A FacilitatedFractionationof Stable Urinary Estrogensbefore Their AutomatedFluorometricAssay

Urine samples to be assayed for estrone, estradiol-17, and estriol are processed as usual: hydrolysis, ether extraction, and saponification, as well as partition between toluene and alkali. The samples are purified further, fractionated, and assayed by connecting columns containing Sephadex LH-20 to the sample line of an automated system for fluorometric assay. With this procedure, a sample can be fractionated and assayed every 45 mm. Additional Keyphrases Sephadex chromatography #{149}acid hydrolysi8 of estro-gen conjugates #{149}Celite #{149}AutoAnalyzer #{149}total estro gene in pregnancy urine diagnostic aid We have developed an automated system for fractionation and simultaneous assay of the three most abundant urinary estrogens (estrone, estra-diol-17$, and estriol1) because of increased requests for such fractionations. It was therefore necessary to replace our previous (1) purification and fractiona-tion steps-which involved countercurrent dis-tribution, magnesium silicate (Florosil) chromatog-raphy, and methylation of the estriol fraction-with a faster system. Acid hydrolysis and a pre-liminary purification by extractions are now fol-lowed by pumping the extract through a Sephadex LH-20 column and directly into the AutoAnalyzer system. The Sephadex also removes some impuri-ties. The net result is a simple procedure for frac-tionation and assay of estrogens that is adequate for routine analysis, and yields a fractionation every 45 mm. From the Section of Research in Endocrinology and Metabo