The isolation from naturally-occurring substances, synthesis, separation and determination of the Δ 1-3,4-trans and Δ 6-3,4-trans isomers of tetrahydrocannabinol, the physiologically active component of marijuana and hashish, have been recently reported 1-6. We wish to report here our determinations of the ratio of these two isomers present in naturally-occurring substances.
Author: Melvin LERNER, Judith T. ZEFFERT
Pages: 53 to 59
Creation Date: 1968/01/01
The isolation from naturally-occurring substances, synthesis, separation and determination of the Δ 1-3,4-trans and Δ 6-3,4-trans isomers of tetrahydrocannabinol, the physiologically active component of marijuana and hashish, have been recently reported [ 1] - [ 6] . We wish to report here our determinations of the ratio of these two isomers present in naturally-occurring substances.
The isomers were determined by gas chromatography of seized samples of marijuana and hashish obtained from United States Customs laboratories. Pure standards of Δ 1-3,4-trans and Δ 6-3,4-trans-tetrahydrocannabinol were used for reference.
Experimental
The marijuana samples were stored in a freezer; they were air-dried before use. A sample weight of 0.75 g was placed in 15 ml of petroleum ether and extracted for three hours. Hashish and "red oil" (hashish concentrate) samples were dissolved in petroleum ether to a concentration of 5 mg/ml. The pure standards were diluted with petroleum ether to a concentration of 1 mg/ml.
A F & M Model 400 gas chromatograph equipped with a flame ionization detector and a ball and disc integrator for quantitative measurements were used. A supplementary electrometer was the Keithley Model 417 High Speed Picoammeter. Digital readout of the ball and disc integrator was accomplished by a Beckman Universal Eput and Timer, Model 7360. The samples were run on a 2% OV-17 (phenyl methyl silicone on 100 mesh Gas-Chrom-Q. Applied Science Laboratories) column at 210°C with helium as the carrier gas. Under these conditions the Δ 1 and Δ 6 trans isomer peaks were dearly separated by approximately two and a half minutes with a sharp return to the base line between the peaks. No Δ 6 was detectable in the gas chromatogram of the Δ 1 reference sample, nor was any Δ 1recorded in the gas chromatogram of the Δ 6reference sample.
Several other columns were tried. Most noteworthy are the QF-1 and the XE-60 columns. They both gave a 3. 5 minute retention time separation as well as having the lowest operating temperatures of all the columns tried - 170°C for the 2% QF-1 and 195°C for the 2 % XE-60. (Low column temperature was the most important criterion for a column next to good base line separation because, according to Taylor (3), there was a" demonstrably facile heat isomerization" of the Δ 1 to the Δ 6.) 1 Although the QF-1 and XE-60 columns were excellent for work with the pure standards, they did not satisfactorily separate the Δ 6 isomer from cannabidiol, which is present in naturally-occurring samples, and these columns were, therefore, not used in our work. Columns of SE-30, GE-949, cyclohexane dimethanol succinate, a mixture of neopentyl glycol succinate and Carbowax 4000 MS, and a mixture of neopentyl glycol succinate and QF-1 were also tried, but appeared to have no advantage over the OV-17. The 2% SE-30 column at 200°C gave a separation which did not return to the base line between peaks. The mixtures of neopentyl glycol succinate and Carbowax 4000 MS and neopentyl glycol succinate and QF-1 had to be run at temperatures that were at the upper temperature limit of the column materials.
A derivative method described by Claussen, Borger and Korte [ 7] at the University of Bonn was also tried. Pyridine, hexamethyldisilazane and trimethylchlorosilane, in a ratio of 10:2:1, were added to samples, which were then allowed to stand for an hour at room temperature. The samples were run on an SE-30 column, but the trimethylsilyl derivatives of the Δ 1 and Δ 6 isomers were not clearly separated by this procedure.
The marijuana samples consisted of ten seizures from the Los Angeles Customs laboratory and four from the New Orleans Customs laboratory, all presumed to be of Mexican origin. On the average, the samples, when run "as is " - stems, leaves and seeds included - contained 1.2 % tetrahydrocannabinol by weight; 0.4% of the total tetrahydrocannabinol was the Δ 6 isomer.
1Y. Gaoni and R. Mechoulam (J. Am. Chem. Soc. 88, 5673 (1966)) have observed none of this isomerization occurring up to 300°C.
