Fractionation and Analysis of Marijuana Smoke Condensate

MSC is a highly complex matrix containing several thousand compounds that may vary over several orders of magnitude (4). A liquid-liquid fractionation scheme (5,6) allowed the separation of these components into different classes of compounds (i.e., acidic, basic, and neutral: nonpolar, polar, and polyaromatic hydrocarbons; see Fig. 1).

In 1975, Jones and Foote (7) reported acids, phenols, and bases that were chemically separated from the smoke condensate of 2638 marijuana cigarettes and semi-quantitatively analyzed by GC and GC/mass spectrometry (MS). The analysis of the basic fraction (1.47 g, 4.8% of total MSC hydrochlorides) was carried out by GC/FID using a packed column (10 ft. x 1/8 in., 28% Pennwalt 223 + 4% KOH on chromosorb R, 80-100 mesh). While no fore-column was used for the GC/MS analysis, a glass fore-column was used for GC/MS analysis with the first 2 in. packed with powdered soda lime to liberate the amines and the remaining 5 in. packed with ascarite to absorb water. The phenolic fraction (0.96 g, 4.6% of total MSC) was analyzed as the TMS derivative by GC/thermal conductivity detector using a packed column (5% OV-17 on Diatoport S, 60-80 mesh). The acidic fraction (1.57g, 7.5% of total MSC) was esteri-

Stamboom Van Cleopatra Vii
Fig. 1. Fractionation scheme for marijuana smoke condensate.

fied with boron trifluoride-methanol (BF3-MeOH, 14%, V/V) to the corresponding methyl esters and analyzed by GC/FID using a packed column (2% OV-17 on GasChrom Q, 80-100 mesh). The neutral fraction (17.4 g, 83.1% of the total MSC) was not analyzed.

Van Den Bosch et al. (8) reported on the constituents of MSC generated from 640 cigarettes hand-rolled from Mexican marijuana (A9-THC content 1.29%). The condensate was fractionated into basic (0.3 g), phenolic (1.6 g), acidic (0.3 g), and neutral (6.9 g) fractions. The neutral fraction was further purified by column chromatography using silica gel and a step-gradient mobile phase consisting of n-hexane, n-hexane-benzene, benzene, ether, and methanol. The different fractions were analyzed by GC and GC/MS using a glass column (200 x 3 mm id) packed with 3% OV-17 on chrompak SA (80-100 mesh) or a glass capillary column containing OV-101.

Zamir-ul Haq et al. (9) identified and quantitatively determined the ^heterocyclic carbazole, indole, and skatole in MSC using GC, MS, and liquid scintillation spectrometry. The dry condensate was partitioned between hexane and methanol/water. The hexane fraction was subjected to column chromatography to yield a fraction enriched in the above-mentioned compounds. Qualitative analysis was carried out by GC/FID/MS using a glass column (6 ft x 2 mm) packed with 3% Silar 5CP on Gas

Chrom Q. For the quantitative analysis, separate experiments were done using individual radiolabeled carbazole, indole, and skatole as internal standards. The operational losses of carbazole, indole, and skatole were quite different from each other, and thus none of the internal standards could be used for the quantitation of the other components. The average amounts of carbazole, indole, and skatole were 89 ± 3, 826 ± 4, and 597 ± 7 ^g/g of fresh condensate, respectively. The effect of aging of the condensate was studied by analysis of a composite of all samples collected every 8 weeks for 2 years. The data showed a decrease in the levels of carbazole and indole, whereas levels of skatole increased on standing.

The previously described solvent partition method (Fig. 1; ref. 6) was used by Merli et al. (10) to separate the basic fraction of Mexican MSC. Enrichment of some trace components was accomplished with high-performance liquid chromatography on an aminosilane-bonded Porasil C (11). The analysis of this fraction was carried out by capillary GC/MS using a glass capillary column (50 m x 0.25 mm id) etched with gaseous HCl at 400°C and statically coated with UCON 50-HB-2000 stationary phase. Kalignost or benzyltriphenyl phosphonium chloride was added directly to the stationary phase solution in order to form a 10% addition to the amount of polymer phase used. The method allowed the identification of more than 300 nitrogen-containing compounds. The authors pointed to the fact that certain compounds of the hydrogen-donor nature, e.g., indole and carbazole derivatives, may end up in the polar neutral fraction (12) while using this solvent partitioning scheme. In addition, the comparison of MSC with that of tobacco (prepared and characterized by the same methodology) revealed that there are both qualitative and quantitative differences between the two condensates.

Further analysis of the basic fraction of marijuana and tobacco smoke condensates was carried out by Novotny et al. (13) using capillary GC/MS. The use of thermostable Superox-coated glass capillary column (Superox-4, 15 m x 0.25 mm id) allowed for the elution of relatively large nitrogen-containing compounds. The use of short columns allowed the elution of larger nitrogen-containing molecules in a reasonable time without sacrificing the peak resolution needed for the subsequent mass spectral investigations. Marijuana and tobacco smoke condensates showed qualitative similarities with a number of alkylated pyridine and quinoline derivatives, aza-in-doles, and aza-carbazoles; however, quantities of these components in both condensates were quite different.

