A study of the colorimetric estimation of oestradiol-17β, oestradiol-17α, oestrone, oestriol and 16-epioestriol by the Kober reaction

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Abstract
The influence of various reaction conditions on the color production by five naturally occurring estrogens in the Kober reaction was studied. Optimum conditions for the formation of the yellow color from estradiol-17[beta], estrone and estriol were almost identical with those of Brown (1955), whereas those for the conversion of yellow into pink differed considerably. The differences are most likely due to the different commercial types of sulphuric acid used in the two methods. Transformation of yellow color into the pink color is aided by (a) decreasing the sulphuric acid concentration, (b) increasing the heating time, (c) the presence of oxidizing agents and (d) ultraviolet light. Excess of these influences results in fading of the final pink color. The pink-color complex once formed under optimum conditions from both pure crystalline compounds and urinary fractions is stable for at least 24 hr. in the absence of bright light. Urinary contaminants reduce the chromogenicity of phenolic steroids. The effects are small and reproducible in a very narrow range when the color reaction is used in connexion with the Brown purification procedures.