The hashish samples were taken from two different sources. One source, labelled " Hashish A ", was of unknown origin and consisted of large flat blocks covered with a dark brown hard crust. The insides of the blocks were moist and soft and were a lighter, greenish-brown. The outside of the blocks had a total tetrahydrocannabinol content of 1.9% and 4.5% of this was the Δ 6 isomer. The lighter-coloured insides contained 8% total tetrahydrocannabinol and 2% of this was the Δ 6 isomer. The other source, labelled " New Orleans Hashish ", consisted of small thin flat blocks of granular or sandy-looking material. The blocks varied in colour from deep brown to almost a sand colour. The total tetrahydrocannabinol content of the lighter blocks was 1.2% and 9% of this was the Δ 6 isomer. In the darker blocks, there was 0.7 % tetrahydrocannabinol by weight, and of this 35 % was the Δ 6 isomer.
Two red oils were analysed. One was newly made from a block of Hashish A. It was found to be 31% tetrahydrocannabinol, 3% of which was the Δ 6 isomer. The other red oil was about four years old and contained 0.8 % tetrahydrocannabinol; 60 % of its tetrahydrocannabinol content was the Δ 6 isomer.
The results are condensed hereunder:
|
Sample |
Total tetrahydrocannabinol as a percentage of the sample weight |
Δ 6-3, 4-trans isomer as a percentage of the total tetrahydrocannabinol |
|---|---|---|
|
Fresh marijuana, Mexican #1
|
1.3 | 0.4 |
|
Fresh marijuana, average of 14 Mexican
|
1.2 | 0.4 |
|
Fresh marijuana, range of 14 Mexican
|
0.8-1.4
|
0.1-1.2
|
|
Hashish A
|
||
|
Outside
|
1.9 | 4.5 |
|
Inside
|
8.0 | 2.0 |
|
New Orleans hashish
|
||
|
Light
|
1.2 | 9.0 |
|
Dark
|
0.7 | 35.0 |
|
New "red oil"
|
31.0 | 3.0 |
|
Old "red oil"
|
0.8 | 60.0 |
|
Smoked new "red oil"
|
-
|
9.0 |
|
Smoked Mexican marijuana #1
|
-
|
0.6 |
DISCUSSION
The fact that the New Orleans hashish was no stronger in tetrahydrocannabinol content than fresh marijuana may be explained by Levine's hypothesis that hashish undergoes an ageing process in which tetrahydrocannabinol degrades to cannabinol [ 8] . As expected, the New Orleans hashish was found to contain a much greater percentage of cannabinol than the hashish A.
There seems to be a pattern in the variability of the percentage of the Δ 6 isomer in the different samples; there is a tendency for the percentage of Δ 6 to increase with age or apparent age. The older red oil contained a greater percentage of Δ 6 than the newer. There was a greater percentage of Δ 6 in the darker crust of the hashish A than in the lighter inside and there was a greater percentage of Δ 6 in the darker pieces of the New Orleans hashish than in the lighter pieces. The fresh marijuana contained the smallest percentage of Δ 6 of all the samples studied. This is in agreement with Hively's work in which he found that the Δ 1 isomerized to the Δ 6 at room temperature [ 9] . It is, of course, possible that part of the change in the isomer ratios is due to the preferential oxidation of Δ 1. Taylor has speculated that "in view of this demonstrably facile heat isomerization, it seems possible that the physiological effects attendant upon smoking of hashish, now ascribed to the Δ 1 isomer, may in actuality be due to the Δ 6 isomer (3). To test whether smoking would actually affect the isomerization of the Δ 1 to the Δ 6 isomer, a smoking machine was designed in which a vacuum was controlled to simulate puffing, and the resulting smoke passed through glass tubing contained in a beaker of ice. Regular cigarette tobacco soaked with new red oil and a sample of Mexican marijuana were both rolled into cigarettes and smoked by the machine. The condensed smoke was run on the 2 % OV-17 column. It was found that the proportion of the Δ 6 isomer had increased, but conversion was by no means complete. For the sample containing regular cigarette tobacco impregnated with new red oil the Δ 6 isomer had increased from 3 % of the total tetrahydrocannabinol before smoking to 9 % after smoking. For the marijuana cigarette the Δ 6 isomer had increased from 0.4 % of the total tetrahydrocannabinol before smoking to 0.6 % after smoking.
There has been much interest in the smoking of marijuana and hashish, not only concerning the isomers but also concerning the physiological effects. From our gas chromatograph results, we are able to calculate an average dose of tetrahydrocannabinol. An addict customarily smokes three cigarettes an evening, each cigarette weighing approximately 300 mg and containing 1.2 % tetrahydrocannabinol. Assuming 50 % absorption, three cigarettes would represent a total dose of approximately 5 mg of tetrahydrocannabinol.
