or above the 15 µg/L cutoff during the lowest dosing session of 0.39 mg/day. For the higher 0.47 mg/day dosing session, two of seven participants had positive test results. The mean numbers of specimens with THCCOOH at or above the 15 µg/L cutoff were 3.1 and 2.4 for the 0.39 and 0.47 mg/day doses, respectively. Approximately one-third of the specimens were positive after the 7.5- and 14.8-mg daily doses. With the exception of the lowest dose, mean times for first positive urine specimens were <24 h. Generally there were intermediate negative specimens before the last positive specimens for all four doses. Three participants had a positive specimen after the last 0.39 mg/day dose, and one after the last 0.47 mg/day dose. The longest times for a last positive test were 110.5 h after the 7.5 mg/day dose and 84.2 h for the 14.8 mg/day dose. First negative times for the high doses ranged from 0.3 to 91.6 h.
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Table 4. GC/MS THCCOOH quantification data for 15 µg/L cutoff.
The 0.39 and 0.47 mg/day doses produced maximum urine THCCOOH concentrations (cmax) of 7.3—38.2 and 5.4—31 µg/L, respectively, with mean times to the highest THCCOOH concentration (tmax) of 99.9 and 85.9 h, respectively. The mean cmax for the 7.5 mg/day dose (145.7 µg/L) was larger than that of the 14.8 mg/day dose (116.0 µg/L). The highest THCCOOH concentration was 436 µg/L, after a daily dose of 7.5 mg, and the lowest cmax at the 14.8 mg/day dose was 19.0 µg/L. The mean tmax values were 52.1—119 h for the 7.5 mg/day dose and 46.0—157.4 h for the 14.8 mg/day dose.
immunoassay performance characteristics
Immunoassay semiquantitative and GC/MS quantitative results (n = 4381) were used to calculate TP, FP, FN, and TN values. These data determined the sensitivity, specificity, and efficiency of each immunoassay (Table 5 ). The two high doses produced the majority of TP specimens. There were 528 TP specimens with the Emit II assay and slightly fewer (n = 517) with the DRI assay. The CEDIA assay identified far fewer TP specimens (n = 397), but had a higher number of TN results (n = 3716). The Emit II and DRI assays had 3526 and 3658 TN specimens, respectively.
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Table 5. Cannabinoid immunoassay performance at 50 µg/L cutoff.1
The Emit II assay provided the highest mean sensitivity of 84%, with the DRI and CEDIA assays having values of 82.5% and 62%, respectively. Mean specificity values were all >90%; the CEDIA assay was the most specific at 99.3%, followed by the DRI assay at 97.8% and the Emit II at 94.7%. The three assays also produced mean efficiency values >90%, in part because of the high number of TN values. The DRI assay had the highest overall efficiency at 95.2%, with the CEDIA and Emit II assays having efficiencies of 93.9% and 92.8%, respectively.
tp detection rates
The detection rate data for TP specimens, those specimens positive at a 50 µg/L immunoassay cutoff and a THCCOOH concentration at or above 15 µg/L by GC/MS, are shown in Table 6 . The mean TP detection rates for the Emit II and DRI assays at the lowest dose, 0.39 mg/day, were 0.2% and 0.1%, respectively. One participant had 1.6% and 0.8% TP urine specimens with the Emit II and DRI assays, respectively. Three of the remaining six participants had urine samples with THCCOOH concentrations at or above the GC/MS cutoff, but none was positive by immunoassay. The 0.47 mg/day dose produced TP in two participants, but only with the Emit II assay. TP detection rates for these two participants were 1.9% and 1.0%, for a mean TP detection rate of 0.4%. Administration of the two low doses produced no TP tests with the CEDIA assay. Mean detection rates for the 7.5 mg/day dose were 35.9%, 34.8%, and 29.0%, respectively, for the Emit II, DRI, and CEDIA assays. The 14.8 mg/day dose had lower mean detection rates than the 7.5 mg/day dose: rates for the Emit II, DRI, and CEDIA immunoassays were 28.1%, 27.9%, and 22.5%, respectively.
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Table 6. TP1 detection rate data.
