Ll-Hydroxy-A9-THC

Figure 6. GLC (FID) of an extract of 2 mL of plasma containing A9-THC, Peak I (200 ng/mL), and ll-hydroxy-A9-THC, Peak II (200 ng/mL), before (A) and after (B) reversed-phase HPLC separation of both cannabinoids over a range of predetermined collection volumes.

After 24 hours, 3-5 ng/ml of A9-THC were still found in the plasma. Our results for ll-hydroxy-A9-THC are probably the most accurate data yet reported in man. The concentration of this active metabolite (Figure 6) was only 2-3 ng/ml at peak levels declining at a slower rate than A9-THC to 0.5 ng/ml after 24 hours. Although A9-THC is readily converted to 11-hydroxy-A9-THC in the liver (3), only small quantities find their way into the blood. 

We attempted to approach the determination of ll-hydroxy-A9-THC in the same way. Preliminary experiments showed that ll-hydroxy-A9-THC was not very soluble in Brodie s solvent and the metabolite was unstable to methylation with trimethylanilinium hydroxide. 

There was one further problem, namely the 1-0-ethyl-ll-hydroxy-A9-THC was susceptible to pyrolysis at the elevated temperatures of the ion source. This resulted in irreproducible mass spectra. Silylation of the allylic alcohol functionality overcame this difficulty and the resulting electron impact fragmentation pattern was quite simple showing only one major peak, base peak at m/e = 327. The trimethylsilyl ion appeared at m/e =73 (7). 

This was interesting because there are several monohydroxylic metabolites of A9-THC. If we were monitoring, say, the molecular ion, we would detect all of these metabolites. Since 327 results from a loss of C-ll, this ion is specific for the derivative of ll-hydroxy-A9-THC. This is probably a minor point, because the various monohydroxylated metabolites have been shown to be separable by gas chromatography. Nevertheless, it does result in a highly specific determination for the most debated metabolite. 

We carried out a study in the dog to determine the formation of ll-hydroxy-A9-THC from A9-THC. In the first stage we injected 11-hydroxy-A9-THC in order to determine the beta phase half-life of the metabolite the half-life was approximately 1.5 hours. However, when A9-THC was administered to the same dog either orally or intravenously the metabolite was not detectable. 

In order to answer some of these questions we embarked on a second study. The purpose of this study was to show that A9-THC was present in the circulation of an animal and as a consequence, ll-hydroxy-A9-THC appeared in the plasma. For this study we chose the rabbit as our animal model. Several workers have shown that the rabbit oxidizes A9-THC at the C-ll position and also that rabbit liver microsomes oxidize A9-THC at the C-ll position and also that rabbit liver microsomes oxidize A9-THC to ll-hydroxy-A9-THC. We were thus assured that we would not be dealing with a species where there was doubt about oxidation at C-ll (3). ... 

A simple study on the simultaneous extraction of radio-labeled A9-THC and ll-hydroxy-A9-THC once again... 

Having determined the most appropriate extraction from plasma we investigated the simultaneous derivati-zation of A9-THC and ll-hydroxy-A9-THC. We have claimed that ethylation of 11-hydroxy-A9-THC proceeded by phase transfer catalysis (7). However, it is known that quaternary ammonium hydroxides are capable of catalyzing alkylations with alkyl iodides in aproptic solvents (8). Furthermore, we had not demonstrated that A9-THC could be derivatized under the same conditions as ll-hydroxy-A9-THC. We found that the minimum requirement for the reaction to proceed is the presence of water, which probably increases the degree of ionization of the quaternary ammonium hydroxide. However, in order for the reaction to go to completion, at least 0.1N NaOH is necessary. This supports the contention that this derivatization is, to some extent, a phase transfer catalyzed alkylation. 

We had shown that it was possible to extract both A9-THC and ll-hydroxy-A9-THC from plasma with toluene. The next question was the problem of lipophiles. Again we found that in order to remove interferences to A9-THC determinations it was necessary to fractionate the toluene extract with Claisen s alkali in order to obtain the phenol fraction of plasma. We found that the extractions of A9-THC from toluene with Claisen s alkali is not quantitative, but it is reproducible. The loss of A9-THC in this reaction however, is compensated for by removal of interferences which were the limiting factors in the single extraction step. 

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. 

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