Determination of dehydroisoandrosterone and 17-ketosteroids.

Methods for the quantitative determination of steroids have been developed, based on their specific chemical or physicochemical properties. The Zimmermann reaction (1) and the Kober reaction (2) have been found useful in the quantitative determination of certain individual steroids occurring in urine. Modifications of these reactions have been reported by Callow et al. (3), Holtorff and Koch (4), and others (5, 6). These reactions give characteristic colors with specific absorption maxima when pure compounds are used. The chromogenic impurities in urinary extracts make a correction factor necessary when these methods are employed. The color produced by the interaction of alcoholic HzS04 and certain steroids has been used for the estimation of testosterone (7) and dehydroisoandrosterone (8, 9). A color reaction between various steroids and the anthrone reagent has been described in a recent publication from this laboratory (10). The reaction of the anthrone reagent with urinary steroid extracts and a method of computing the concentration of dehydroisoandrosterone and total 17ketosteroids are reported here. When the individual absorption spectral curves of each of several components of a mixture of substances are known, the proportional amounts of each of the substances may be determined from the spectral characteristics of the mixture. The reaction of the anthrone reagent with androsterone and dehydroisoandrosterone yields absorption spectral Curves A and B respectively (Fig. 1) as well as Curve D resulting from the mixture of androsterone and dehydroisoandrosterone. Spectral absorption maxima for the constituents are defined in the region of 415 to 420 and 470 to 475 mp. Since the contours of the absorption spectral curves of the components are established, optical density measurements are required at only two wave-lengths to resolve the composition of any mixture of these constituents, provided the materials conform to Beer’s law when examined at the designated wave-lengths. The colored solutions produced by the re-