Reductive amination-assisted quantitation of tamoxifen and its metabolites by liquid phase chromatography tandem mass spectrometry

Abstract

Tamoxifen, a hormonal therapy drug against estrogen receptor-positive breast cancer, can be metabolized by cytochrome P450 enzymes such as CYP3A4 and CYP3A5, and converted to N-desmethyltamoxifen, which is subsequently, metabolized by CYP2D6 and inverted to form 4-hydroxy-N-desmethyltamoxifen (endoxifen). Conventional mass spectrometry (MS) analyses of tamoxifen and its metabolites require isotopic internal standards (ISs). In this study, endoxifen and N-desmethyltamoxifen amine groups were modified by reductive amination with formaldehyde-D2 to produce new metabolite molecules. Both endoxifen and N-desmethyltamoxifen generated their corresponding D2-methyl modified analogs. This method is expected to simplify MS detection and overcome the difficulty in selecting adequate ISs when tamoxifen metabolites are analyzed by absolute quantification. It identified tamoxifen, D2-methyl modified endoxifen, and D2-methyl modified N-desmethyltamoxifen with a linearity ranging from 2 to 5000 ng/mL with correlation coefficient (R(2)) values of 0.9868, 0.9849, and 0.9880, respectively. Furthermore, this reductive amination-based method may enhance the signal intensities of D2-methyl modified N-desmethyltamoxifen and endoxifen, thus facilitating the MS detection.

Keywords: Absolute quantification; Endoxifen; N-Desmethyltamoxifen; Reductive amination; Tamoxifen.