The multicomponent analysis of estrogens in urine by ion exchange chromatography and GC-MS--II. Fractionation and quantitation of the main groups of estrogen conjugates.

A method for the metabolic profiling of estrogen conjugates in urine is described. It mainly involves protection of carbonyl functions by ethoximation, solid extraction on Sep-Pak C18 cartridges, a number of ion exchange chromatographic steps and quantitation by capillary GC or GC-MS. The acetate form of DEAE-Sephadex is used to initially separate estrogen conjugates into four groups; unconjugated, monoglucuronides, monosulfates and double conjugates. Monoglucuronides are further subfractionated to A- and D-ring glucuronides by carbodiimide methylation of the carboxylic functions and chromatography on the free base form of DEAE-Sephadex. Double conjugates are subfractionated to disulfates and sulfoglucuronides by solvolysis and chromatography on the acetate form of DEAE-Sephadex. After the appropriate enzymatic hydrolysis or solvolysis procedures the liberated free estrogens are purified and fractionated by a series of anion exchange chromatographic steps. Finally, following trimethylsilyl ether derivatization estrogens are analysed by capillary GC or GC-MS. The method permits the quantitation of the main conjugates of all the important estrogen metabolites including catechol estrogens. The method is precise, the sensitivity depending on the quantitation mode employed GC or SIM GC-MS. The method was applied to seven late pregnancy urines the values of which are presented.