ANALYSIS OF URINARY STEROIDS FOLLOWING GLUCURONIDASE HYDROLYSIS. I. EXTRACTION AND FRACTIONATION*

Abstract

WE have presented a preliminary description of methods for the extraction and separation of urinary corticosteroids (1,2). With the development of glucuronidase hydrolysis (3) and the consequent increased yield of corticosteroid, along with methods for paper partition chromatography (4, 5, 6), the possibility of routinely determining patterns of corticosteroids present in two- to three-day collections of human urine seemed feasible. It soon became evident that a considerable array of substances was revealed by paper chromatography of glucuronidase-hydrolyzed urines and a systematic effort at the resolution of the steroid mixture was initiated. In this paper we present the techniques evolved for the pattern analysis. The chief features of the method now routinely employed are: (a) glucuronidase hydrolysis followed by methylene chloride extraction and separation of a crude neutral lipid, (b) fractionation of the neutral lipid by silica gel chromatography into three major “steroid” eluates, and (c) paper partition chromatography of the three major “steroid” fractions, conducted so as to effect adequate separation of the component substances. In this paper we also present data on a number of urine extracts which illustrate the separations achieved and the types of substances observed.