Extensive but unrealistic use of. Cannabio shampoo can cause drug-free hair to test positive for. CBD and CBN but not for the primary psychoactive drug THC.
Journalof AnalyticalToxicology,Vol. 23, September1999
Are CannabinoidsDetected in Hair After Washingwith Cannabio Shampoo? Vincent Cirimele, Pascal Kintz, Carole Jamey, and Bertrand Ludes Institut de M6decine L6gale, l I rue Humann, 67085 Strasbourg, France
Introduction
Abstract Today, cannabis plants are used in shampoo preparations, in foodstuffs (e.g., oils, noodles, crackers, etc.), and in beverages (e.g., tea). These products often contain < 1% Ag-tetrahydrocannabinol (THC) in order to eliminate psychoactive effects, but some of them can include 1 to 3% of THC. Gas chromatography-mass spectrometry (GC-MS) analysis of Cannabio shampoo revealed the presence of THC (412 ng/mL) and two constituents of cannabis plants, cannabidiol (CBD, 4079 ng/mL) and cannabinol (CBN, 380 ng/mL). In order to verify if normal hygiene practices with Cannabio shampoo can result in positive tests for cannabinoids in hair, three subjects washed their hair with this shampoo once daily for two weeks. After this period, hair specimens were collected. In the three hair specimens, THC, CBD, and CBN were never detected within their limits of detection, 0.05, 0.02, and 0.01 ng/mg, respectively. We concluded that the use of Cannabio shampoo during normal hygiene practices cannot be considered as a source of potential contamination of hair. In a second experiment, drug-free hair specimens (200 mg) were incubated in 10 mL water/Cannabio shampoo (20:1, v/v) for 30 min, 2 h, and 5 h. After incubation, hair strands were washed with water and separated into two portions. One portion was extracted directly; the second was decontaminated with methylene chloride and then extracted. After an incubation period of 30 min, the analysis of hair by GC-MS did not reveal the presence of THC, CBD, and CBN in hair, regardless of whether the hair was decontaminated. After an incubation period of 2 h, specimens tested positive for CBD (0.11 ng/mg without decontamination and 0.10 ng/mg with decontamination) and CBN (0.02 ng/mg without decontamination and 0.02 ng/mg after decontamination). After an incubation period of 5 h, specimens tested positive for CBD (0.25 ng/mg without decontamination and 0.14 ng/mg after decontamination) and CBN (0.02 ng/mg without ddcontamination and 0.02 ng/mg after decontamination). In all cases, THC was never detected. Extensive but unrealistic use of Cannabio shampoo can cause drug-free hair to test positive for CBD and CBN but not for the primary psychoactive drug THC.
Ag-Tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) are three constituents that can be currently isolated from Cannabis spp plants. Their identification in decontaminated hair indicate exposure to cannabis. CBD and CBN, like THC, are present in cannabis smoke, and the potential contamination of hair by external sources of drugs, which would generate false positives, was presented as the major limitation in hair analysis, particularly for the interpretation of forensic results (1). Today, cannabis plants are used in shampoo preparations, foodstuffs (e.g., oils, noodles, crackers, etc.), or beverages (e.g., tea, etc.). Usually, these products contain < 1% THC in order to eliminate psychoactive effects, but some of them can include I to 3% THC (2). Recently, two papers reported marijuana-positive urine tests after consumption of hemp seeds in food products (3) or ingestion of hemp seed oil (4). Cannabio shampoo (Swihtco, Mauss, Switzerland) was submitted to our laboratory to verify if normal hygiene practices can induce positive results for cannabinoids in hair.
Materials and Methods Chemical reagents All chemicals were provided by Merck (Darmstadt, Germany): methylene chloride, n-hexane, ethyl acetate, and cyclohexane were high-performance liquid chromatographic grade; all others were analytical grade. The deuterated internal standard (THC-d3) was purchased from Radian (Austin, TX). CBD and CBN were obtained from Sigma (St. Louis, MO). Cannabio shampoo was donated by Dr. C. Giroud (Institute of Legal Medicine, Lausanne, Switzerland). Material for examination Three subjects (two male and one female), recruited from our laboratory personnel, washed their hair with this shampoo daily for two weeks (normal hygiene practices). After this period, hair specimens were collected from the vertex posterior region of each
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Journal of Analytical Toxicology, Vol. 23, September 1999
subject and stored in dry tubes at room temperature. Before the study, a hair strand of each subject was collected to be used as reference. Samples (approximately 100 mg) were decontaminated twice ifl 5 mL methylene chloride, for 2 min at room temperature and extracted as described in our previously published procedure (5). In the second experiment, a drug-free hair specimen (200 rag) was incubated in 10 mL water/Cannabio shampoo (20:1, v/v) for 30 min, 2 h, and 5 h. After the incubation, hair strands were washed fivetimes with water (10 mL) and separated into two portions. One portion was extracted directly, and the second was decontaminated with methylene chloride as described and then extracted. In vitro experiments were made in duplicate.
in the presence of 50 ng of THC-d3. After cooling, the homogenate phase was extracted with 5 mL n-hexane/ethyl acetate (9:1 v/v). The organic phase was evaporated to dryness, and the dry extract was dissolved in 20 HLof cyclohexane.
