Analysis of estrogen sulphate mixtures in pharmaceutical formulations by reversed-phase chromatography.

The relative retention times of estrogen sulphates for reversed-phase, ion-pair and argentous chromatography have been determined. Detector wavelengths of 200 nm, 200 nm and 270 nm were used, respectively. Samples are prepared by dissolution of pharmaceutical formulations in the high-performance liquid chromatography (HPLC) eluent. With reversed-phase chromatography, the estrone, equilin and 17 alpha-dihydroequilin estrogen sulphates are resolved from other estrogen sulphates in 20 min. Any traces of free estrogens which might be present are eluted too slowly for the formation of chromatographic peaks. With ion-pair chromatography, any hydrolyzed estrogens would be eluted before the sulphates with the formation of resolved chromatographic peaks. With argentous chromatography the retention times of the estrogen sulphates varied by nearly a factor of four. The utility of HPLC for the analysis of estrogen sulphates in pharmaceuticals is discussed.