EFFECT OF WATER ON THE M-DINITROBENZENE REACTION IN THE DETERMINATION OF CRYSTALLINE ANDROGENS BY THE EVELYN PHOTOELECTRIC COLORIMETER1,2

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Abstract
The presence or absence of water influences the colorimetric reaction of androsterone and dehydroisoandro-sterone or various mixtures of the 2 with m-dinitrobenzene in an alkaline alcoholic soln. Dehydroisoandrosterone yields more color per unit of wt. than androsterone (1.24 to 1) when the reaction is carried out in 2N aqueous KOH. When the color reaction is carried out in absolute alc., however, these androgens give the same amt. of color per unit wt. of substance. In the latter circumstance, mixtures of the 2 androgens may be treated as simple solns. of one or the other substance from a chromogenic viewpoint. This rule does not apply to solns. in which aqueous KOH is used in the reaction mixture. In the latter case, if the "total andro-gen" of a mixture of the 2 is reported in mg. equivalents of androsterone, an error will appear which will depend in part on the difference in chromogenic properties of the individual androgens. Hence, the error will be proportional to the amt. of dehydroisoandrosterone in relation to the androsterone present in the mixture. Another factor which may play an important role in magnifying this error in the detn. of the total androgen in a mixture of these substances is the total amount of each androgen in soln. In the interests of greater accuracy each of the androgens should be present in soln. in amts. which can be detd. from their respective calibration curves in accordance with Beer''s law. The results obtained by the use of aq. KOH in the m-dinitrobenzene reaction are higher in individual instances than those reported for corresponding groups of patients by Callow, Callow and Emmens who used only absolute alc. in the reaction mixture; both series of results, however, lie within the same range. Consequently the differences in the results obtained by these 2 technics are not significant from a clinical viewpoint. From a chemical standpoint, however, the use of alcoholic KOH represents a refinement in technic and leads to more nearly quantitative results. The universal adoption of the Wu and Chou-Callow method in one of its acceptable modifications is urged in order to standardize the colorimetric procedure for the detn. of the ketosteroid content of urine extracts.