A9-THC and its metabolites

The cause for the differences has not been determined but these results prompted Mechoulam to warn that in the analysis of A9-THC and its metabolites it may not be possible to simply exchange plasma of different species but that each may, in fact, be different (3). 

The final analytical method for the simultaneous determination of A9-THC and its metabolites consists of the following sequence the cannabinoids are extracted from plasma with toluene they are then back extracted from toluene into Claisen s alkali the Claisen s alkali is diluted with water, tetrahexyl ammonium hydroxide is added and the alkaline solution is extracted with methylene chloride containing ethyl iodide. The overall recoveries were 45% for A9-THC and 83% for 11-hydroxy-A9-THC. External standards (l-0-ethyl-A9-THC and l-0-ethyl-ll-hydroxy-A9-THC) were added to the methylene chloride phase followed by a small amount of Florosil, which absorbed the tetra-hexylammonium hydroxide and tetrahexylammonium iodide. The methylene chloride was decanted and evaporated. 

A9-THC and its main metabolites are detected and quantified in forensic samples. Determination of these compounds in human beings is needed to make decision on abuse of A9-THC-containing drugs by individuals. A careful interpretation of the results is very important to avoid fallacies with regard to the behavior of individuals. The Cannabis influence factor (GIF), for example, is an useful tool for distinguishing between acute and chronic intake of A9-THC [98]. 

Cannabinoid analytical work in our laboratory currently consists of two major areas of activity 1) quantitative analysis of A9-THC in samples of physiological fluids submitted by outside researchers under a NIDA program, and 2) development of an ultra-sensitive method for simultaneous quantitation of A9-THC and its major metabolites in body fluids. 

The primary active component of cannabis is A9-tetrahy-drocannabinol (THC), which is responsible for the greater part of the pharmacological effects of the cannabis complex. A8-THC is also active. However, the cannabis plant contains more than 400 chemicals, of which some 60 are chemically related to A9-THC, and it is evident that the exact proportions in which these are present can vary considerably, depending on the way in which the material has been harvested and prepared. In man, A9-THC is rapidly converted to 11-hydroxy-A9-THC (5), a metabolite that is active in the central nervous system. A specific receptor for the cannabinols has been identified it is a member of the G-protein-linked family of receptors (6). The cannabinoid receptor is linked to the inhibitory G-protein, which is linked to adenyl cyclase in an inhibitory fashion (7). The cannabinoid receptor is found in highest concentrations in the basal ganglia, the hippocampus, and the cerebellum, with lower concentrations in the cerebral cortex. 

As it can be observed in Fig. 2, three out of the 16 investigated compounds, namely, heroin, lysergic acid diethylamide (LSD), and its metabolite 2-oxo, 3-hydroxy-LSD (O-H-LSD), were not detected in any wastewater sample. Two other target analytes, 6-acetyl morphine (6ACM) and A9-tetrahydrocannabinol (THC), were only present in influent wastewaters and with low detection frequencies. The most ubiquitous compounds, present in all influent and effluent wastewater samples analyzed, were the cocaine metabolite benzoylecgonine, and the amphetamine-like compounds ephedrine (EPH) and 3,4-methylenedioxymethamphetamine (MDMA or ecstasy). Cocaine, cocaethylene (CE, transesterification product of cocaine formed after the joint consumption of cocaine and ethanol), and morphine (MOR) were detected in all influent, but not in all effluent wastewaters (see Fig. 2). 

A detailed discussion of many alternate methods for the quantitative determination of A9-THC and some of its metabolites has been presented previously (9). This paper will deal with determination of A9-THC, 11-hydroxy -A 9 -THC and cannabinol in blood with one eXtraC-... 

A plasma calibration curve for ll-nor-A9-THC-9-carboxylic acid, 5a, is shown in Figure 9. There was reasonable linearity from 1.0-50 ng/ml plasma with detection limits of 0.5 ng or less per ml. Figure 10 presents similar data for a urine calibration curve. The method showed reasonable linearity between 2.0-100 ng/ ml urine. Figure 11 presents pharmacokinetic data. for plasma levels of a human volunteer, BS, over a 0.5 hour to 48 hour period comparing A9-THC and 11-nor acid levels after a dose of 5.0 mg of A9-THC by the intravenous route. Both parent compound and acid metabolite exhibited a biphasic elimination pattern although the levels of the acid did not fall as rapidly as parent compound. Elimination of the acid metabolite 5a in urine is shown in Figure 12. It is evident that urinary elimination proceeded rapidly as 80% of the total 11-nor-acid excreted was eliminated in the urine during... 

Before entering into the study we felt that it would be useful to have a method that would determine both parent drug and metabolite simultaneously. There were two reasons for this first, the logistics of the work would be greatly improved since only one determination would have to be carried out per sample of plasma second, our original hypothesis was that, by exploitation of the chemistry of the phenol groups we could determine A9-THC and most of its metabolites. 

The HPLC result reported here suggests a transfer of A9-THC into the breast milk of a chronic cannabis user. The calculated level of 0.26 yg/ml extrapolates to 26 yg per average 100 ml feeding which corresponds to 0.05-0.10% of the mother s estimated THC intake. It should be noted that this analysis was on a single sample and has not yet been confirmed by off-line spectral characterization. The identification of A9-THC in the milk must therefore be regarded as tentative. Future work requires multiple sample analyses from both average and chronic users. The milk should be analyzed for both A9-THC and metabolites. 

THC undergoes metabolic degradation in the liver, where it is hydroxylated to 11-hydroxy tetrahydrocannabinol (THC-llOH). The latter, still with psychoactive activity, is oxidized to A9-THC-COOH, an inactive metabolite which is conjugated as 1 l-nor-A9-tetrahydrocannabinol-9-carboxy-glucuronide (A9-THC-COOH-glu), more hydrophilic metabolite and therefore easily excreted in the urine [32],... 

The basic objective of this investigation was to establish sensitive methodology which would not depend on radio-labeling for the quantitative estimation of A9-THC, its primary metabolite 11-hydroxy-A9-THC (3), and cannabinol, which has been reported to be a metabolite of A9-THC in the rat (14). This objective has been realized, utilizing GLC-MS with a variety of techniques and instruments. In addition, the quantitative estimation of 11-nor-A9-THC-9-carboxylic acid has been accomplished. Several aspects of our results merit further discussion. 

A9-THC is the major psychoactive constituent of Cannabis. Its detection and quantitation pose a difficult analytical problem because of its low concentration in biological fluids. Much work has been done on the identification and quantitation of A9-THC, its metabolites and cannabinoids by standard methods such as radio-immunoassay (1,2), gas chromatography, either alone (3-6) or coupled with mass spectrometry (7,8) and fluorometry (9-15). All these methods endeavor to satisfy two major criteria specificity and sensitivity. 

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