A SIMPLE QUANTITATIVE COLORIMETRIC METHOD FOR ESTROGENIC STEROIDS

Abstract

MANY colorimetric methods for determining natural estrogens have been described, most widely used of which is the reaction described by Kober (1931) (4). Although supposedly specific for estrogenic steroids, this reaction has been shown by Mather (1940) (6) to give similar positive tests with five times as much androsterone. Kober (1931) (4) noted that a red color was obtained upon the addition of concentrated sulfuric acid to estrone, heating, diluting with water and reheating. However, an intense greenish fluorescence was also present which interfered with the quantitative colorimetric determination by visual methods. He found that addition of phenol quenched the fluorescence and permitted a more accurate determination of the red color. The Kober test is a two phase reaction. The first is heating with phenolsulfonic acid, which produces a yellow colored solution, and the second phase is dilution with water or dilute sulfuric acid and reheating, which produces a red colored solution. Various modifications have been described by Cohen and Marrian (1934) (3), Cartland et al. (1935) (2),Pincus etal. (1936) (7), Venning et al. (1937) (9), Kober (1938) (5), Bachmann (1939) (1) and Szego and Samuels (1943) (8).