Ag-Tetrahydrocannabivarin as a Marker for the Ingestion, of Marijuana versus Mannol : Results of a Clinical Study

Transcription

1 I I Ag-Tetrahydrocannabivarin as a Marker for the Ingestion, of Marijuana versus Mannol : Results of a Clinical Study Mahmoud A. EISohly I, Harriet dewit 2, Stephen R. Wachtel 2, Shixia Feng 1, and Timothy P. Murphy 1 7 EISohly Laboratories, Incorporated, 5 Industrial Park Drive, Oxford, Mississippi and 2Department of Psychiatry, The University of Chicago, MC 3077, South Maryland Avenue, Chicago, Illinois I Abstract Ag-Tetrahydrocannabinol (THC), the main psychologically active ingredient of the cannabis plant (marijuana), has been prepared synthetically and used as the bulk active ingredient of Marinol, which was approved by the FDA for the control of nausea and vomiting in cancer patients receiving chemotherapy and as an appetite stimulant for AIDS patients. Because the natural and the synthetic THC are identical in all respects, it is impossible to determine the source of the urinary metaholite of THC, 11-nor-A% tetrahydrocannabinol.9.carboxylic acid (THC-COOH), in a urine specimen provided in a drug-testing program. Over the last few years there has been a need to determine whether a marijuana positive drug test is the result of the ingestion of marijuana (or a related product) or whether it results from the sole use of Marinol. We have previously proposed the use of Ag-tetrahydrocannabivarin (THCV, the C3 homologue of THC) as a marker for the ingestion of marijuana (or a related product) because THCV is a natural component of most cannabis products along with THC and does not exist in Marinol. We have also reported that THCV is metabolized by human hepatocytes to 11-nor-Ag-tetrahydrocannabivarln-9- carboxylic acid (THCV.COOH); therefore, the presence of the latter in a urine specimen would indicate that the donor must have used marijuana or a related product (with or without Marino]). in this study, we provide clinical data showing that THCV-COOH is detected in urine specimens collected from human subjects only after the ingestion of marijuana and not after the ingestion of Marinol (whether the latter is ingested orally or by smoking). Four subjects (male and female) participated in the study in a threesession, within-subject, crossover design. The sessions were conducted at one.week intervals. Each subject received, in separate sessions and in randomized order, an oral dose of Marinol (15 rag), a smoked dose of THC (16.88 mg) in a placebo marijuana cigarette, or a smoked dose of marijuana (2.11% THC and 0.12% THCV). Urine samples were collected and vital signs were monitored every 2 h for a 6-h period following drug administration. Subjects were then transported home, were given sample collection containers and logbooks, and were instructed to record at home the volume and time of every urine collection for 24 h, and once a day for the remainder of a week (6 days). Subjects were also instructed to freeze the urine samples until the next session. All urine samples were analyzed by GC-MS for THC.COOH and THCV-COOH using solid-phase extraction and derivatization procedure on RapidTrace and TBDMS as the derivative. The method had a limit of detection of 1.0 ng/ml and 1.0 ng/ml for THCV-COOH and THC-COOH, respectively. Introduction Ag-Tetrahydrocannabinoi (THC), the main psychologically active ingredient of the cannabis plant (marijuana) has been prepared synthetically and used as the bulk active ingredient of Marinol, which was approved by the FDA for the control of nausea and vomiting in cancer patients receiving chemotherapy and as an appetite stimulant for AIDS patients. Because the natural and the synthetic THC are identical in all respects, it is impossible to determine the source of the urinary metabolite of THC, 11-nor- Ag-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), in a urine specimen provided in a drug-testing program. Over the last few years there has been a need to determine whether a marijuana-positive drug test is the result of the ingestion of marijuana (or a related cannabis product) or whether it is the result of the sole use of Mafinol. Because marijuana and other cannabis-related products do contain other cannabinoids along