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delta1-Tetrahydrocannabinol, delta6-tetrahydrocannabinol, cannabinol, cannabidiol and several of their monooxygenated derivatives have been separated from each other by a combination of liquid, thin-layer and gas chromatography. Retention volumes (on Sephadex LH-20), Rf values and retention times can be recorded, and may provide guidance in the separation and identification of these known cannabinoids.

Marihuana cigarettes containing 1.32%, 1.97%, and 2.54% delta 9-tetrahydrocannabinol (THC) were smoked by six experienced marihuana users at weekly intervals in a double-blind cross-over design under laboratory conditions. Puff duration, number of puffs taken, duration of inhalation holding, interval between puffs, and duration of smoking were recorded for each cigarette smoked. The portion of each cigarette remaining after smoking was weighed and analyzed to determine THC content. Subjective ratings of the "high" achieved and the heart rate acceleration induced by smoking the marihuana were measured. The plasma concentrations of THC and of its principle metabolite, 11-nor-delta 9-THC-9-carboxylic acid (9-carboxy THC), were determined by radioimmunoassay of blood samples drawn at frequent intervals for 6 hr. The results indicate that, irrespective of the potency of the marihuana, the pattern of smoking was much the same. The magnitude of the subjective high, heart rate acceleration, THC, and 9-carboxy THC plasma concentrations were proportional to potency. This dose response was particularly clear between the 1.32% and the 2.54% cigarettes. Peak plasma concentrations of THC consistently occurred 7 to 8 min after initiation of smoking and declined thereafter despite continued smoking for another 6 to 10 min. Peak subjective high and peak heart rate acceleration occurred several minutes after the end of smoking and at a considerable interval after maximal THC plasma concentrations were reached.

Using a simple injection adapter of our own design, the tetrahydrocannabinol content of marlhuana was determined by gas chromatography. The injection method obviates the need for preliminary extractions and allows analyses to be conducted on milligram quantities in less than thirty minutes.

A tritium based radioimmunoassay for delta9THC and its metabolites has been developed for the use of investigators studying the epidemiological, medical, clinical, and research aspects of cannabis use. The assay is sufficiently sensitive to detect cannabinoids in the urine of marijuana smokers for several days after their last exposure to the drug. The results obtained from a 28 day study indicate that the assay reflects the administration and removal of oral doses of THC. The specificity of the antisera, as determined in cross reactivity studies, allows not only the assay of metabolites in biological samples without interference from other drugs, but also the evaluation of extracts of other kinds of samples which may contain unmetabolized delta9THC. The technique of radioimmunoassay has many advantages over other methods of analysis. It is simple to perform and can be readily applied to the rapid analysis of large numbers of samples. It can be used in the direct analysis of physiological fluids and other biological samples which ordinarily have to be processed before other techniques can be applied. The method is non-destructive abd obviates the need to use radiolabelled drugs in man during metabolic and other studies. This radioimmunoassay has been designed with particular emphasis on ease of use by other investigators. We anticipate that it will prove useful to investigators and scientists for determining the absence, or presence and amount, of THC metabolite in a biological specimen, for epidemiologists in determining the full extent of cannabis use and to the medical/clinical community for establishing the minimum effective dose of delta9THC for each patient. The widespread application of a single method of analysis should also remove a great deal of the controversy surrounding marijuana studies performed to date.