ACKNOWLEDGEMENTS
We want to thank Dr. Richard L. Hively for his gift of pure Δ 1-3,4-trans and Δ 6-3,4-trans-tetrahydrocannabinols; Dr. Raphael Mechoulam for his gift of cannabigerol which was used in our separation studies; and Dr. K. E. Fahrenholtz for suggesting the use of the XE-60 and the mixed neopentyl glycol succinate columns.
Gaoni, Y. and Mechoulam, R., J. Am. Chem. Soc. 86, 1646 (1964).
002Mechoulam, R., and Gaoni, Y., J. Am. Chem. Soc. 87 , 3273 (1965).
003Taylor, E. C., Lenard, K., and Shvo, Y., J. Am. Chem. Soc. 88 , 367 (1966).
004Hively, R. L., Mosher, W. A. and Hoffman, F., J. Am. Chem. Soc. 88 , 1832 (1966).
005Fahrenholtz, K. E., Lurie, M., and Kierstad, R. W., J. Am. Chem. Soc. 88 , 2079 (1966).
006Korte, F., Sieper, H., Tira, S., U.N. Bulletin on Narcotics , XVII, 35, 1965.
007Claussen, U., Borger, W., and Korte, F., Ann. Chem . 693, 158 (1966).
008Levine, J., J. Am. Chem. Soc. 66 , 1868 (1944).
009Hively, R. L. and Hoffmann, F. W., Edgewood Arsenal Technical Report, EATR 4002, July 1966.
LSD ABUSE IN THE UNITED KINGDOM
In the opinion of Bewley (1968), LSD has not been used as widely in Great Britain as in the United States. The extent of this type of use ("taking a trip") is not certain, possibly 1-5 per 100,000 population. ( Proc. Roy. Soc. Med. 61 (2), p. 175-7 (1968).)
In 1966 Bewley had suggested LSD abuse at the rate of less than 1 per 100.000 population. ( Bulletin on Narcotics, Vol. XVIII, No. 4, 1966.)
The Bureau of Drug Abuse Control, of the U.S. Food and Drug Administration, reports that narcotic pedlars are selling a new cannabis concoction called "SMASH".
Marijuana is reported to be cooked with acetone to obtain oil of cannabis; the oil is then added to hashish to form a tar-like material, which is then rolled into small pellets and smoked, ,
"SMASH" is said to be made in Mexico. (F.D.A. "Micro-gram", January 1968.)
Three cases of intoxication from such abuse have been reported by the John Peter Smith Hospital, Forth Worth, Texas. The three patients admitted to a psychiatric ward had been eating a mixture of stramonium and belladonna extract (a powder to be ignited and the fumes inhaled to relieve asthmatic attacks) in order to get "high".
The use of belladonna to produce hallucinations was a well-known practice of witchcraft in various primitive societies and in the Middle Ages in Europe. This practice is now said to have been "renewed" within "Hippie" communities.
A new quinazoline derivative - 3 - (? - dimethylaminoethyl) - 2 - thio - 2,4 - [1H, 3H] -quinazoline - dione - possesses appreciable narcotic antagonistic properties. The pharmacological experiments, reported by J. Weis, show that this new analgesic does not belong to the "morphine type" or "antiphlogistic" analgesics. The morphine anta-gonistic activity was investigated in the tail-flick test in mice and compared to the activity of chlordiazepoxide. Appreciable antagonism to morphine was observed with doses of 10 mg/kg, while chlordiazepoxide was active in 5 mg/kg doses. Some psychopharmacological tests were carried out and it appears that the new substance may be a psychopharmacological agent with a new profile of action. (Acta Pharmacologica et Toxicologica, 1967, Supl. 4, Vol. 25.)
Connel has reviewed the problem of amphetamine dependence in Great Britain. He separated the two largest groups of amphetamine abusers: (a) the older age group, often a tired, obese and depressed housewife, and (b) the adolescent group.
For the first (older age) group it was proved by Brandon and Smith that 85.5 % of the amphetamine takers were women. The adolescent group of amphetamine abusers appeared around 1960 and built up to major proportions by 1963. Analysing the recent situation Connel states that some of those who have been taking amphetamines as "week-enders" (usually in the form of amphetamine-barbital mixture) have now pro-gressed to the hard drugs, and are taking heroin and cocaine. (Proceedings of the Royal Society of Medicine, Vol. 61, No. 2, Feb. 1962.)
Suicide by "pain-killing and sleep-causing substances" increased from 5 % of all suicides in 1950 to 12 % in 1964, reports the United States Public Health Service's National Center for Health Statistics. About three-fourths of these deaths were said to involve the use of barbiturates.
Suicide is one of the 12 leading causes of death in the United States. An estimated 20,588 suicides - an average of 56 a day - were committed in 1964. (Journal of the American Medical Association, Vol. 202, No. 6, November 6, 1967.)
