The analysis of urinary 17-oxo steroids by gradient elution

Abstract

A new method for the quantitative analysis of the 17-oxo steroid fraction of urine is described which avoids the formation of artifacts during hydrolysis of conjugates and which gives improved separation of individual 17-oxo steroids. The hydrolysis of the 17-oxo steroids glucuronides is achieved with B-glucuronidase prepared from limpets (Patella vulgata) and, subsequently, that of 17-oxo steroid sulfates by continuous extraction of an acidified aqueous solution with ether. The separate chromatographic analysis of material liberated by these processes has given information on the form of conjugation of individual 17-oxo steroids. The application of the gradient-elution principle to the Dingemanse system for the chromatographic analysis of 17-oxo steroid mixtures has given improved separation, particularly of the 11-oxygenated 17-oxo steroids, all of which have been found in normal urine. The identity of the separated 17-oxo steroids was established by infrared spectroscopy and by the application of paper chromatography to the free 17-oxo steroids and to the complexes which these compounds form with m-dinitrobenzene. Results obtained by the analysis of urine samples from a group of normal subjects and from patients suffering from various forms of adrenal disfunction are presented.