Separation of Six Urinary 17-Ketosteroids by Two-Dimensional Thin-Layer Chromatography; Control Values and Response to ACTH

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Abstract
Two-dimensional thin-layer chromatography on aluminum oxide provides a simple, rapid, yet sensitive method for the separation and identification of individual urinary 17-ketosteroids. By employing 2 solvent systems in which the steroid compounds exhibit different mobilities, it is possible to separate not only the 11-oxygenated 17-ketosteroids but also such closely related compounds as dehydroepiandrosterone, androsterone and etiocholanolone on 1 single plate within 1 hr. The solvent systems giving the best results are 3.5 % methanol in benzene and equal volumes of ethyl ether and ethyl acetate. Good separation is obtained with pure standards as well as urine extracts. As little as 1 μg of material can readily be detected, so that 25–50 ml of urine is adequate for fractionation of the major urinary 17-ketosteroids. Recoveries of pure compounds as well as standards added to urine exceed 80%. The urinary excretion of the 6 major 17-ketosteroid metabolites in 6 normal males and 12 normal females, during a control period and following a standard 2-day ACTH test by the procedure described, yielded results which compare well with data obtained by more complicated methods employing column or sequential paper chromatography.

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