Some errors in the colorimetric estimation of oestriol, oestrone and oestradiol by the Kober reaction

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Abstract
Solvent residues interfere with production of color by estriol, estrone and estradiol-17-beta in the reaction with the quinol: sulfuric acid reagents of Brown (Jour. Endocrin., 8, p. 196, 1952). There are 3 different kinds of interference with the color reaction. The 1st (failure to form the initial yellow complex; type I inhibition) applies only to estriol and is due to diminished reducing power of the reagent as a result of sulfonation of the quinol. This has been overcome by addition of quinol immediately before color development The other 2 types of interference depend upon the fact that the 2d stage of the Kober reaction, involving a conversion of the yellow complex into pink, appears to be an oxidation which, if excessive, causes fading. Under the conditions descr. by Brown (1952) the conversion of yellow into pink and the stability of the latter are influenced by the degree of illumination of the laboratory, size of the tube used and degree of stirring. Elimination of the final dilution stage, the use of larger-bore tubes and modification of the conditions for the 2d stage removed these variable factors. The opt. reaction conditions depend upon the make of analytical reagent grade sulfuric acid used; this variable can be eliminated by inclusion of trace amts. of sodium nitrate and quinone in the reagent. The modified reaction is unaffected by solvent residues and gives color intensities which are reproducible and obey Beer''s law over a suitable range of concn.