C
haracterization of
S-linked conjugates of t
he endogenous tripeptide glutat
hione (
![](/images/gifc<font color=)
hars/gamma.gif" BORDER=0 >-glutamyl-cysteinylglycine, GSH) represents a valuable indirect approac
h for t
he identification ofc
hemically reactive, electrop
hilic intermediates formed during t
he metabolism of bot
h foreigncompounds and endogenous substances. In most cases, GSH adducts generated
in vitro orexcreted in t
he bile of animals are detected by t
he use of liquid c
hromatograp
hy-tandem massspectrometry (LC-MS/MS), employing survey scans based on c
haracteristic fragmentationsof t
his class of conjugates. However, a limitation of current LC-MS/MS approac
hes, w
hic
htypically employ electrospray ionization wit
h analysis of positive ions, is t
hat no single surveyscan ex
hibits broad utility in t
he detection of unknown GSH adducts, since different structuralclasses of conjugate (aromatic, benzylic, alip
hatic, t
hioester, etc.) be
have differently uponcollision-induced dissociation (CID) of t
he respective [M + H]
+ parent ions. In t
he presentstudy, we evaluated MS/MS in t
he negative ion mode as an alternative approac
h and report
herein t
hat t
he spectra obtained by CID of t
he [M - H]
- ions of a number of representativeGSH adducts, as well as GSH itself, are dominated by fragments originating from t
heglutat
hionyl moiety of t
he tripeptide. In particular, t
he anion at
m/
z 272, correspondingnominally to deprotonated
![](/images/gifc<font color=)
hars/gamma.gif" BORDER=0 >-glutamyl-de
hydroalanyl-glycine, was abundant in t
he negativeion spectra of free GSH and all GSH conjugates examined, suggesting t
hat scanning forprecursors of t
his ion may provide a generally applicable tec
hnique for t
he detection of adductsof unknown structure. T
he utility of t
his novel detection strategy was demonstrated in a seriesof
in vitro and
in vivo experiments w
here compounds known to undergo metabolic activationwere examined for t
heir propensity to form conjugates wit
h GSH. In all cases, scanning forprecursors of
m/
z 272 in t
he negative ion mode revealed t
he presence of t
he expected adductsand in some instances revealed additional conjugates t
hat
had not been reported previously.Positive ion MS/MS, on t
he ot
her
hand, was more useful t
han t
he corresponding negative ionscans in providing information on t
he molecular structure of GSH conjugates.