Group separation of steroid conjugates by DEAE-Sephadex anion exchange chromatography

Abstract

A stepwise elution system was devised for the separation of different groups of steroid conjugates using the acetate form of DEAE-Sephadex A-25 in methanol. Seven groups of steroids were eluted in the following order: unconjugated steroids, glucuronides of neutral steroids, estrogen A-ring glucuronides, estrogen D-ring glucuronides, monosulphates of neutral steroids, monosulphates of estrogens, and mixed and double conjugates of neutral steroids and estrogens. The advantage of this system is its ability to separate conjugates of neutral steroids from the corresponding estrogen conjugates. Thus, basic anion exchangers, which transform catechol and ring-D estrogens, do not have to be used to separate neutral and phenolic steroids after hydrolysis. When applied after an appropriate extraction step, the separation method is useful for analysis, e.g. by capillary GLC and GC/MS, of estrogen profiles in different types of biological material. In the case of urine, where estrogens are present almost exclusively as conjugates, high yields are expected of all known estrogen metabolites including the labile ones.