14C DANS

The method we have developed for the detection and quantitation of A9-THC in plasma also utilizes dansyl technology, but our methodology is based on the use of 14C DANS-Cl and, for quantitative determination, on the use of 3H2 A9-THC as an internal standard (19-22). ... 

The main steps for detection are 1) Extraction of A9-THC from plasma 2) Esterification by 14C DANS-Cl 3) Purification by TLC 4) Elution of the 14C DANS-A9-THC spot and measurement of i4C activity. 

In a hemolysis tube, 60 nmoles 14C DANS-C1 solution in 60 yl acetone, and 15 yl 0.5 M Na2CC>3 buffer are added either to 50 yl of the acetone solution (extraction) or to a solution of 3.18 nmoles A9-THC (yield study). Simultaneously with each test, a blank is made up under the same conditions, but without A9-THC. The tubes are stoppered, wrapped in aluminum foil and incubated for one hour at 40°C. After cooling, 18 nmoles of unlabeled DANS A9-THC (carrier),... 

The maximum yield of 14C DANS-A9-THC (22.5 0.6%) is obtained in 120 minutes, but as early as the fifteenth minute the rate of esterification is considerably slowed. A comparative study conducted for 24 hours at room temperature did not show how the yield could be significantly increased. The reaction time does not therefore have much effect on the dansylation yield. Consequently, it is preferable to operate at maximum temperature for a brief period, i.e. for one hour at 40-45°C. 

Experience has shown that 14c DANS-C1 excess is a predominant factor in esterification. Whereas at preparative scale five times more DANS-C1 (in moles) are sufficient to obtain a DANS A9-THC yield of about 60%, this does not suffice to obtain an acceptable yield in the case of microsynthesis. A study of the effects of 14c DANS-C1 excess in relation to A9-THC (r = i4c DANS/C /A9-THC) shows that the yield varies in three ways ... 

The yield therefore depends very much on the 14C DANS-Cl excess, since a large excess of labeled reagent cannot lie used. On the other hand, the lower the A9-THC concentration, the higher the ester yield, which is conductive to satisfactory detection sensitivity. ... 

The influence of the concentration of the reagents was checked under the optimal conditions described above, by varying the reaction volume but keeping the amounts of A9-THC and 14C DANS-C1 constant. 

The aim of hydrolysis is to destroy 14C DANS-C1 excess vtftich has not reacted by turning it into a salt which, under our chromatographic conditions, remains at the origin and therefore does not contaminate the 14C DANS A9-THC spot. TLC performed on a synthetic extract before and after hydrolysis shows that ... 

The separation between the different dansyl-can-nabinoids can be obtained by the use of the one or two-dimensional TLC. Autoradiographies showed that the major difficulty is to have a good separation between dansyl-cannabinoids and 14C dansyl breakdown products which show several spots which are formed under the experimental conditions. All cannabinoids tested show one spot except CBD which give two spots. 14C DANS-neutral cannabinoids give a satisfactory separation in a one-dimensional TLC using the cyclo-hexane/ethyl acetate (95 5) (Fig. 9) and also in a two-dimensional TLC with cyclohexane/ethyl acetate (95 5) for the first migration followed by hexane/ benzene (60 40) for the second (Fig. 10). The best separation for the 14C DANS-ll-OH-A8 and A9-THC was obtained using the cyclohexane/acetone triethylamine (70 25 5) system (Fig. 11). 

The result given in Table IX confirm the observations made when the effects of 14C DANS-Cl excess were studied, i.e. that the proportionality between the amount of A9-THC and the response was only verified between 1000 and 50 ng. 

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