Sparicino et al. (3) analyzed the strongly mutagenic fraction of MSC, produced from high-dose marijuana (A9-THC, 4.4%) under constant draft mode, by GC/MS. A capillary column (60 m, packed with DB-1701) was used. Approximately 200 compounds were identified. About half of this total were amines; with about half of these being aromatic amines. Pyrazines, pyrimidines, pyrroles, pyridines, and isoxazoles were the predominant compound classes. Some alkylated pyrazoles and pyrazines, as well as an alkylated benzimidazole, were detected in very large amounts.

Chemical ionization/MS was used to quantify noncannabinoid phenols in MSC (14). The methylene chloride-soluble material of the smoke condensate generated from 100 cigarettes prepared from female Mexican marijuana was fractionated between saturated aqueous sodium bicarbonate and then with 0.1 N aqueous sodium hydroxide solution. The aqueous sodium bicarbonate and sodium hydroxide solutions were acidi fied, extracted with ether, and analyzed as their TMS derivatives. A stainless steel column (3 m, 1% OV-17 on 100/120 mesh Gas-Chrom Q) and FID were used.

A capillary GC/MS method was developed by Maskarinec et al. (15) for the analysis of organic acids and phenols in MSC. The methodology used consisted of solvent partitioning (6), selective fraction enrichment by gel chromatography, followed by conversion of sample components to volatile methyl ester/ether derivatives for GC. A glass capillary column (20 m x 0.25 mm id) coated with free fatty acid phase was used, and it provided adequate resolution required for the MS investigation of the sample components. GC profiles of the acidic fractions obtained from Mexican (100 cigarettes, A9-THC, 2.8%; 6.25 mg acid/cigarette) and Turkish marijuana (100 cigarettes, A9-THC, 0.3%) and standard tobacco (prepared from equal weight, 2.05 mg acid/cigarette) smoke condensates were compared and indicated both qualitative and quantitative changes in the constituents of chromatographic profiles. Forty-nine components were identified in the acidic fraction of Mexican MSC.

Analysis of the polynuclear aromatic hydrocarbon fraction (see Fig. 1; ref. 6) of marijuana and tobacco smoke condensates was carried out with a combination of chromatographic and spectral methods (16). Selective enriched extracts were further purified by liquid chromatographic methods and analyzed by capillary GC/MS using a capillary column (11 m x 0.26 mm id) coated with SE-52 methyl phenyl silicone as a stationary phase. Approximately 150 polynuclear compounds in each smoke material type were quantitated and tentatively identified as to parent ring structures and type of alkyl substituents. Further identification of methyl derivatives of polynuclear aromatic hydrocarbons in air particulates, tobacco, and MSCs was accomplished by chromato-graphic separation into fractions of similar ring types and analysis using nuclear magnetic resonance (17). The positions of substitution in the rings were identified from the methyl chemical shifts. For the lower relative molecular mass fractions of anthracene-phenanthrene and fluoranthene-pyrene, the smaller number of methyl derivatives made identification possible from nuclear magnetic resonance alone. For mixtures containing benz[a]anthracene and chrysene derivatives, additional GC/MS was required. Overnight accumulation of Fourier transform spectra allowed approx 20-^g amounts of single constituents to be measured in 0.5- to 1.5-mg fractions.

The analysis of the neutral constituents (polar and nonpolar) of the smoke condensates of Mexican marijuana and standard tobacco (obtained according to Fig. 1) was carried out using GC/MS (18). Because the constituents of the polar neutral fraction were mostly nonvolatile, silylation facilitated a partial characterization of this fraction. A glass capillary column (50 m x 0.25 mm id) coated with 0V-101 methyl silicone fluid was used. In total, more than 130 neutral smoke components were characterized. It is to be pointed out that the comparison of the chromatographic profiles of the nonpolar fractions for marijuana and tobacco indicated some similarities, but also qualitative and quantitative differences in their terpenic compositions. The authors noted that peaks eluting in the temperature range of 120-160°C represent fairly unique components of marijuana smoke. Terpenes of these and similar structures have previously been found in the unburned marijuana samples (19) and are believed to be responsible for the characteristic odor of marijuana and its smoke. The components of the polar neutral fraction of both marijuana and tobacco smoke condensates revealed considerable similarity between the two materials. The only notable differences are the expected presence of nicotine and main cannabinoids in tobacco and marijuana smoke, respectively. The profiles of phenolic substances in tobacco and marijuana were qualitatively and quantitatively similar. A summary of the acidic, phenolic, nonpolar neutral, polar neutral and polynuclear aromatic hydrocarbons is presented in Table 1.

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