Determination of pyrrolizidine alkaloid metabolites from mouse liver microsomes using tandem mass spectrometry and gas chromatography/mass spectrometry

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Abstract
The rapid and sensitive identification and quantification of important pyrrolizidine alkaloid metabolites using tandem mass spectrometry (MS/MS) and gas chromatography/mass spectrometry (GC/MS) is described. Identifications of N‐oxide and hydrolytic metabolites of the pyrrolizidine alkaloids senecionine and monocrotaline in extracts of mouse hepatic microsomal incubations were accomplished by comparing collisionally activated decomposition/mass‐analyzed ion kinetic energy spectra of specific ions from microsomal extracts with spectra obtained from synthetic standards of suspected metabolites. Trace amounts of the toxic metabolite dihydropyrrolizine (DHP) were observed by GC/MS of trimethylsilyl (TMS) derivatives, but the amounts present in hepatic microsomal extracts were below the MS/MS limit of detection. Quantitative determinations of senecionine N‐oxide were performed by fast atom bombardment MS/MS. Suppression of N‐oxide ionization by other substances in the extracts was judged to be minimal. The TMS derivatives of the metabolites senecic acid, monocrotalic acid and DHP were quantified using capillary GC/MS. Results from the study demonstrate that the relative contributions of the three major pathways of pyrrolizidine alkaloid metabolism (N‐oxidation, hydrolysis and oxidation to pyrrolic compounds) can be assessed using a single analytical instrument and minimal sample preparation.