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Discussion
TOP
Abstract
Introduction
Materials and Methods
Results
Discussion
References
There continues to be potential for oral cannabis abuse and an increasing likelihood of THC ingestion from medications or food products that may produce positive urine drug tests for cannabinoids. The need for deterring and detecting cannabis abuse continues to be an important component in drug treatment and medical screening, along with judicial, workplace, and military forensic urine drug-testing programs. This study investigated excretion pharmacokinetics after administration of oral THC. This included evaluating the extent to which ingestion of hemp oil and dronabinol could produce a positive urine test for cannabinoids. In the course of the investigation, we evaluated the performances of three commercial cannabinoid immunoassays. This study's data indicate that it is possible but unlikely that a person would test positive for urinary cannabinoids after ingestion of hemp oil. This is valid as long as mandated cannabinoid screening and confirmation cutoffs are used, the THC concentrations in hemp oils remain at their present low values, and individuals take hemp oil according to manufacturers' recommendations.
There are many relevant pharmacologic factors, such as dose, route, and vehicle, and physiologic factors, such as metabolism and excretion, that influence drug concentrations in the circulation. Pharmacologic factors have a significant influence on the amount of drug absorbed into the bloodstream. Perez-Reyes et al. (7) described the efficacy of five different vehicles used in the administration of 35 mg of THC containing 50 µCi of tritium-labeled THC in gelatin capsules. Glycocholate produced the highest THC plasma values, followed by sesame oil. However, there was considerable variability in peak concentration and rate of absorption, even when drug was administered in the same vehicle.
Oral THC bioavailability was estimated to be 10—20% by Wall et al. (8). In their study, participants were dosed with either 15 mg (women) or 20 mg (men) of THC, a percentage of which was radiolabeled, dissolved in sesame oil and contained in gelatin capsules. One drawback of studies with radiolabeled THC is the inability to differentiate labeled THC in plasma from its labeled metabolites, which leads to overestimation of drug concentrations. Possibly a more accurate assessment of oral bioavailability, which used GC/MS to quantify THC in plasma samples, was reported by Ohlsson et al. (25). They estimated a 6% bioavailability after a 20-mg THC dose ingested in a cookie. Slow rates of absorption and low THC concentrations were observed. Low oral THC bioavailability may be attributable to poor absorption, degradation by stomach acid, and/or biotransformation to metabolites during first passage through the liver.
Unlike the inhalation route, THC absorbed from the gastrointestinal system has considerable first-pass metabolism in the liver. Cytochrome P450 enzymes, primarily CYP3A4 and CYP2C9 and -11 (26), oxidize THC into numerous metabolites. The C-11 allylic methyl group may be oxidized to 11-hydroxy-tetrahydrocannabinol (11-OH-THC), a pharmacologically active metabolite. The plasma concentration of 11-OH-THC is ~10% of that of the parent drug after smoking or intravenous infusion of THC, whereas the relative 11-OH-THC plasma concentration increases to 50—100% after oral administration (8). Similar physiologic and pharmacodynamic effects, relative to smoked marijuana, have been observed after ingestion of THC, although THC plasma concentrations were lower after oral ingestion (25). An increase in the equipotent metabolite concentration is a possible explanation for comparable effects. 11-OH-THC may undergo further oxidation to THCCOOH, thereby inactivating the drug before it reaches the heart for systemic distribution. Most of the THCCOOH is rapidly conjugated to a water-soluble glucuronic acid. This glucuronide metabolite is the primary urinary metabolite of THC (27). Immunoassays for the detection of cannabinoids are designed to be specific for THCCOOH with less cross-reactivity to other cannabinoid metabolites (28)(29). After hydrolysis and extraction, THCCOOH is the primary urinary cannabinoid analyte quantified by GC/MS.
The individual TP detection rate data (Table 6Up ) provide useful examples of variability after THC ingestion. The detection rates for participant G were approximately five times those of participant N. Variability in THC concentrations found in smoking studies have been attributed to smoking topography variables such as number of puffs, depth of inhalation, and hold time; therefore, an experienced smoker may attempt to control the degree of pharmacologic effects by titrating the amount of drug absorbed. No such control is available via the oral route, but the variability persists. Other factors that may play a part are age, disease state, body weight, and metabolic differences. One variable that is unique to the oral route and that could affect drug absorption is co-ingestion of food. During this study, participants were allowed unrestricted access to food and drink. In addition, doses were given with meals to simulate common usage conditions. It is possible that controlling dietary intake could have reduced interindividual variability in urinary cannabinoid concentrations.