with THC, it was thought that ingestion of any of these natural materials would result in the presence of other cannabinoid metabolites with THC metabolites. One such cannabinoid that is very similar in structure to THC and occurs in most drug-type cannabis products, albeit to a much lower extent than THC, is Ag-tetrahydrocannabivarin (THCV) (1-3). We have previously proposed the use of THCV (the C3 homologue of THC) as a marker for the ingestion of marijuana (or a related product) because THCV does not exist in Marinol (4). We have also reported that THCV is metabolized by human hepatocytes to THCV-COOH, the C3 homologue of THC-COOH; there- Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission, 565

2 Journal of Analytical ToxicoJogy, Vol. 25, October 2001 fore, the presence of THCV-COOH in a urine specimen would indicate that the donor must have used marijuana or a related product with or without Marinol (4). THCV-COOH was synthesized in our laboratory (EISohly Laboratories, Oxford, MS), and its chemical structure was determined by spectroscopic means and by comparison to its Cs homologue (THC-COOH) (5). In this study, we provide clinical data showing that THCV-COOH is detected in urine specimens collected from human subjects only after the ingestion of marijuana and not after the ingestion of Marinol (whether the latter is ingested orally or by smoking). Materials and Methods Drugs Marinol capsules (Roxane Laboratories, Inc., Columbus, OH) were administered in opaque, hard gelatin capsules (size 00) with dextrose as filler. THC cigarettes were prepared by lacing placebo marijuana cigarettes (obtained from the National Institute on Drug Abuse [NIDA]) with 16.9 mg of synthetic THC (dronabinol) by injecting the dronabinol dissolved in ethanol along the length of the cigarette. Regular marijuana cigarettes were obtained from the NIDA supply and were analyzed to contain 2.11% THC and 0.12% THCV. Each cigarette was rolled in an additional piece of opaque purple cigarette paper with a grape aroma to partially blind the subjects to the drug condition. Table I. Drug Codes* Subject Code Subject 1 (SP) VA Smoked marijuana Oral Marinol Smoked THC Subject 2 (WB) VC Smoked THC Oral Marinol Smoked marijuana Subjects Participants were male and female marijuana users (N = 4) recruited from the surrounding community through posters and newspaper advertisements. Initial inclusion criteria included use of marijuana/hashish for at least i year, use of marijuana/hashish at least 10 times in their lifetime and use of marijuana/hashish in the prior 2 months, at least a high school education, native English speaker, and a body mass index in the range of kg/m 2. Qualified individuals completed questionnaires regarding their health and psychiatric symptomatology (6) and underwent a psychiatric interview (7), an electrocardiogram, and a physical examination. Three females and one male participated. They were aged between 19 and 27 years; two were Caucasian, one was African American, and one was Asian. They weighed between 120 and 160 lbs. They consumed 2-6 alcohol drinks per week and 3-20 caffeinated beverages. None were cigarette smokers. The study was approved by the Institutional Review Board of The University of Chicago. Before participating, subjects attended an orientation session to obtain written informed consent and to review the experimental procedures and dependent measures. The consent form stated that the purpose of the experiment was to investigate the breakdown products of cannabinoids (marljuana) that can be measured in urine. The consent form stated that the subjects would either receive a capsule that contains Marinol (an FDA-approved cannabinoid) or smoke a cigarette that contains either marijuana or THC alone. The consent form listed side effects that these drugs could induce. Subjects were instructed not to smoke marijuana for one week prior to and following each session and to refrain from other drug use for 24 h prior to each session. Subjects were instructed to minimize their consumption of caffeinated beverages and nicotine for 24 h prior to each session. After completing the study, subjects were debriefed and paid for their participation. Subject 3 (DFL) VD Oral Marinol Smoked THC