ABSTRACT:The therapeutic benefits of the antidepressant nefazodone have been hampered by several cases of acute hepatotoxicity/liver failure. Although the mechanism of hepatotoxicity remains unknown, it is possible that reactive metabolites of nefazodone play a causative role. Studies were initiated to determine whether nefazodone undergoes bioactivation in human liver microsomes to electrophilic intermediates. Following incubation of nefazodone with microsomes or recombinant P4503A4 in the presence of sulfydryl nucleophiles, conjugates derived from the addition of thiol to a monohydroxylated nefazodone metabolite were observed. Product ion spectra suggested that hydroxylation and sulfydryl conjugation occurred on the 3-chlorophenylpiperazine-ring, consistent with a bioactivation pathway involving initial formation of p-hydroxynefazodone, followed by its two-electron oxidation to the reactive quinone-imine intermediate. The formation of novel N-dearylated nefazodone metabolites was also discernible in these incubations, and 2-chloro-1,4-benzoquinone, a by-product of N-dearylation, was trapped with glutathione to afford the corresponding hydroquinone-sulfydryl adduct. Nefazodone also displayed NADPH-, time-, and concentration-dependent inactivation of P4503A4 activity, suggesting that reactive metabolites derived from nefazodone bioactivation are capable of covalently modifying P4503A4. A causative role for 2-chloro-1,4-benzoquinone and/or the quinone-imine intermediate(s) in nefazodone hepatotoxicity is speculated. Although the antianxiety agent buspirone, which contains a pyrimidine ring in place of the 3-chlorophenyl-ring, also generated phydroxybuspirone in liver microsomes, no sulfydryl conjugates of this metabolite were observed. This finding is consistent with the proposal that two-electron oxidation of p-hydroxybuspirone to the corresponding quinone-imine is less favorable due to differences in the protonation state at physiological pH and due to weaker resonance stabilization of the oxidation products as predicted from ab initio measurements.

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We report herein, facile metabolite identification workflow on the anti-depressant nefazodone, which is derived from accurate mass measurements based on a single run/experimental analysis. A hybrid LTQ/orbitrap mass spectrometer was used to obtain accurate mass full scan MS and MS/MS in a data-dependent fashion to eliminate the reliance on a parent mass list. Initial screening utilized a high mass tolerance ( approximately 10 ppm) to filter the full scan MS data for previously reported nefazodone metabolites. The tight mass tolerance reduces or eliminates background chemical noise, dramatically increasing sensitivity for confirming or eliminating the presence of metabolites as well as isobaric forms. The full scan accurate mass analysis of suspected metabolites can be confirmed or refuted using three primary tools: (1) predictive chemical formula and corresponding mass error analysis, (2) rings-plus-double bonds, and (3) accurate mass product ion spectra of parent and suspected metabolites. Accurate mass characterization of the parent ion structure provided the basis for assessing structural assignment for metabolites. Metabolites were also characterized using parent product ion m/z values to filter all tandem mass spectra for identification of precursor ions yielding similar product ions. Identified metabolite parent masses were subjected to chemical formula calculator based on accurate mass as well as bond saturation. Further analysis of potential nefazodone metabolites was executed using accurate mass product ion spectra. Reported mass measurement errors for all full scan MS and MS/MS spectra was <3 ppm, regardless of relative ion abundance, which enabled the use of predictive software in determining product ion structure. The ability to conduct biotransformation profiling via tandem mass spectrometry coupled with accurate mass measurements, all in a single experimental run, is clearly one of the most attractive features of this methodology.

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These data are consistent with data reported from clinical studies and indicate that a single-dose regimen would be at least as effective as the same dose administered over several days.