Abstract
Introduction
Experimental Gas chromatographic conditions
Extraction and analysis of marihuana plant material
Collection and analysis of smoke samples
Marihuana placebo
Results and discussion. Cannabinoid composition of the marihuana plant extracts
Lot 2-70, Ref. No. 2-PF-114
FIGURE 4
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FIGURE 10
FIGURE 11
FIGURE 12
FIGURE 13
Author: G. W. KINZER, R.L. FOLTZ, R.I. MITCHELL, BATTELLE, E. B. TRUITT
Pages: 41 to 54
Creation Date: 1974/01/01
Cannabis of known origin was used to prepare cigarettes. The cigarettes were smoked by machine under conditions which simulated the smoking patterns of a typical marihuana smoker. Both the smoke condensate and the marihuana plant extract were analysed by gas chromatography and mass spectrometry. It was found that approximately 50 per cent of the Δ 9-tetrahydrocannabinol in the marihuana was destroyed during the smoking process. Analysis for the cannabinoid acids was achieved by trimethylsilylation followed by gas chromatography. The trimethylsilylated cannabinoids were identified on the basis of their mass spectra. Some of the Δ 9-tetrahydrocannabinolic acid and the cannabidiolic acid were found to survive the smoking process.
Smoking is the most common method of human consumption of marihuana. Knowledge of the composition of marihuana smoke is therefore important to an understanding of the pharmacology and toxicology of marihuana. Previous studies with tobacco have shown that considerable variation in the composition of the tobacco smoke can occur unless the various parameters affecting the combustion are carefully controlled (1). To control these parameters and simulate the human marihuana smoking habits (puff profile) a smoking machine was constructed with which it is possible to vary the puff volume, puff duration, and interval between puffs. The machine was programmed to duplicate as nearly as possible an average of the smoking patterns obtained with a pneumotachometer from volunteers at the Addiction Research Center in Lexington, Kentucky.
Two strains of the female plant of Cannabis sativa were used in this study. Both were obtained from the National Institute of Mental Health and were grown, harvested and manicured at the University of Mississippi from Mexican seeds (Ref. No. 2-PF-115) and Turkish seeds (Ref. No. 2-PF-114).
Previously reported studies on the composition of marihuana smoke have apparently utilized plant material having an undocumented origin and did not attempt to simulate the smoking patterns typical of a marihuana smoker (2,3).
Smoke condensate and extracts of the butts were analysed directly by gas chromatography. Portions of the samples were also treated with bis-(trimethylsilyl)-trifluoroacetamide and the cannabinoids analysed by gas chromatography as their trimethylsilyl derivatives. The latter analysis is necessary to detect the cannabinoid acids, since the free cannabinoid acids are decarboxylated in the injection part of the gas chromatograph.
To determine whether there is significant interconversion of the major cannabinoids during smoking, measured quantities of synthetic Δ 8- and Δ 9-tetrahydrocannabinol (THC) and cannabidiol (CBD) were deposited on placebo marihuana. The resulting spiked marihuana was subjected to the same smoking and analysis procedure described for the natural marihuana.
The gas chromatograph used was a Varian-Aerograph Model 1200. The column was 10 feet x 1/8 inch O.D. stainless steel packed with 2 per cent OV-17 coated on 80/100 mesh Gas Chrom Q. The column temperature was 230 oC. The temperature of the glass-lined injector was 250 oC and the detector temperature was 275 oC. The nitrogen, hydrogen, and air flows were 30, 30, and 300 ml/min respectively.
Extracts of marihuana were obtained for analysis by a modified Lerner procedure (4). Approximately 1 g of manicured marihuana was covered with 40 ml of reagent grade chloroform and kept in a refrigerator at 20 oC for one hour, with intermittent shaking at 10 minute intervals. The extracted plant material was removed by suction filtration on a glass-frit and then washed with chloroform. The filtrate was adjusted to a volume of 50 ml. A 2 ml aliquot was concentrated to a volume of 200 Δ 1 under a stream of nitrogen at room temperature. A measured quantity of the internal standard, Δ 4-androstene-3,17-dione, was added and the solution analysed for free cannabinoids by gas chromatography. Parallel analyses were performed on trimethylsilylated mixtures prepared by treating 50 Δ1 of the above solution with 50 Δ 1 of bistrimethylsilyl trifluoroacetamide (BSFTA) containing 1 per cent trimethylchlorosilane.
Marihuana cigarettes were fabricated using a low cost and commercially available cigarette-making machine. The marihuana was first passed through a 10-mesh screen to remove rough stems and seeds; then conditioned by storing at room temperature and 72 per cent humidity. The fabricated reefers contained 0.8 g of marihuana and were 8.1 mm in diameter and 60 mm in length. The pressure drop through these reefers is approximately 2.0 inches of water at a flow rate of 17.5 cc/second.
Smoke samples from the two varieties of marihuana were obtained using a negative pressure smoking machine. The machine was programmed to simulate an average of the smoking patterns obtained with a pneumotachometer from five experienced volunteers at the Addiction Research Center at Lexington, Kentucky. Three consecutive puffs, each of 2 seconds' duration, were taken per minute and puff volume was 40 ml. The smoke analyses were performed in duplicate.
Five cigarettes were smoked to a butt length of about 3 mm for each analysis. The mainstream smoke was collected in two 40 ml spiral traps connected in series and cooled in a bath of Dry Ice-trichloroethylene. A Cambridge filter was placed between the traps and bellows. Side stream smoke was collected by suspending a large filter holder containing absolute glass filter paper over the cigarette. Air flow through the filter provided by a mechanical vacuum pump was adjusted so that the sidestream smoke particulate was collected on the filter without increasing the burning rate of the marihuana cigarette.
