19-Nortestosterone phenylpropionate was found to color yellow when heated with sulfuric acid, and addition of acetone changed this color to reddish violet, which had absorption maximum at 585mμ. This color reaction was tested with various kinds of steroid and Δ⁴-3-ketosteroids all showed good coloration of reddish violet to blue, having absorption maxima in the range of 580-610mμ. Limit of detection of 19-nortestosterone phenylpropionate by this method was 0.4μg./0.05ml.
Examinations were made on the colorimetric determination using this color reaction and a method was established for quantitative determination of 19-nortestosterone phenylpropionate. At the same time, application of this method for oily injections was examined. It was found that solution of this sample in isooctane and extraction with 90% methanol resulted in elimination of interference by vegetable oils, giving a good result. Recovery of synthetic sample by this method was 100.3% (n=8) and precision of measurement was σ=0.61% (n=8).
Colorimetric Procedure: To 1ml. of the test solution (chloroform solution containing about 40μg./ml. of 19-nortestosterone phenylpropionate) placed in a 10ml. volumetric flask, 0.8ml. of sulfuric acid is added, shaken vigorously for 1 minute, and heated in a boiling water bath for 10 minutes. The flask is cooled in ice, while adding 25ml. of acetic acid with shaking, and 4ml. of acetone is added with shaking. The whole volume is brought exactly to 10ml. with chloroform and allowed to stand for 5 minutes. Absorbance of this solution is measured at 585mμ. A blank test is carried out with the same amount of the same reagent and in the same manner, using 1ml. of chloroform in place of the test solution.
In the case of oily injection, the sample is dissolved in isooctane, extracted with 90% (v/v) methanol, and methanol is evaporated from the extract. Benzene is added to its residue, the solvent is evaporated, and the residue is dissolved in chloroform. This chloroform solution is submitted to the same procedure as above.