Smoked marijuana Subject 4 (AB) VE Smoked THC Oral Marinol Smoked marijuana * THCV protocol no Design The study utilized a three-session, within-subject, crossover design. The sessions were conducted at one-week intervals. Each subject received, on separate sessions and in randomized Table II. GC-MS Analysis of THC-COOH and THCV-COOH in Urine Validation Data Ions monitored (m/z) R t (quant. ion LOD LOQ Compound (rain)* underlined) Ion ratios (ng/ml) (ng/ml) THC-COOH , / , /515 = 0.29 THC-COOH-d , /521 = 1.13, N/A N/A 578/521 = 0.33 THCV-COOH , 4_8_Z, /487 = 0.90, /487 = 0.3O THCV-COOH-d , /493 = 0.90, N/A N/A 550/493 = 0.29 tinearity range (ng/ml) N/A N/A Precision (% CV, n = 6) 19 ng/ml N/A 6 ng/ml NIA * DB-I column (I0 m x 0.18-ram i.d. with 0.4-pro film thickness), operated at 200~ (0.5 rain) to 280~ at 30~ and held for 8.83 rain. 566

3 order, either an oral dose of Marinol (15 mg), a smoked dose of THC (16.98 rag) in a placebo marijuana cigarette, or a smoked dose of marijuana (2.11% THC and 0.12% THCV/800 mg cigarette = mg THC and 0.96 mg THCV). Procedure Experimental sessions were conducted in the Human Behavioral Pharmacology Laboratory at The University of Chicago Hospitals. Upon arrival for each session, a technician assessed baseline vital signs using a Digital Blood Pressure Table III. GC-MS Analysis of Specimens Collected from Subject I (SP) Total THCV-COOH THC-COOH Sample Date Time volume (ml) (ng/ml) (ng/ml) (Subject smoked one marijuana cigarette containing 2.11% THC and 0.12% THCV) I 7/21/00 9:20 am /21/00 11:00 am /00 1:00 pm :30 pm :00 pm /00 11:00 pm /22/00 7:00 pm :00 pm :00 pm I :00 pm /00 7:00 pm :00 pm (Subject administered one 15-mE dose of Marinol) /11/ /11/ /11/ /11/00 35* 8/ /11 O0 29 8/12/ /13/ /4/00 9:40 am 8/4/00 11:10 am 8/4/00 1:15 )m 8/4/00 3:40 )m 8/4/00 9:00 )m 8/5/00 7:00 ]m :00 )m :00 )m :00 )m :00 )m O II :05 am 2:00 ~m 2:40 am 3:15 am 5:00 am 9:00 am * Specimen numbered out of sequence Monitor HEM-706 (Omron Healthcare, Inc., Vernon Hills, IL), and subjects provided a baseline urine sample that was also used for drug and pregnancy screening. Breath-alcohol level was determined using an Alco-Sensor III hand-held breathalyzer (Intoximeters, Inc., St. Louis, MO) to verify that each subject was ethanol-bee. Following these baseline measures subjects either ingested a capsule containing Marinol with 100 ml of water or smoked a marijuana cigarette using a paced puff procedure. Urine samples were collected and vital signs were monitored every 2 h for a 6-h period following drug administration, and were then transported home. Subjects were given sample collection containers and a logbook and were instructed to record the volume and time of each urine collection for 24 h, and once a day for the remainder of a week (6 days) at home. Subjects were instructed to freeze all the urine samples until their next session. Urine analysis All urine specimens collected in this study were analyzed by gas chromatography-mass spectrometry (GC-MS) for THC-COOH and THCV-COOH following the procedure developed herein and described. Reagents and standards 11-nor-Ag-THC-9-COOH (100 pg/ml methanol) and ll-nor-ag-thcv-9-cooh (100 pg/ml methanol) were obtained from E1Sohly Laboratories, Inc. and diluted with methanol to working solutions of 2 IJg/mL each. THC-COOH-d6 (100 IJg/mL methanol) and THCV-COOH-d6 (100 pg/ml methanol) were obtained from EISohly Laboratories, Inc. and diluted with methanol to working solutions of 5 mg/ijl and 1.5 ljg/ml, respectively. The TBDMS silylating reagent N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide with 1% TBDMSCI (97%, # ) was obtained from Sigma-Aldrich (St. Louis, MO). All solvents were ACS grade. Instrumentation A Hewlett-Packard (HP, Palo Alto, CA) 5890A GC interfaced with an HP 5970A mass selective detector and coupled with a 7673 autosampler was used for sample analyses. The GC-MS was fitted with a 10-m DB-1 column (one half of a J&W Scientific, Folsom, CA, 20-m x 0.18-ram i.d., 0.4- pm film thickness column). The injector was operated in the splitless mode. The GC operating conditions were as follows: initial temperature of 200~ (0.50 rain), raised to 280~ at 30~ and held for 8.83 min. Selected ion monitoring mode (SIM) was used to acquire ions at m/z 413, 515, and 572 for THC-COOH and m/z 419, 521, and 578 for THC-COOH-d6 as well as m/z 487, 385, and 544 for THCV-COOH and m/z 493, 391, and 550 for THCV-COOH-d6. The electron multiplier 567

