Optimised separation of E- and Z- isomers of tamoxifen, and its principal metabolites using reversed-phase high performance liquid chromatography

Abstract

A reversed phase isocratic high-performance liquid chromatographic method is reported in which a formal structured procedure, the solvent selectivity triangle, was applied to predict the mobile phase composition giving baseline resolution of the clinically important triphenylethylene antioestrogenic agent (Z)-tamoxifen, its principal (Z)-metabolites, and also the clinically relevant (E)-geometric isomers of tamoxifen and 4-hydroxytamoxifen. The technique of solvent selectivity triangle was used to select the optimal organic modifier parameter for use with a Hichrom ODS 1 column, to achieve baseline separation of six triphenylethylene solutes. The detection system utilised post-column ultraviolet irradiation to convert solutes into their respective photocyclisation products, followed by fluorescence detection ( λ ex=254 nm, λ em=360 nm), and the low detection limit for tamoxifen in serum of 0.1 μM. The optimal mobile phase composition was determined to be methanol–acetonitrile–water–trichloroacetic acid (50:31:18.9:0.1, v/v, pH 2.9). A single stage liquid–liquid extraction method for determination of triphenylethylene drugs in serum was developed. Reproducible recoveries for the (Z)-geometric isomers of tamoxifen (84±3%) and its principal metabolites including Metabolite Y (94±3%), N-desmethyltamoxifen (94±3%) and 4-hydroxytamoxifen (92±3%) were achieved, though more variable results were obtained for their corresponding (E)-geometric isomers (71±7% and 70±10%, respectively).