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

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 (lambda[ex] = 254 nm, lambda[em] = 360 nm), and the low detection limit for tamoxifen in serum of 0.1 microM. 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).