4 voltage was set to 200 V above the standard autotune value. The carrier gas was helium at a linear velocity of 34 cm/s. Extraction All specimens were extracted in batches containing an unextracted standard, a three-point calibration curve, a negative control, and a positive control (18.2 ng/ml THC-COOH and 10 Table IV. GC-MS Analysis of Specimens Collected from Subject 2 (WB) Total THCVoCOOH THC-COOH Sample Date Time volume (ml) (ng/ml) (ng/ml) 36 8/22/00 t:45 pm /22/00 3:15 pm /22/00 4:30 pm /22/00 6:45 pm /22/00 7:45 pm /23/00 5:25 am /23/00 9:00 am /23/00 11:45 am /23/00 12:20 pm /24/00 12:15 pm /25/00 7:30 pm /26/00 8:10 pm /27/00 1:30 pm /28/00 1:20 pm (Subject administered one 15-mg dose of Marinol) 50 8/30/00 9:00 am /30/00 9:40 am /30/00 10:20 am /30/00 11:30 am /30/00 11:45 am /30/00 2:25 pm /31/00 6:15 am /31/00 9:15 am /31/00 12:15 pm /1/00 1:00 pm /2/00 l:00 pm /3/00 1:15 prn /4/00 12:20 pm (Subject smoked one marijuana cigarette containing 2.11% THe and 0.12% THCV) 63 9/7/00 12:30 pm /7/00 1:30 pm /7/00 2:03 pm /7/00 4:00 pm /7/00 6:30 pm /8/ am /8/00 8:15 am /8/00 9:40 am 170 7, /8/00 12:30 pm /9/00 11:00 pm /10/00 11:59 pm /I 1/00 12:20 pm /12/00 12:50 pm /13/00 11:45 am ng/ml THCV-COOH). The equivalent of 50 ng/ml of THC- COOH-d6 (401JL of the 5 I~g/mL THC-9-COOH-d 6 working solution) and 15 ng/ml of THCV-COOH-d6 (40 IJL of the 1.5 IJg/mL THCV-COOH-d6 working solution) were added to 4-mL aliquots of all samples in the batch. The three calibrators were spiked with 11-nor-bg-THC-9-COOH at 6 ng/ml, 15 ng/ml, and 50 ng/ml (12 IJL, 30 IJL, and 50 1JL of the 2 tjg/ml THC-COOH working solution, respectively) and 11-nor-b9-THCV-9 - COOH at 2, 6, and 15 ng/ml (4 1JL, 12 IJL, and 30 IJL of the 2 IJg/mL THCV-COOH working solution, respectively). After spiking, all samples were vortex mixed for 30 s. Samples were hydrolyzed with 0.5 ml of I0N KOH at room temperature for 15 min, then acidified with 0.55 ml concentrated HCI to ph 1. Samples were extracted on a Zymark (Hopkinton, MA) RapidTrace workstation through CleanScreen 200-rag extraction columns (CS THC020 United Chemical Technologies, Inc., Bristol, PA, Part CSTHC203). The extraction process was the same as outlined in the CleanScreen manual on Automated Methods, Zymark RapidTrace. The ehates were evaporated to dryness under nitrogen at 50~ Derivatization To the residue of the extracted samples TBDMS (50 IJL) was added. The samples were capped and placed in an oven at 70~ for 15 rain. After the samples had cooled to room temperature, 50 tjl of ethyl acetate was added. The samples were vortex mixed and transferred into inserts of properly labeled GC vials. tinearity, limit of detection (LOD), limit of quantitation (LOQ), and precision Certified blank urine was combined with THC- COOH (2 IJg/mL) and THCV-COOH (2 tjg/ml) to prepare extended points (samples) at levels above and below the multipoint calibration curves (6, 15, and 50 ng/ml for THC-COOH, and 2, 6, and 15 ng/ml for THCV-COOH). The THC-COOH samples ranged from 0.5 ng/ml to 1000 ng/ml and contained 50 ng/ml of the internal standard (THC-COOH-d6). The THCV-COOH samples ranged from 0.5 ng/ml to 1000 ng/ml and conrained 50 ng/ml of the internal standard (THCV- COOH-d6). Included in each batch was an unextracted standard, a multi-point calibration curve, a negative control and a positive control. The LOD was defined as the lowest concentration of THC-COOH or THCV-COOH where the identity of the drug was established (ion ratios within 20% of the calibration curve averaged ion ratios as well as the retention time and relative retention time within + 2% and + 1%, respectively, of the 15-ng/mL calibrator retention time and relative retention time) regardless of the accuracy of quantitation. The LOQ was defined as the lowest 568

