Methods for the estimation of grouped urinary steroids by quantitation as 17-ketosteroids.

Abstract

ABSTRACT Methods for the estimation of urinary total 17-ketosteroids and 17-hydroxycorticosteroids, both total and 11-deoxy and 11-oxy fractions are described. It was found that for the hydrolysis of 17-ketosteroid conjugates in a boiling water bath better results were obtained by boiling the material for 7 minutes with hydrochloric acid 4 mol/l than for 35 minutes with hydrochloric or sulphuric acid 1 mol/l. The 17-hydroxy-corticosteroids were estimated using the reaction sequence – borohydride reduction, periodate oxidation, basic hydrolysis, partition chromatography and quantitative estimation of the 17-ketosteroids produced. Basic hydrolysis of the formates produced by periodate oxidation was complete at pH 13, but not at pH 9. Quantitation of the 17-ketosteroids was carried out by a modified Zimmermann reaction, extraction of unionised colourless forms of the reaction products, conversion of these into quinoid forms and measuring the extinction at 530 nm. KOH 4 mol/l, methanol as the reaction medium and incubation for 35 minutes at 13–15° C were found to be optimal for colour development. A two phase system consisting of a water phase with KOH concentrations of less than 0.16 mol/l and a pure dichloromethane phase produced optimal conditions for the extraction of the unionised stable reaction products of androsterone, aetiocholanolone, dehydroepiandrosterone and 11-hydroxy-aeticcholanolone. The concentration of the methanol in the water phase was 20% (v/v). Excellent specificity was obtained for the technique used, when evaluated by the absorption spectra for urinary 17-ketosteroids and by comparing the values obtained with and without purification by partition chromatography. It is concluded that the methods are simple, reliable and suitable for routine analysis.