At present, many commercial cannabinoid immunoassay systems are designed with a 50 µg/L cutoff. In 1994, the US Department of Health and Human Services lowered the required initial immunoassay test cutoff from 100 µg/L to its present value. This brought the mandated cutoff in line with the cutoff established by the Department of Defense in 1992. Administrative cutoffs are established after taking into account many factors, such as goals of the drug-testing program, capabilities of the analytical methods, data from pharmacologic studies, and desired detection time windows. Detection rate data from the present study demonstrate that it is unlikely that a urinary cannabinoid immunoassay will be positive at the 50 µg/L cutoff because of consumption of hemp oils with lower THC concentrations. One participant of seven had two positive samples after the 0.39 mg/day dose by the Emit II assay and one positive by the DRI assay. The time to first positive was >100 h, after the individual had received 15 doses over 5 days. Three participants had positive specimens at the 0.47 mg/day dose, with the first positive samples occurring between 30 and 87 h.
The two higher doses, 7.5 and 14.8 mg/day, produced numerous positive specimens. At the 7.5 mg/day dronabinol dose, one participant produced a specimen positive by all three immunoassays within 2 h of the first dose, which was the first void after dosing. The longest time to first positive at this dose was 52.8 h by the CEDIA assay and 10.6 h by the Emit II assay. The mean time to first negative specimen was shorter for the 14.8 mg daily dose than for the dronabinol dose. Time to first negative for the dronabinol dose was 15.1 (CEDIA) to 83.9 h (Emit II), whereas the range for the higher dose was 2.6 (CEDIA) to 53.9 h (Emit II). As in previous studies (5)(11)(30), the last positive specimen did not necessarily occur before the first negative. In our study, at the 7.5 mg/day dose, all participants had at least one negative sample before the last positive; only one individual had no positive samples after the first negative sample at the 14.8 mg/day dose. Fluctuation of urinary THC concentrations at or around the cutoff may be highly dependent on the individual's state of hydration. These data also demonstrate that an individual may absorb enough drug from hemp oils containing high THC concentrations to produce a positive sample by the first urine void.
There are commercial cannabinoid immunoassays designed with cutoffs of 20 and 100 µg/L. The 20 µg/L cutoff is used in workplace settings, such as nuclear power plants, where higher sensitivity is desired, yielding a broader detection window. The 100 µg/L cutoff continues to be used in some workplace testing programs, possibly because of contractual obligations. This study found higher detection rates and larger detection time windows with the 20 µg/L cutoff, similar to other published results (30)(31)(32). At this lower cutoff, three participants produced positive specimens with the 0.39 mg/day dose and all but one of seven participants had positive samples at the 0.47 mg/day dose. As expected, increasing the cutoff to 100 µg/L decreased detection rates considerably with no FP specimens at the lower dosing concentrations. These results demonstrate that detection rates and times can vary dramatically depending on choice of immunoassay and cutoff value.
All three immunoassays in this study provided acceptable performance at the 50 µg/L screening cutoff. Overall efficiencies were >90%, with the DRI system having the highest efficiency of 95.2%. The objective of the initial screening test is to identify TP specimens with a minimum number of unconfirmed positive results. To accomplish this task, high immunoassay sensitivity must be balanced with high specificity to reduce the number of specimens that require the expensive and time-consuming GC/MS confirmation. The results from this study indicate a difference in sensitivity between the immunoassays, with the Emit II being the most sensitive (528 TP results) and the CEDIA the least sensitive (397 TP results). The Emit II produced the fewest number of TN results, reflecting its higher sensitivity but lower specificity. Conversely, the CEDIA produced the largest number of TN specimens, providing the highest specificity. The results for the DRI immunoassay were consistently between those of the Emit II and CEDIA systems, indicating a more efficient balance between specificity and sensitivity. Huestis et al. (30) reported similar performance variability among six cannabinoid immunoassays.