5 concentration of THC-COOH or THCV-COOH where the identity of the drug was established and the quantitation was within Table V. GC-MS Analysis of Specimens Collected from Subject 3 (DFL) 20% of the nominal concentration. Precision was calculated as the coefficient of variance (%) for the analysis of multiple samples (n --- 6) for THCV-COOH at 6 ng/ml and THC- COOH at 19 ng/ml. Total THCV-COOH THC-COOH Sample Date Time volume (ml) (ng/ml) (rig/rot) (Subject administered one 15-rag dose of Marinol) 78 8/17/00 11:00 am 1 O /17/00 11:20 am :45 am /17/00 1:10 pm /17/00 3:00 pm :30 pm :30 pm :50 pm :45 am I :45 am :45 pm :30 pm :45 pm /21/00 8:45 pm :00 pm :00 pm I /24/00 11:lOam /24/00 12:30 ~m /24/00 1:20 ~m 1 O /24/00 1:50 )m /24/00 3:50 )m /24/00 5:15 )m :00 ~m /24/00 11:40 ~m /25/00 9:00 am /25/oo 10:00 am O4 8/25/00 6:00 pm /26/00 4:00 pm /27/00 8:00 pm /28/00 5:00 pm /29/00 4:00 pm / :30 pm I (Subject smoked one marijuana cigarette containing 2.11% THC and 0.12% THCV) II :30 am III :15 am :40 am :50 pm :30 pm :00 pm :30 pm :00 am :00 pm /27/00 11:00 pm /28//00 11:30 pm /29/00 11:00 pm :00 pm /1/00 9:30 pm Results and Discussion The question of whether the presence of THC- COOH in the urine of an individual could be explained on the basis of the sole use of Marinol, has been of interest to the forensic drug testing community. A reliable test to distinguish between marijuana (or other related cannabis products) use and the use of Marinol, was needed to support (or counter) the claim that the positive marijuana urine was the result of the use of the prescription drug Marinol. In a previous communication (4), we reported that the detection of THCV-COOH in urine along with THC-COOH would indicate the ingestion of a marijuana-related product with or without Marinol. This conclusion was based on the following facts: (1) THCV (the C3 homologue of THC) is a natural product that exists only in cannabis plants with THC; (2) Marinol is synthetic THC that does not contain THCV; (3) THCV is metabolized by human hepatocytes to its carboxylic acid metabolite, THCV-COOH, just like THC; (4) the derivative of the fragmentation of THCV-COOH was found to be identical to that of THC-COOH with the only difference in the masses of the fragments corresponding to the shorter side chain (28 ainu less), an observation previously reported by Foltz (8); (5) we have synthesized THCV-COOH, proved its identity, and confirmed its fragmentation, ion ratios, and retention times; and (6) we have shown that plasma samples collected in a clinical trail involving the oral ingestion of Marinol did not contain THCV-COOH and that only THC-COOH was detected. The purpose of this study was to carry out a clinical trial in which human subjects were to ingest oral Marinol, smoke THC spiked into placebo cigarettes, or smoke regular marijuana cigarettes which contained both THC and THCV, followed by collection of urine specimens after each session for GC-MS analysis. Four subjects (male and female) participated in the study in a three-session, within-subject crossover design at one-week intervals, which allowed sufficient time to reach baseline drug levels in urine between sessions. Approximately equal doses of THC equivalent were administered in each session (see Experimental section). Urine specimens were collected at Time 0 (prior to dosing) and at approximately 2-h intervals for 569

6 6 h. The subjects were then allowed to void at will, and samples were collected from each void for 24 h and then once per day for the rest of the week. All urine volumes were recorded, and all samples were frozen until analyzed by GC--MS for THC-COOH and THCV-COOH. The samples were submitted to the laboratory with code numbers only, and codes were not revealed until after the analyses were completed. Table I shows the drug codes for the four subjects delineating which drug was administered in each of the three weeks. The GC-MS procedure was based on solid-phase extraction using the Clean Screen Cartridges on the RapidTrace automated extraction system. The procedure was validated for both THC- COOH and THCV-COOH using the hexadeutero-derivatives of Table Vl. GC-MS Analysis of Specimens Collected from Subject 4 (AB) Total THCV.COOH THC-COOH Sample Date Time volume (ml) (ng/ml) (ng/ml) 124 9/25/O /25/ / / / /26/ /26/ /27/O /28/O /29/ /30/ /1/00 12:30 pm 2:30 pm 4:30 pm 6:30 pm 8:15 am 3:20 :~m 10:20 ~m 3:00 ~m 4:15 ~m 4:36 ~m 5:30 ~m 3:55 ~m (Subject administered one 15-mg dose of