Complete profiling and characterization of in vitro nefazodone metabolites using two different tandem mass spectrometric platforms

Abstract

This paper describes the complete profiling and characterization of in vitro metabolites of the antidepressant agent nefazodone (NEF) generated by human liver microsome (HLM). Two new metabolic pathways (biotransformation) for NEF have been discovered by the characterization of three new metabolites, including two new metabolites (M24, M25) formed due to the N-dealkylation reaction that occurred between the triazolone and propyl units, and one new metabolite (M26) formed due to the O-dearylation reaction that occurred on the phenoxyethyl unit. These metabolites were initially detected by a 4000 Q-Trap instrument and then confirmed by exact mass measurement using an LTQ-Orbitrap. Both instruments proved to be capable of providing complete in vitro metabolite information in a single liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis, although each had its advantages and disadvantages. One noticeable disadvantage of the 4000 Q-Trap was the reduced quality of isotopic pattern in the enhanced mass scan (EMS) spectrum when it was used as survey scan to trigger multiple dependent product ion scans. The problem was especially exacerbated for minor metabolites with low signal intensity. On the other hand, the LTQ-Orbitrap maintained excellent isotopic pattern when used as a full scan survey scan. Twenty-six metabolites were detected and identified. The formation of these new metabolites was also confirmed by analyzing duplicate incubations at different time points.