A method for the quantitative analysis of 17-ketosteroid mixtures.

Neher and Wettstein (1) have recently published the details of a paper chromatographic method for the separation of weakly polar steroids, with a heptane-phenyl Cellosolve solvent system. Savard (2) has described the separation of 17-ketosteroids on paper with ligroin as the mobile phase and propylene glycol as the stationary phase. Starting from the basic work of these authors, a method has been developed for quantitative analysis of 17-ketosteroid mixtures. It has been designed chiefly for application to the analysis of the principal 17-ketost.eroids of urine, although it is not limited to urinary mixtures. The least polar components, such as A2cor 3, -androstene-17-one and 3/3-chloro-A6-androstene-17-one, have been resolved with the heptane-phenyl Cellosolve system. A heptane-propylene glycol system has been employed to separate the more polar components, such as androsterone: etiocholane-3cu-ol17-one, 11-ketoandrosterone, and etiocholane-3a-ol-11,17-dione. The paper chromatographic systems will not resolve a mixture of androsterone and Ag(11)-androstene-3a-ol-17-one or one of etiocholane-3a-ol-17one and Ag(n)-etiocholene-3~-ol-17-one. However, the Ag(“)-unsaturated compounds can be oxidized to the respective epoxides with perbenzoic acid, as suggested by Lieberman et al. (3). The mobilities of the epoxides in the heptane-propylene glycol system are sufficiently different from the mobilities of androsterone and etiocholane-3a-ol-17-one to afford complete resolution. Perbenzoic acid oxidation of the androsterone and etiocholane3cu-ol-17-one zones obtained from urine extracts by paper chromatography makes possible the quantitative determination of androsterone, etiocholane3cr-ol-17-one, Ag(11)-androstene-3~-ol-17-one, and Ag(n)-etiocholene-3cr-ol-17one. These Ag(ll)-unsaturated steroids are artifacts which arise from corresponding 1 lp-hydroxy compounds. Thus this analysis gives a measure of the concentration of 1 lb-hydroxyandrosterone and etiocholane-3cr, 1 lpdiol-17-one originally present in the urine.