CHROMATOGRAPHIC PROCEDURE FOR THE DETERMINATION OF URINARY CORTICOSTEROIDS & C19STEROIDS

Abstract

A comprehensive procedure for the routine analysis of urinary steroids is described. It consists of: (A) A simple partition chromatographic column operated so as to separate 3 fractions predominantly composed of (a) C19O2 steroids or androgen metabolites (e.g., androsterone and dehydroepiandrosterone) and C21O2 steroids (e.g., pregnanediol); (b) 11-oxygenated C19O3 steroids (e.g., 11-keto-etiocholanolone) and C21O3 metabolites (e.g., pregnanetriol); and (c) all corticosteroids, including corticosterone, tetrahydrocortisol and more polar metabolites. (B) Application to each fraction of 2 or more assays, each specific for certain groupings attached to carbon 17; two of these determinations[long dash]17-ketosteroids present after oxidation with CrO3 (CrO3-KS) and formaldehydogenic steroids (FS)[long dash]measure almost all types of adrenocortical steroid metabolites. The column consists of synthetic aluminum silicate as the supporting phase, and 50 per cent ethanol as the stationary phase. It is developed by a "stepwise gradient" employing hexane containing increasing proportions of chloroform. The column and assays can be used for urine extracts representing from one to six hours'' collection, or up to about 10 mg of a steroid mixture. It runs completely in one working day, and avoids large volumes of solvents. Eluates are sufficiently pure to yield accurate titers in various colorimetric assays specific for different side-chains or groupings. Thus, the excretion of several types of metabolites can be determined without complete separation. In the corticosteroid fraction of the urine from normal subjects, 24-hour excretion values were about 7 mg of 21-hydroxy (formaldehydogenic) steroids, 6-7 mg of total 17-hydroxy steroids (CrO3-KS), 2 mg of 17,20,21-triols (such as cortols) and 2.5 mg of Silber-Porter chromogens. Examples of other readily obtainable information in a variety of human subjects are presented.