On-line high-performance liquid chromatographic-electrospray ionization mass spectrometric method for the study of...

An on-line high-performance liquid chromatographic (HPLC)-electrospray ionization mass spectrometric (ESI-MS) method has been developed and optimized for the study of tamoxifen metabolism. Metabolism in mouse liver microsomes was chosen to demonstrate the applicability and superiority of the method, since mice metabolize tamoxifen faster and produce more metabolites than rats or humans. Mouse liver microsomal preparations were incubated with tamoxifen in the presence of NADPH and MgCl2. The metabolites formed were separated and analyzed by the optimized HPLC-ESI-MS system. The separation was performed on a Res Elute-BD column (5 microns particle size, 250 x 4.6 mm I.D.) with 70% (v/v) methanol in 0.5 M ammonium acetate as the mobile phase. A total of eleven metabolites have been detected, some of which have not been previously reported. The metabolites identified are: tamoxifen N-oxide, N-desmethyltamoxifen, 4-hydroxytamoxifen, 4'-hydroxytamoxifen, 4-hydroxytamoxifen N-oxide, 4'-hydroxytamoxifen N-oxide, 4-hydroxy-N-desmethylamoxifen, 4'-hydroxy-N-desmethyltamoxifen, 3,4-dihydroxytamoxifen, 3,4-epoxytamoxifen and 3,4-epoxytamoxifen N-oxide.