Utility of multivariate analysis in support of in vitro metabolite identification studies: retrospective analysis using the antidepressant drug nefazodone

Abstract

1. The utility of multivariate analysis in in vitro metabolite identification studies was examined with nefazodone, an antidepressant drug with a well-established metabolic profile.

2. The chromatographic conditions were purposefully chosen to reflect those utilized in high-throughput screening for microsomal stability of new chemical entities.

3. Molecular ion, retention time information on groups of human liver microsomal samples with/without nefazodone was evaluated by principal component analysis (PCA). Resultant scores and loadings plots from the PCA revealed the segregation and the ions of interest that designated the drug and its corresponding metabolites. Subsequent acquisition of tandem mass spectrometry (MS/MS) spectra for targeted ions permitted the interrogation and interpretation of spectra to identify nefazodone and its metabolites.

4. A comparison of nefazodone metabolites identified by PCA versus those found by traditional metabolite identification approaches resulted in very good correlation when utilizing similar analytical methods. Fifteen metabolites of nefazodone were identified in β-nicotinamide adenine dinucleotide phosphate (NADPH)-supplemented human liver microsomal incubations, representing nearly all primary metabolites previously reported.

5. Of the 15 metabolites, eight were derived from the N-dealkylation and N-dephenylation of the N-substituted 3-chlorophenylpiperazine motif in nefazodone, six were derived from mono- and bis-hydroxylation, and one was derived from the Baeyer Villiger oxidation of the ethyltriazolone moiety in nefazodone.

Keywords::

• Cytochrome P450
• nefazodone
• principal component analysis (PCA)
• multivariate analysis
• liquid chromatography-tandem mass spectrometry (LC/MS/MS)