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Optimization of an isotope dilution gas chromatography/mass spectrometry method for the detection of endogenous estrogen metabolites in urine samples.

A gas chromatography/mass spectrometry (GC/MS) method for the simultaneous quantitation of ten estrogen metabolites in human urine was optimized. The method consists of initial enzymatic hydrolysis of the estrogen conjugates using beta-glucuronidase followed by solid-phase extraction (SPE) on Sep-pak C18 columns and further sample purification by ion-exchange chromatography on QAE-Sephadex cartridges in the acetate form. QAE-Sephadex cartridges in the borate form were used to separate estrogens into two fractions: one fraction containing estrogens lacking vicinal cis-hydroxyls (Fr 1) and another containing estrogens possessing vicinal cis-hydroxyls (catecholestrogens; Fr 2). Finally, following O-trimethylsilyl ether derivatization, the estrogens were analyzed by GC/MS in the selected ion monitoring mode. Estrogens were quantitated using deuterated internal standards, which were added to the samples at the initiation of the work-up procedures. After addition to estrogen-low male human samples the standards showed good chromatographic linear response and reproducibility. A reduction in the number of steps and improvements in the robustness of the work-up procedures were achieved. The modified method described is less complex, amenable to use with commercially available SPE columns and fulfils all the reliability criteria, resulting in highly specific and accurate results.