In a collaborative work, six chemical fractions obtained from smoke of cannabis, prepared by the Department of Biological Chemistry, University of Athens, were assayed pharmacologically at the Department of Psychobiology, Escola Paulista de Medicina, Brazil. The chemical fractions, obtained through burning cannabis mixed with a special tobacco through a water pipe similar to that used by Greek smokers, were: fraction IS composed of water-soluble compounds, fraction IIS made up of water-insoluble substances that condensed at the top of the water phase due to their low solubility; and fraction IIIS, of water-insoluble highly volatile substances, corresponding to the part that reaches the human lungs. Fractions IB, IIB and IIIB were placebo fractions obtained from burning only the special tobacco, called "tubeki", which is mixed with cannabis by the Greek smokers. The six fractions were assayed pharmacologically, in a blind fashion, using the following methods: barbiturate sleeping time in mice, catatonia in mice, corneal reflex in rabbits (Gayer test), and spontaneous motor activity in mice. Fraction IS, and its placebo fraction IB, were inactive in all methods employed, revealing no water-soluble active substances in cannabis. Fraction IIS revealed to be the most active as it induced significant effects in all 4 methods used; its IIB placebo fraction was inactive. Fraction IIIS, composed of the only substances that reach the human lungs, was moderately active; it produced significant effects on barbiturate sleeping time of mice and on the corneal reflex of rabbits, but was inactive in inducing catatonia and in decreasing spontaneous motor activity of mice. These results indicate that devices such as the water pipe used by Greek smokers retain active principles from cannabis.

Pyrolysis gas chromatography coupled with infrared identification of eluted peaks confirms that paraquat is pyrolyzed into chloromethane and 4,4'-dipyridyl at smoking temperatures and above. This reaction occurs at 610 degrees C to completion in small amounts in an inert atmosphere. The toxicity of 4,4'-dipyridyl remains to be determined. Pyrolysis of contaminated marijuana also produces the same two products, although detection at low limits is difficult with this procedure.

Numerous methods for the detection of urinary metabolites of marijuana constituents are available. Documentation of the sensitivity and specificity of these tests is needed before the determination of a pair of screening-confirmation tests can be made. This study used 88 clinical specimens to evaluate five commercially available marijuana metabolite methods and one new gas chromatography/mass spectrometry (GC/MS) method. The EMIT-d.a.u. test was found to have 2 to 3% unconfirmed positives when compared to the other methods evaluated. The new thin layer procedure, TOXI-LAB, was not as sensitive as the EMIT-d.a.u. procedure for some specimens, but proved to be a good confirmation for the EMIT-d.a.u. with elimination of all "unconfirmed positives." The Abuscreen (Roche) and the EMIT-st assays were positive for samples that contained larger amounts of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (11-nor-delta 9-THC-9-COOH). The Immunalysis-radioimmunoassay (RIA) was positive for all samples found positive by the GC/MS method, but the concentrations found by the two assays did not correlate. The GC/MS method was developed to use the same extraction as the thin layer procedure and provides confirmation for all procedures except 2 to 3% of the positive EMIT-d.a.u. results.

This paper reviews the applications of high-pressure liquid chromatography (HPLC) to forensic problems, and discusses some of the developments that have taken place in the use of the technique in the Metropolitan Police Laboratory. Preparation of octadecyltrichlorosilane-modified silica is described and some of the chromatographic characteristics of this material are investigated. Applications of HPLC to the analysis of cannabis, opium alkaloids, amphetamine-related materials, LSD and polynuclear hydrocarbons are described.

Regenerating pieces of planarian worms are able to absorbe insoluble substances deposited on the base of the vessels in which they are cultivated. This biological test was used to study the toxic effects of the essential oil of Cannabis sativa L. The hydrocarbons such as pinene (alpha and beta), caryophyllene and so on were not toxic. On the contrary caryophyllene oxide was highly toxic. It was not possible to detect any protection by 5-hydroxytryptamine as it was the case against delta 1-tétrahydro-cannabinol and cannabidiol.

Components of the sterol fraction of tobacco and marijauna were resolved as trimethylsilyl derivatives by gas chromatography with glass capillary columns. Ten phytosterols in tobacco and five in marijuana were identified by comparisons of their retention with authentic compounds on three different stationary phases and through mass-spectral data.

