the fast blue salt B test which involves the fast blue salt B reagent which gives various colors with the phenolic cannabinoids, and the Grlic test which involves the use of hydrogen peroxide and sulfuric acid.84,85

It should be emphasized that it is difficult to say whether or not a sample being tested contains active psychotomimetic substances through the use of most of these chemical tests and false-positive reactions are not uncommon.

(2) microscopic tests-Cannabis sativa plants bear numerous hairs of two types that are useful in the identification of this plant, if it is in the powdered or crushed form-i.e., as one would find in a marihuana cigarette. The first of these types of hairs is known as a cystolith since it contains a crystal of calicum carbonate in the basal portion of the hair (see Figure 3). If a mineral acid is added to a microscopic preparation of a suspected sample of marihuana and the calcium carbonate crystal in the basal portion of the hair decomposes with effervescence (carbon dioxide), this is good evidence for the presence of marihuana in the sample. Until just recently, it was thought that the unicellular cystoliths of C. sativa were unique to this species and therefore offered a positive means of identification. However, a recent comprehensive study of hundreds of species of plants has revealed that similar cystoliths occur in many others."

On the other hand, most of the literature fails to point out that in addition to the presence of unicellular cystoliths on C. sativa plants, there are also characteristic resin-producing multicellular grandular hairs (see

CH

XV cannabipinol

Figure 3). Observation of both types of hairs in a single sample of suspected marihuana is positive evidence that C. sativa is present in the sample.

A precaution on the use of the microscopic method for identifying marihuana samples is that the method is of no value if the sample contains hashish, which of course would not contain any appreciable amounts of cellular elements for identification.

(3) thin-layer chromatography–The technic of thin-layer chromatography of a petroleum ether extract from a suspected Cannabis sativa-containing sample offers a rapid, simple, inexpensive and accurate assessment of the specific cannabinoid content of the sample, both from a qualitative as well as from a semi-quantitative point

of view. Following a rapid development of the sample on Kieselgel G plates impregnated with N,N-dimethylformamide and using cyclohexane as the eluent, the various cannabinoids can be separated into well-defined spots. Subsequent treatment of the chromatogram with fast blue salt B reagent results in the production of several colors for the separated components. Tetrahydrocannabinol under these conditions forms a scarlet color, cannabinol is violet, cannabidiol and cannabidiol carboxylic acid are orange, cannabichromene is brownish-violet and cannabicyclol is brick red. The R, values of the separated components offer additional evidence for their identification.30 The technic offers the simplest, most accurate and most practical means for the identification of marihuana samples.

(4) gas chromatography-A final method for the identification of C. sativa samples is to examine extracts from them by means of gas chromatography. Several studies37-41 have been reported recently using this technic, and it appears to be useful. However, the instrument is expensive, the technic requires some degree of skill for its operation and maintenance and reference samples are a necessity. summary

(1) Cannabis sativa plants grown in temperate zones-and especially in the northern part of the United States-have not been shown to contain appreciable amounts of tetrahydrocannabinols or any other psychotomimetic principles.

(2) The long-established opinion that male Cannabis sativa plants are devoid of psychotomimetic substances is incorrect.

prior to 1967 must be considered of questionable value. variability of cannabinoids

Although exhaustive and definitive studies have not been reported, it is generally recognized that Cannabis sativa plants grown in temperate zones or areas with short light days produce lower concentrations of resin than plants grown in tropical or subtropical zones. There is a fair amount of evidence that the resin from the former plants is higher in cannabidiolic acid and cannabidiol with little, if any, tetrahydrocannabinols. Plants from the latter areas, on the other hand, have little cannabidiolic acid, and larger amounts of the tetra