Marinol) , /2/00 1:00 pm /2/00 3:00 pm 70 0, /2/00 4:00 pm /2/00 7:00 prn /2/00 11:23 pm /3/00 5:00 pm 120 0, /4/00 6:20 pm /5/00 5:00 pm /6/00 5:30 pm /7/00 5:30 prn 60 0, /8/00 5:30 prn 120 0,0 (Subject smoked one marijuana cigarette containing 2.11% THC and 0.12% THCV) both compounds as internal standards and TBDMS as the derivative. Table II shows the details of the validation procedure with LOD, LOQ, linearity range, and precision. The results of the GC-MS analysis of the urine samples collected from the four subjects over the three-week period and following the three different dosing sessions are shown in Tables III-VI. The data show that, in all cases, THCV-COOH was detected only during the week after the session in which marijuana containing THCVwas smoked ( for Subject I and for Subjects 2-4). No THCV-COOH was detected in any of the urine specimens collected from any of the subjects post ingestion of Marinol, or smoking of THC spiked in placebo marijuana, This shows conclu- 0.0 sively that the presence of THCV-COOH in a urine sample is proof that the individual must have ingested marijuana (or a related cannabis product) which contains THCV along with THC. Further examination of the data shows that the concentration of THCV-COOH in the urine relative to the concentration of THCV in the 2.8 smoked marijuana is much higher than the 16.2 relative concentration of THC-COOH, in the 27.0 same urine samples, to the concentration of 21.9 THC in the smoked cigarette. This could be lo.6 explained on the basis of the difference in 13.4 polarity between THC and THCV, the latter 9.1 being more polar. This could result in either 6.0 higher degree of bioavailability of THCV rela- 2.8 tive to THC or that the excretion of THCV- 3.3 COOH in urine occurs to a higher degree than 1.8 THC-COOH or a combination of these two factors. It is also of interest to note that the urine levels of THC-COOH after oral ingestion of THC are much higher than those resulting from the smoking of equal THC doses. 3, , /10/00 3:00 pm / :00 pm , /10/00 7:00 pm , /10/00 9:00 pm /11/00 12:15 pm 120 9, /11/00 6:00 pm /12/00 6:30 pm , /13/00 6:15 pm /14/00 6:00 pm /15/00 6:45 pm , /16/00 6:30 pm Conclusions The results of this study lead to the following definitive conclusions: THCV-COOH is only excreted in human urine as a result of the ingestion of a product containing hg_ THCV; THCV-COOI-I is not excreted in human urine as the result of the sole ingestion of Marinol by either the oral route or by smoking; the presence of THCV-COOH in a urine specimen is a positive indication that the individual providing the specimen must have ingested a cannabis-related product; THCV appears to have a higher rate of bioavailability than THC and is also excreted faster and to a higher degree in urine; and the urinary levels of THC- COOH are much higher as the result of oral ingestion of THC than smoking an equal dose. 570

7 References I. R. Brenneisen and M. ElSohly. Chromatographic and spectroscopic profiles of cannabis of different origins: part I. J. Forensic Sci. 33: 1385-I 404 (I 988). 2. C.E. Turner, K. Hadley, and RS. Fetterman. Constituents of Cannabis sativa L. Vl. Propyl homologs in samples of known geographical origin. J. Pharm. $ci. 62:1739-I 741 (1973). 3. E.W. Gill. Propyl homologue of tetrahydrocannabinoh its isolation from cannabis, properties and synthesis. J. Chem. Soc. (C): (1976). 4. M.A. EISohly, S. Feng, T.P. Murphy, S.A. Ross, A. Nimrod, Z. Mehmedic, and N. Fortner. Ag-Tetrahydro-cannabivarin (69- THCV) as a marker for the ingestion of cannabis versus Marinol J. Anal. Toxicol. 23: (1999). 5. M.A. EISohly, S. Feng, T.P. Murphy, A.W. Warrington, S. Ross, A. Nimrod, Z. Mehmedic, and N. Fortner. Identification and quantitation of 11-nor-Ag-tetrahydrocannabivarin-9-carboxylic acid, a major metabolite of Ag-tetrahydrocannabavirin. J. Anal. Toxicol. 25: (2001). 6. L. Derogatis. SCL-90-R Manual II, Clinical Psychiatric Research, Diagnostic and Statistical Manual of Mental Disorders, 4th ed. American Psychiatric Association, Washington, D.C., R. Foltz. Analysis of urine specimens for cannabinoids to distinguish the ingestion of Marinol from smoked THC. Proceedings of the California Association of Toxicologists, November, 1996, pp