The analysis of urinary 17-oxo steroids by gradient elution.

The present work was initiated by the observation of Reifenstein, Homburger & Dobriner (1950) that 3a: 1 1l-dihydroxy-5p-androstan-17-one (1 l,B-hydroxyaetiocholanolone), a minor component of the urinary 17-oxo steroids, was significantly associated with neoplastic growth. The re-examination of this problem has led to the development of a micro method for the quantitative separation and determination of the individual compounds which comprise the 17-oxo steroid fraction of urine. Callow, Callow & Emmens (1938) first applied the Zimmermann (1936) reaction with m-dinitrobenzene and ethanolic potassium hydroxide for the determination and isolation of androgen metabolites from urine. Extension and development of this work led to the isolation and characterization of a large number of urinary steroids, many of which were 17-oxo steroids (Dobriner, Lieberman & Rhoads, 1948a; Lieberman, Dobriner, Hill, Fieser & Rhoads, 1948; Lieberman, Fukushima & Dobriner, 1950). In contrast to the large-scale work of the American investigators, Dingemanse, Huis in't Veld & De Laat (1946) described a method for the analysis of the 17-oxo steroid fraction which could be applied to a urine sample smaller than a 24 hr. sample. The fraction was adsorbed on alumina and the individual compounds were eluted from the alumina with benzene containing increasing concentration of ethanol; the ethanol concentration was increased stepwise. This method in its original form and in the many modifications to which it gave rise (Robinson & Goulden, 1949; Zygmuntowicz, Wood, Christo & Talbot, 1951; Pond, 1951) has been of great value in the study of 17-oxo steroid metabolism. The essentials of a reliable method for the analysis of the urinary 17-oxo steroid fraction are quantitative extraction, complete separation of compounds, unequivocal identification and precise determination. The existing methods do not fulfil these requirements and, in particular, are unsuited to the determination of the 11-hydroxy-17-oxo steroids. Three main improvements have been introduced. The 17-oxo steroids are excreted almost exclusively in the conjugated form as glucuronides