A second generation of Cannabis plants has been grown from seeds produced by first generation plants which were raised in the United Kingdom of Great Britain and Northern Ireland from seeds taken from imported cannabis of known geographical origin and chemistry. The gross physical appearance of the second generation plants, in general, resembled their parents and the cannabis produced therefrom was not usually distinguishable from that derived from first generation plants. Although cannabinoid patterns were, in some cases, similar to those of both parents and original seedstock cannabis, there were many exceptions. The yields of cannabis were significantly higher than from first generation plants, but the tetrahydrocannabinol contents were lower. Although higher than in the original imported cannabis, the tetrahydrocannabinolic acid to tetrahydrocannabinol ratios in cannabis from second generation plants were lower than in cannabis produced from first generation plants.

Although the use of ethanol, marijuana, and other drugs may be detrimental to driving safety, this has been established by direct epidemiological evidence only for ethanol. In this study, the incidences of detection of ethanol (and other volatile substances), delta-9-tetrahydrocannabinol (THC), barbiturates, cocaine and benzoylecgonine, opiates, and phencyclidine were determined in an inclusive population of 600 verified single-vehicle operator fatalities that occurred in North Carolina in 1978 to 1981. The incidence of detection of amphetamines and methaqualone were determined for drivers accepted for study during the first two years (n = 340) and the last year (n = 260), respectively. Blood concentrations of 11-nor-deta-9-tetrahydrocannabinol-9-carboxylic acid (9-carboxy-THC) were determined in THC positive drivers. EMIT cannabinoid assays were performed on blood specimens from all drivers accepted for study during the third year, and the feasibility of using the EMIT cannabinoid assay as a screening method for cannabinoids in forensic blood specimens was investigated. The incidence of detection of ethanol (79.3%) was far greater than the incidences determined for THC (7.8%), methaqualone (6.2%), and barbiturates (3.0%). Other drugs were detected rarely, or were not detected. Blood ethanol concentrations (BECs) were usually high; 85.5% of the drivers whose bloods contained ethanol and 67.8% of all drivers had BECs greater than or equal to 1.0 g/L. Drug concentrations were usually within or were below accepted therapeutic or active ranges. Only a small number of drivers could have been impaired by drugs, and most of them had high BECs. Multiple drug use (discounting ethanol) was comparatively rare. Ethanol was the only drug tested for that appears to have a significantly adverse effect on driving safety.

The identification and the quantitative estimation of cannabis constituents is important for forensic purposes. High resolution gas-chromatography gives better results than gas-liquid chromatography with packed columns, as it shows a better resolution and higher number of constituents. Different quantitative values were found with the two chromatographic procedures. High-pressure liquid chromatography revealed the presence of cannabinoid acids in fresh cut influorescences of cannabis plants. The ratio acid/neutral cannabinoid may be useful in supplying information about the age of the cannabis preparations.

The results from these urine studies and a few other isolated extracts we have run may be generalized as follows: The hexane fractions showed low levels of delta9-THC (ca. 10 ng/ml) with marginal Confidence Indices (25-30), but both the quantities and the K-scores were well above the values seen in extracts of control urines; the hexane fractions showed similar levels of CBN (ca. 12-25 ng/ml) with significant K-scores (60-80); the ether-extractable neutral fraction contained 8alpha, 11-dihydroxy-delta9-THC (25-150 ng/ml) with low conficdence and sometimes a suggestion of 8beta-hydroxy-delta9-THC (15 ng/ml) with a marginal identification; and the E-II fraction (ether-extractable, weak acids) usually contained a strong indication (CONF 60-95) of delta9-THC-11-oic acid (100-200 ng/ml.