To establish a standard for comparing immunoassay performance and to have quantifiable data to compare urinary excretion after oral THC, all urine specimens were analyzed by GC/MS to determine THCCOOH concentration. All doses, with the exception of placebo, produced specimens with THCCOOH >15 µg/L. Interestingly, the 7.5- and 14.8-mg daily doses had nearly equal numbers of specimens above the 15 µg/L GC/MS cutoff despite an almost twofold difference in dose. The most striking disparity between the two doses is in the cmax results. Although statistically insignificant (Student t-test, P >0.05), the mean cmax of 145.7 µg/L for the 7.5 mg/day dose and 116.0 µg/L for the 14.8 mg/day dose yields an inverse dose response. The mean tmax for all doses was consistent, ranging between 85.9 and 103.5 h. These times indicate that the highest urinary concentrations occurred after 12—15 THC doses on days 4—5 of treatment. With the last positive specimen times exceeding 72 h, these data demonstrate that multiple doses of hemp oils with high THC concentrations may produce GC/MS-positive specimens as long as 3 days after last ingestion.
One participant had a quantifiable THCCOOH specimen at the beginning of the placebo dose, immediately after the initial washout phase. The 3.5 µg/L THCCOOH urine concentration was therefore a result of cannabis use before ward admittance. Before starting the first dosing session, a participant's urine needed only to have been negative by immunoassay at a 10 µg/L cutoff.
Previous studies on the occurrence of positive urine samples after hemp oil ingestion examined results after a single dose or a single dose per day for several days (16)(17). The two most recent reports on hemp oil ingestion and urine cannabinoid concentration were not controlled clinical studies (14)(19). In a report by Leson et al. (19), 15 individuals self-administered single daily doses of liquid hemp oil with concentrations ranging from 0.09 to 0.6 mg of THC over a 10-day period. A total of 11 urine specimens were collected from each individual over a 40-day period, with the first morning void specifically not collected. Urine samples were tested by a RIA for cannabinoids (Immunalysis Direct RIA Kit). None of the participants ingesting daily THC doses between 0.09 and 0.45 mg produced positive urine samples at the 50 µg/L cutoff. One specimen, from one of three participants ingesting the 0.6-mg THC daily dose, did screen positive by immunoassay, but GC/MS analysis quantified the THCCOOH concentration at 3.0 µg/L. The authors also reported a cmax of 5.2 µg/L in a specimen after the high dose, which did not screen positive. In the present study, every urine sample was collected to allow for a full evaluation of the potential for positive urine samples. Many of the cmax specimens (15 of 28) occurred in the early morning hours, times not included in the previous study.
Bosy and Cole (14) reported urine cannabinoid results after a daily 15-g hemp oil dose for 7 days. Six hemp oils with THC concentrations ranging from 11.5 to 117.5 µg/g were evaluated, each in a single participant. Urine samples were collected just before dosing and at 6 h post-ingestion. Fluorescence polarization immunoassay and Kinetic Interactions of Microparticles in Solution (KIMS) immunoassay systems with 50 µg/L cutoffs were used to test specimens. Participants ingesting low-dose hemp oils (97.2 and 172.5 µg of THC) had immunoassay results well below the cutoff. Positive specimens occurred by the third and fourth days after medium doses of THC in hemp oil (540 and 546 µg of THC); however, specimens tested negative within 24 h after ingestion ceased. The high-dose hemp oil (1762.5 µg of THC), ingested by one participant, produced a positive urine test on the first day of dosing and tested negative within 72 h of last ingestion. The investigators also reported a peak THCCOOH concentration of 48.7 µg/L after the 1762.5 µg/day dose. The results from the studies by Leson et al. (19) and Bosy and Cole (14) are in general agreement with the data presented here, especially with results from our low doses. The 0.39 and 0.47 mg/day doses produced GC/MS-positive specimens in four and two of seven participants, respectively. The respective cmax means were 19.8 and 12.2 µg/L. These data show that even low doses of THC can produce urine specimens with THCCOOH concentrations >15 µg/L; however, multiple doses appear to be required.