X

OH

cannabidiol

hydrocannabinols, 4.30

To point out the importance and significance of this information, it has been shown that the constituents of Cannabis sativa change on standing, and that these changes are more rapid in tropical areas than in temperate zones. Cannabidiolic acid, an inactive psychotomimetic substance, is transformed into the inactive cannabidiol which is then converted to the active tetrahydrocannabinols, and then into the inactive cannabinol (see Figure 2). It can be seen that almost any batch of Cannabis sativa can have a quite variable chemical composition, depending on the geography of the source material, the age of the plant when harvested, the age

of the harvested sample and the storage conditions, as well as other factors.30

While the tetrahydrocannabinols are generally claimed to be the active psychotomimetic principles of marihuana, they do not necessarily have to be present in the dried plant material or resin in order for a person to experience psychotomimetic effects when smoking this sample. Proof of this has recently been advanced by Claussen and Korte who have clegantly shown that precursors such as tetrahydrocannabinol carboxylic acid readily decarboxylate during the smoking process to form the active tetra hydrocannabinols. Cannabidiolic acid and cannabidiol are not cyclized to the active tetrahydrocannabinols during smoking. Also, the heat labile double bond in the tetrahydrocannabinols is only very slowly isomerized during the smoking process. About 98 percent of the cannabinoids present in marihuana are destroyed by the smoking process,31

identification of marihuana

There are several methods that have been used for the detection of Cannabis sativa in cigarettes or when admixed with other plant materials. They are-

(1) chemical tests-Several cheinical tests have been developed for the identification of C. sativa. Perhaps the most widely used of these is the Beam test, which simply involves extraction of the suspected sample with petroleum ether, evaporation of the solvent and the addition of a few drops of alcoholic potassium hydroxide to the residue. The production of a violet color is taken as evidence that the sample contains marihuana. The reaction is a test for the presence of the inactive cannabidiol and cannabidiolic acid and other constituents of C. sativa do not give a positive test, including the tetrahydrocannabinols. A number of plants other than C. sativa will give false-positive reactions to this test but it is claimed that these can be eliminated for the most part by shaking the violet color reac tion mixture of a positive test with chloroform and allowing the chloroform and water layers to separate. If the reaction is a false-positive one, the violet color will not migrate into the chloroform layer 32.33

Other tests that have been applied are the Ghamravy test which involves the use of dimethylamino benzalde hyde and sulfuric acid; the Duquenois-Negm test which involves vanillin, acetaldehyde and ethanol; the modified Duquenois-Negm test which involves hydrochloric acid followed by extraction with chloroform;

(3) Two common constituents of Cannabis sativa-i.e., cannabidiolic acid and cannabichromene-are sedative principles. The assessment of any effect of the constituents on the body during the smoking of marihuana cigarettes has not been established.

(4) Chemical tests for detecting marihuana are unreliable and they do not establish the presence of C. sativa psychotomimetic principles. Falsepositive reactions also can be expected with the tests. A trained microscopist can identify C. sativa in crushed or powdered marihuana samples by detecting the cystolith unicellular, nonglandular hairs in addition to typical multicellular glandular hairs. This method cannot be used if hashish is the type of marihuana present. The best means for identifying C. sativa in suspected marihuana samples is by thin-layer chromatography.

(5) A total of 15 cannabinoid constituents of C. sativa have been isolated in pure form and their structures determined. Eight of these have not been tested for psychotomimetic activity.

(6) The most important psychotomimetic agent in C. sativa is (-)-A1transtetrahydrocannabinol but the

(-)-A1(6) -transtetrahydrocannabinol isomer, also active, is present in a much lower concentration.

(7) A good animal assay for detecting the psychotomimetic acavity of C. sativa is lacking and this has hindered research in this area.

necessarily mean that we will soon know whether or not marihuana smoking can be considered a safe practice.

(9) In light of our present day knowledge, legalization of marihuana under government control and distribution does not appear to be a realistic possibility. There is adequate and overwhelming data available that marihuana samples-even those from plants grown in the same geographic area-are so variable in their chemical composition that standardization of the drug would be next to impossible. One must remember that not only must the active psychotomimetic tetrahydrocannabinols be taken into consideration as active principles but the opposite acting sedative principles cannabidiolic acid and cannabichromene cannot be discounted as contributing to the overall activity. Chemical standardization of each batch of marihuana could be carried out with little difficulty but adjustment of the biological potency of high or low psychotomimetic activity drug would be next to impossible. On this ground alone, a standard marihuana activity preparation could not be made available for general use.

There are no clear-cut and simple answers to the marihuana controversy, and based on the direction in which current research is headed, the situation will become worse before it becomes better.

One can expect that the Pandora's box of marihuana will remain closed for some time to come.

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Tetra

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