The smoke condensate was removed from the traps by washing with chloroform, followed by a rinse with ethanol. Filter and butt samples were extracted with ethanol. These solutions were concentrated at room temperature under reduced pressure. The concentrated solutions were dissolved in 2 ml of chloroform and analysed from cannabinoids before and after trimethylsilylation using the procedure described for the marihuana plant extract analyses.
Marihuana was extracted for a period of 14 days with ethanol in a Soxhlet extractor. The resulting placebo material had a negligible cannabinoid content when analysed by the modified Lerner Procedure previously described. Spiked placebo marihuana containing 0.19 per cent Δ 9-THC was prepared by thoroughly mixing the placebo marihuana and a heptane solution of synthetic Δ 9-THC and evaporating the solvent under reduced pressure. Placebo marihuana samples containing 0.31 per cent Δ 8- THC and 1.6 per cent CBD were similarly prepared. The placebo marihuana and the spiked placebo samples were used to prepare cigarettes which were smoked as previously described and the smoke and butts collected and analysed.
Cannabinoid occur in the plant in both the free and carboxylic forms. Fetterman and co-workers(5) have shown that the acid form decarboxylates quantitatively when gas chromatographed and the resulting analysis represents the combined quantity of the free and acid forms. In out work cannabinoid acids were determined by the method of Fetterman, et al. In this procedure the cannabinoid acid content is expressed as the difference between the concentration of the free cannabinoid before and after trimethylsilylation of the sample.
Gas chromatograms of the underivatized plant extracts are shown in figures 1 and 2. Table 1 lists the calculated plant content of CBD, Δ 9-THC and cannabinol (CNB). The marihuana sample obtained from the female plant grown from Mexican seeds has a Δ 9-THC content of 2.0 per cent and is typical of the more potent strains of Cannabis sativa. The high CBD content (1.5 per cent) in the female plant cultivated from Turkish seeds is typical of hemp plants grown for their fiber.
Chromatograms of the trimethylsilylated plant extracts are shown in figures 3 and 4. The major peaks were identified on the basis of their mass spectra and coincident retention times of authentic material. In the marihuana sample obtained from the female plant grown from Mexican seeds 75 per cent of the major cannabinoid, Δ 9-THC, is present in the acid form. Similarly, approximately 72 per cent of the cannabinoid content of the sample obtained from plants grown from Turkish seeds is present in the acid form. Mass spectra of the trimethlysilyl (TMS) derivatives of the cannabinoids and their carboxylic acid forms have not been reported. In view of their importance in the analysis of marihuana samples, the mass spectra of the TMS derivatives of Δ 8-THC, Δ 9-THC, Δ 9-THC acid, CBD acid and CBN acid presented here (figures 9, 10, 11, 12, 13).
Per cent content of the marihuana |
|||
---|---|---|---|
Cannabinoid |
Relative retention time a |
Female Mexican Lot 3-70, Ref. 2-PF-115 |
Female Turkish Lot 2-70, Ref. 2-PF-114 |
Analysed as free cannabinoids
|
|||
Cannabidiol
|
0.34 |
0.19 ± 0.01b
|
1.50 ± 0.08
|
Δ9-THC
|
0.49 |
1.97 ± 0.16
|
0.17 ± 0.02
|
Cannabinol
|
0.62 |
0.39 ± 0.04
|
0.15 ± 0.02
|
Analysed as Trimethylsilyl Derivatives
|
|||
Cannabidiol
|
0.11 |
Trace
|
0.40 ± 0.02
|
Cannabidiolic acid c
|
0.65 | 0.19 | 1.00 |
Δ9-THC
|
0.22 |
0.48 ± 0.03
|
0.05 ± 0.03
|
Δ9-THC acid d
|
0.60 | 1.49 | 0.12 |
Cannabinol
|
0.31 |
0.05 ± 0.01
|
0.03 ± 0.01
|
Cannabinolic acid
e
|
0.66 | 0.34 | 0.14 |
a Internal standard, Δ4-androstene-3,17-dione = 1.00. Relative retention times are reproducible within ± 0.01.
b The ± values are average deviations based on duplicate determinations.
c The difference in the cannabidiol content based on the free cannabidiol peak and the trimethylsilylated cannabidiol peak.
d The difference in the Δ9-THC content based on the free Δ9-THC peak and the trimethylsilylated Δ9-THC peak.
e The difference in the cannabinol content based on the free cannabinol peak and the trimethylsilylated cannabinol peak.
Smoke condensates from the two varieties of marihuana were analysed by GLC before and after trimethylsilation (see figures 5, 6, 7, 8). In each case the mainstream smoke, sidestream smoke, and the butts were analysed. The data are shown in tables 2 and 3.
The transfer of cannibinoids by the mainstream smoke based on the available cannabinoid in the marihuana cigarette is about 22 per cent. This result is comparable
to that reported by Agurell and Leandes (6) for smokers of Turkish cigarettes. Recoveries of CBD, Δ 9-THC and CBN in smoke and butts were about 50 per cent for the variety of cannabis grown at the University of Mississippi from Mexican seeds. In the case of the cannabis variety obtained from Turkish seeds recoveries were considerably higher for CBD (84 per cent) and Δ 9-THC (117 per cent). Current studies in our Laboratory indicate that CBD can rearrange at elevated temperatures to a material having the same retention time as Δ 9-THC. This could account in part for the apparent high recovery of Δ 9-THC.