Shampooextraction One hundred microliters Cannabio shampoo was extracted with 5 mL n-hexane/ethyl ~cetate (9:1, v/v) in the presence of 50 ng of THC-da. After agitation (20 min at 80 cycles/min) and centrifugation (20 min at 4000 RPM), the organic phase was removed and evaporated to dryness. The dry extract was dissolved in 20 I~Lof cyclohexane before GC-MS analysis.
&leexlradion The protein matrix of the hair (50 mg) was destroyed by incubation in 1 mL 1N sodium hydroxide solution for 10 rain at 95~ Ion 231.00 (1230.70 to 231.70): 3101001.D 9.~3
300000
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CBD
4079 ng/mL
:
250000 200000
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GO-MS analysis Briefly, a 1.5-1JLportion of extract was injected through the HP5-MS capillary column (30 m x 0.25-ram i.d.) of a Hewlett Packard 5890 GC coupled with a Hewlett Packard 5989 B MS Engine. The column temperature was pro(+,-) grammed to rise from an initial temperature of 60~ (1 rnin) to 295~ at 30~ and held at 295~ for the final 6 min. The detector was used in electronic-impact (EI) mode at 70 eV. The detector was autotuned daily with perfluorotributylamine. Mass spectra were recorded in the mass range m/z 200-350.
50000 9 50
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Results and Discussion
Time
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Figure I. SJM chromatogram obtained after extraction of 100 IJL of Cannabio shampoo. THC (m/z 299), CBD (rn/z 231 ), and CSN (ndz 295) were detected at the respectiveconcentrationsof 412, 4079, and 380 nglmL.
350
The GC-MS analysis of Cannabio shampoo revealed the presence of THC, CBD, and CBN at the respective concentrations of 412, 4079, and 380 ng/mL (Figure 1). Cannabinoid concentrations represent r 1% (w/w). In the three hair specimens collected on the laboratory personnel after Cannabio shampoo use for two weeks, THC, CBD, and CBN were never detected within their limits of detection of 0.05, 0.02, and 0.01 ng/mg, respectively.We concluded that the use of Cannabio shampoo during normal hygiene practices cannot be considered as a source of potential contamination of hair. In order to determined the condition required to induce a positivetest in drug-free samples, hair strands were incubated in 10 mL water/Cannabio shampoo (20:1, v/v)for 30 rain, 2 h, and 5 h. After the incubation, hair strands were washed five times with water (10 mL) and separated into two portions. One portion was extracted directly, the second was decontaminated with methylene chloride and then extracted. After an incubation period of 30 rain in Cannabio shampoo, THC, CBDand CBNwere not detected by GC-MS (with or without decontamination) within the limits of detection given before. After an incubation time of 2 h, specimens tested positive for CBD (0.11 ng/mg without
Journal o! Analytical Toxicology,Vol. 23, September1999
decontamination and 0.10 ng/mg after decontamination) and CBN (0.02 ng/mg without decontamination and 0.02 ng/mg after decontamination). After an incubation time of 5 h, specimens tested positive for CBD (0.25 ng/mg without decontamination and 0.14 ng/mg after decontamination) and CBN (0.02 ng/mg without decontamination and 0.02 ng/mg after decontamination). After increasing incubation time from 2 to 5 h, specimens tested positive for CBD and CBN, but THC was never detected within a limit of detection of 0.05 ng/mg. Extensive but unrealistic use of Cannabio shampoo can therefore cause drug-free hair to test positive for CBD and CBN but not for the primary psychoactive drug THC. When samples tested positive (after incubation in Cannabio shampoo), the decontamination step with methylene chloride did not totally eliminate the passive incorporation of CBD and CBN. The presence of THC in a hair sample cannot be explained by the use of a shampoo such as Cannabio. Positive results for CBD and CBN can be only obtained after an unrealistic use of this shampoo.
References 1. D.L. Black and D.A. Kidwell. Decontamination procedures for drugs of abuse in hair: are they sufficient. Forensic Sci. Int. 70: 13-38 (1995). 2. C. Giroud, A. Breillet, M. Augsburger, W. Bernhard, L. Rivier, and R Mangin. "Cannabis sativa ssp. helvetica" ou le feuilleton du cannabis en Suisse. Toxicorama 10:204-205 (1998). 3. N. Fortner, R. Fogerson, D. Lindman, T. Iversen, and D. Armbruster. Marijuana-positive urine test results from consumption of hemp seeds in food products. J. Anal. Toxicol. 21: 476-481 (1997). 4. A. Costantino, R.H. Schwartz, and R Kaplan. Hemp oil ingestion causes positive urine tests for t~9-tetrahydrocannabinol carboxylic acid. J. Anal. ToxicoL 21: 482-485 (1997). 5. V. Cirimele, H. Sachs, R Kintz, and R Mangin. Testing human hair for cannabis. I11.Rapid screening procedure for the simultaneous identification of Ag-tetrahydrocannabinol, cannabinol, and cannabidiol. J. Anal Toxicol. 20:13-16 (1996). Manuscript received July 13, 1998; revision received December 21, 1998.
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