The only previously reported study on oral THC in which all urine specimens were collected for an extended period of time after dosing was conducted by Cone et al. (11). Participants consumed brownies containing marijuana plant material; each brownie contained 22.4 mg of THC, similar to the amount found in a 2.8% THC cigarette. Mean urinary cmax values of 180 and 312 µg/L occurred after single THC doses of 22.4 mg and 44.8 mg, respectively, with individual cmax values of 177—436 µg/L after the higher dose. In a study by Law et al. (9), five individuals ingested meat sandwiches containing cannabis resin with 5 and 20 mg of THC. Urine samples were collected at timed intervals for up to 12 h after consumption and daily thereafter for up to 14 days. After the administration of 20 mg of THC, total urinary cannabinoid concentrations, measured by combined HPLC/RIA analysis, were 185—1063 µg/L 6 h after ingestion. Our study also demonstrate wide ranges in cmax values with the dronabinol dose, yielding a 20-fold difference between the lowest and highest cmax.
Dronabinol is synthetic THC dissolved in sesame oil and delivered in a gelatin capsule. It has been cleared by the Food and Drug Administration for the symptomatic treatment of nausea and vomiting for cancer patients receiving chemotherapy and as an appetite stimulant for chronically ill patients with wasting syndrome (22). Natural and synthetic THC are chemically identical, undergo similar metabolism, and produce identical metabolites. Studies have been conducted to discover a biomarker to differentiate the occurrence of THCCOOH in the urine from cannabis abuse and from the ingestion of therapeutic THC (33)(34). To date, no definitive analyte has been identified.
Interest in the therapeutic use of cannabinoids has increased dramatically in the past decade, but little research has focused on the excretion pharmacokinetics of oral THC. ElSohly et al. (33) administered a single 15-mg dronabinol dose to four individuals. Every urine specimen was collected for 24 h, and then specimens were collected once a day for 6 days. The cmax values for three of the participants were 188.7—227 µg/L. These results are within the cmax values found with the 14.8 mg/day dose in the present study. Although there have been THC ingestion studies comparing different THC vehicles, with the exception of our study, there have been no studies on the use of different formulations, such as capsule vs liquid. THC delivered in a capsule as opposed to a liquid may allow greater absorption from the gastrointestinal system. Possibly the capsule provides protection against degradation by stomach acids and delivers a large proportion of THC intact to the intestinal tract for absorption.
Oral abuse of cannabis undoubtedly will continue, and the use of orally administered therapeutic cannabinoids probably will increase. Research is needed to determine how synthetic and naturally occurring cannabinoids, administered via different routes, will affect urine drug-testing programs. We have demonstrated that it is possible but unlikely for a urine specimen to test positive at the federally mandated cutoffs for screening and confirmation after hemp oil ingestion, given the lower THC concentrations currently found in hemp oils and the recommendations of manufacturers for once-a-day dosing. In addition, we have provided cmax and tmax values, detection rates and times, and performance data on the three most commonly used cannabinoid immunoassays. Knowledge of an assay's sensitivity, specificity, and efficiency allows the administrators of drug-testing programs to select the most appropriate immunoassay for their program's goals.
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Acknowledgments
We thank David Darwin, Debbie Price, and the clinical staff of the NIDA IRP Research Unit for technical assistance. We are also grateful to Carole Trojan, Erick Fitzer, Daniel Nichols, and the Navy Drug Screening Laboratory Jacksonville's Screening and Confirmation staff for analytical assistance in this project. We would also like to thank the Research Triangle Institute for GC/MS analyses of the hemp oil and the Department of Defense, Department of Transportation, Substance Abuse and Mental Health Services Administration, and NIDA IRP for funding this project.
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Footnotes
1 Nonstandard abbreviations: THC, {Delta}9-tetrahydrocannabinol; THCCOOH, 11-nor-9-carboxy-{Delta}9-tetrahydrocannabinol; GC/MS, gas chromatography—mass spectrometry; IRP, Intramural Research Program; NIDA, National Institute on Drug Abuse; TP, true positive (number of specimens positive by immunoassay and GC/MS analysis); FP, false positive (number of specimens positive by immunoassay and negative by GC/MS analysis); FN, false negative (number of specimens negative by immunoassay and positive by GC/MS analysis); TN, true negative (number of specimens negative by immunoassay and GC/MS analysis); cmax, highest concentration of drug; tmax, time from first dose to highest concentration of drug; and 11-OH-THC, 11-hydroxy-{Delta}9-tetrahydrocannabinol.
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References
TOP
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Source: Urinary Cannabinoid Detection Times after Controlled Oral Administration of {Delta}9-Tetrahydrocannabinol to Humans
