| Abstract
| - Characterization of S-linked conjugates of the endogenous tripeptide glutathione (γ-glutamyl-cysteinylglycine, GSH) represents a valuable indirect approach for the identification ofchemically reactive, electrophilic intermediates formed during the metabolism of both foreigncompounds and endogenous substances. In most cases, GSH adducts generated in vitro orexcreted in the bile of animals are detected by the use of liquid chromatography−tandem massspectrometry (LC−MS/MS), employing survey scans based on characteristic fragmentationsof this class of conjugates. However, a limitation of current LC−MS/MS approaches, whichtypically employ electrospray ionization with analysis of positive ions, is that no single surveyscan exhibits broad utility in the detection of unknown GSH adducts, since different structuralclasses of conjugate (aromatic, benzylic, aliphatic, thioester, etc.) behave differently uponcollision-induced dissociation (CID) of the respective [M + H]+ parent ions. In the presentstudy, we evaluated MS/MS in the negative ion mode as an alternative approach and reportherein that the spectra obtained by CID of the [M − H]- ions of a number of representativeGSH adducts, as well as GSH itself, are dominated by fragments originating from theglutathionyl moiety of the tripeptide. In particular, the anion at m/z 272, correspondingnominally to deprotonated γ-glutamyl-dehydroalanyl-glycine, was abundant in the negativeion spectra of free GSH and all GSH conjugates examined, suggesting that scanning forprecursors of this ion may provide a generally applicable technique for the detection of adductsof unknown structure. The utility of this novel detection strategy was demonstrated in a seriesof in vitro and in vivo experiments where compounds known to undergo metabolic activationwere examined for their propensity to form conjugates with GSH. In all cases, scanning forprecursors of m/z 272 in the negative ion mode revealed the presence of the expected adductsand in some instances revealed additional conjugates that had not been reported previously.Positive ion MS/MS, on the other hand, was more useful than the corresponding negative ionscans in providing information on the molecular structure of GSH conjugates.
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