Quantitation of Toremifene and its Major Metabolites in Human Plasma by High-Performance Liquid Chromatography Following Fluorescent Activation

Abstract

A highly sensitive reverse-phase high-performance liquid chromatography assay utilizing fluorescence activation has been developed for the quantitative analysis of the anti-estrogenic compound toremifene and its major metabolites, 4-hydroxy-toremifene and N-desmethyl-toremifene. Plasma samples containing various quantities of toremifene and its metabolites were spiked with an internal standard (nafoxidine), extracted with 2% n-butanol in hexane, and irradiated with high intensity ultraviolet light (254 nm). Aliquots of the extracted plasma components were then injected onto a C-18 reversed phase column and eluted isocratically with a mobile phase of water and triethylamine in methanol. Fluorescence of toremifene, its metabolites, and internal standard was measured at an excitation wavelength of 266 nm. -The sensitivity of this assay was 8.0, 15.0 and 5.0 ng/mL for toremifene, N-desmethyl-toremifene and 4-hydroxy-toremifene, respectively. Linearity was achieved for the concentration range of 25 to 400 ng/mL for all the compounds, with correlation coefficients of greater than 0.994. The assay presented is highly specific, very sensitive and demonstrates reproducible linearity throughout a wide range of clinically relevant plasma toremifene concentrations.

Key Words:

• Toremifene
• HPLC
• human plasma