Separation of Mixtures of Œstrogens in Urinary Extracts using Gradient Elution Chromatography

Abstract

IN the course of studying the metabolism of œstradiol-4-¹⁴C in human mammary cancer, a method of separating mixtures of œstrogens was developed. The method consists of gradient elution chromatography on silica gel with progressively increasing concentration of ethyl acetate in benzene. Separation of mixtures of œstrogens on columns impregnated with alkali¹ was considered potentially detrimental to labile œstrogen metabolites. Other schemes, such as chromatography of œstrogen methyl ethers on alumina³, were likewise considered inadequate to demonstrate subtle œstrogen metabolic differences without risking either partial decomposition or ease of subsequent identification of metabolites. With the system which has been devised, the separation of a synthetic mixture of œstrogens is complete, and excellent recoveries are obtained. The slight variation in retention volumes is presumably due to lack of temperature control. While the capacity of the columns for separating synthetic mixtures has not been determined, microgram amounts of radioactive œstrogens in urinary extracts weighing 250 mgm. have been successfully separated on 2 gm. columns.