A pharmacokinetic study of the blood plasma levels in man of delta9-tetrahydrocannabinol, 11-hydroxy-delta9-tetrahydrocannabinol and cannabinol has been carried out by means of combined gas chromatographic-mass spectral analysis. In some cases comparison of the data was obtained on the same sample using thin layer chromatography of radiolabeled samples and electron capture gas-liquid chromatography. For the mass spectral studies appropriately deuterium labeled analogs of the previously named compounds were used both as internal standards and as a carrier for the relatively small amounts of nonlabeled drug present in plasma. Blood samples were obtainted at periodic intervals up to 24 hours from volunteers receiving 4-5 mg delta9-THC intravenously. After extraction and "clean-up" by Sephadex chromatography, the extracts were concentrated and subjected to glc-ms in the electron impact (ei) mode or alternatively with a chemical ionization (ci) source, in which case preliminary chromatography could be omitted. In all cases calibration curves were obtained from replicate analyses of spiked plasma containing the internal standard and various quantities of the cannabinoid under analysis. A typical biphasic elimination of the drug was observed with rapid elimination of delta9-THC from the blood over a period of 40 min followed by a much slower elimination up to 24 hours. The experimental data show that 11-hydroxy-delta9-THC is found in the plasma in quantities only about one-twentieth to one-twenty-fifth the values found for delta9-THC. Cannabinol was not found in significant quantities. Good agreement was obtained between the mass spectral analyses and the thin layer chromatography or electron capture gas-liquid chromatographic procedures.

The on-column flash methylation of synthetic cannabidiol, cannabichromene, and cannabivarin by trimethylanillinium hydroxide offers a rapid, simple GLC method for their distinction. The phenolic groups of other cannabinoids are methylated by the procedure described.

Cannabis sativa L. contains more than 420 different constituents. Sixty-one of these constituents are cannabinoids. The electron voltage selected ion monitoring technique was used in the study of this unique group on natural compounds. Mass spectra of each compound were recorded at various eV settings (5.5-21 eV). The electron voltage selected ion recordings were obtained by plotting the relative intensities of characteristic fragment ions against electron voltage. The method was proven useful in the structure analysis of cannabinoids since it requires only minute quantities of sample. It was possible to recognize homologs and to differentiate positional and double bond isomers as well as cannabinoids with the same molecular weight.

Samples taken from 61 slabs of cannabis resin originating in The Lebanon were examined by high performance liquid chromatography. The slabs were derived from seizures of cannabis resin made over a three-year period. From a study of 14 of these samples, taken from bags which bore a number of identical stamp marks, it was shown that there was a correlation between the cannabinoid distribution and the marks. The remaining 47 samples of cannabis resin, all of which were from bags with different marks, were each found to have a unique cannabinoid distribution. Such an examination can therefore be used to aid investigations where evidence of a common origin is sought.

Synthetic and naturally occurring cannabidiol and cannabichromene were distinctly separated without derivation by GLC using a 6% OV-1 column; an artifact of cannabichromene, cannabicyclol, was separated from (minus)-delta9-trans-tetrahydrocanna-bivarin. This procedure is versatile and applicable for the quantitation of Cannabis containing both cannabidiol and cannabichromene. Biological interaction among (minus)-delta9-trans-tetrhydrocanabinol, cannabichromene, and other cannabinoids in natural Cannabis preparations can now be studied. In the phenyl methyl silicone polymer series, cannabidiol precedes ca-nabichromene on columns containing below a 50% phenyl-to-methyl ratio. Columns containing below a 50% phenyl-to-methyl ratio. Columns containing a 50:50 or greater ratio of phenyl to methyl reverse the separation order with cannabichromene preceding cannabidiol.

In order to detect hashish intake, urine, blood and serum were analysed for the main components of hashish, i.e., tetrahydrocannabinol (THC), cannabidiol, cannabinol and the decomposition product of THC, THC-carboxylic acid. After extraction and silylation, the samples were analysed by gas chromatography-mass spectrometry with multiple ion detection. The Emit-st-system is used as a pretest for urine.

We have examined the cannabinoid contents of seedlings from twelve strains of Cannabis of known chemical race. Fourteen days after emergence of the shoot those of the tetrahydrocannabinol (THC) type could be distinguished from those of the cannabidiol (CBD) type. The true leaves of the THC type contained a relatively high content of THC and also contained cannabichromene (CBC), sometimes as the major cannabinoid, whereas the CBD type had much lower amounts of THC and no CBC; CBD being the major component. A strain from China corresponded to the THC type but showed some unusual features. Seeds purchased at seven outlets in Britain were also examined. Only two batches germinated by these proved to be of a THC type and resembled the Chinese strain.