NEW COLOR REACTION FOR CERTAIN URINARY 17-KETOSTEROIDS1

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Abstract
An intense blue color (absorption maximum at 610uu) is developed by andro-sterone and its isomers and by androstenone-17 after heating with concentrated SbCl3 soln. (or amorphous SbCl3) and dilution with glacial acetic acid. Androsterone succinate and androsterone sulfate develop the same intense color. Androstanediol develops qualitatively the same color but in reduced intensity. Dehydroisoandrosterone and its succinate develop a weak yellowish-blue color and pregnandiol a weaker, somewhat similar color. Testosterone, allopregnanolone, epipregnanolone, androstenedione, andro-stanol-3[beta] (?) one, progesterone, cholestenone, and cholesterol develop extremely weak yellow colors. The optimal method for development of the typical stable color requires: a) using a soln. containing 3.8 g. of SbCl3 per 0.9 cc. of glacial acetic acid and 0.1 cc. of acetic anhydride which is added drop by drop in 0.2 cc to the dried steroid prep., b) heating, with shaking for 20 mins. in a boiling waterbath, c) diluting with a soln. containing 95 parts of glacial acetic acid to 5 parts of distilled water, d) incubating in the dark at room temp. for 40-60 mins. When chloroform is used as the diluent, a qualitatively different color develops with androsterone, having absorption maxima at 560 and 620. Dehydroisoandrosterone with chloroform dilution develops a similar, less intense color. Androsterone with acetic anhydride dilution develops a greenish color with absorption maxima at 460 and 610 [mu][mu]. The application of the color reaction to urinary neutral ketonic extracts from the urines of [male][male] and [female][female] gives a color titer less than that of the Zimmerman titer, the largest difference (36-49% less) occurring in the non-alcoholic ketonic fractions.