EFFECT OF WATER ON ACCURACY OF M-DINITROBENZENE REACTION IN THE QUANTITATIVE ASSAY OF URINARY KETOSTEROIDS1

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Abstract
The m-dinitrobenzene reaction for the quantitative detn. of ketosteroids is currently being carried out in the form of a number of modifications of the original Zimmermann procedure which differ fundamentally in only 1 respect, viz., the water content of the reaction mixture. The presence of water in a reaction mixture containing crystalline androsterone and dehydroiso-androsterone interferes to some extent with the accuracy of its assay. A significant error has been detected in the assay of urine extracts for their ketosteroid content by the same method, e.g., doubling the amt. of urine extract used for assay by the aqueous KOH method does not result in double the intensity of color which would be expected on the basis of Beer''s law from a 2-fold increase in the amt. of ketosteroids. This error is reduced to minor proportions if 1.0 cc. instead of 0.2 cc. of a urine extract is used for each colorimetric detn. The deviation from proportionality is obviated by the use of the Wu and Chou-Callow absolute alc. technic. Although a correction factor can be calculated for each urine extract if the aq. KOH method is used, it is urged that the absolute alc. procedure be adopted universally for the detn. of the ketosteroid content of urine extracts in order to standardize this technic and make the results reported by different investigators more readily comparable.

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