spectra of NAc adducts may provide more structural information of
modification site on the drug moiety. Even though fragmentations of the
NAc adducts also result in a neutral loss of 129 Da (Fig. 14.1), the sensitivity of
the constant neutral loss scanning of 129 Da for NAc adducts may not be as
high as that for the GSH adducts. Also, NAc may be less efficient than GSH in
trapping reactive intermediates if glutathione-S-transferase enzymes are
involved in the formation of adducts (Evans et al., 2004).
Both GSH and NAc, are thiol-containing soft nucleophiles, which are
commonly used to trap soft electrophiles (e.g., quinones and epoxides). On the
contrary, cyanide ion is a hard nucleophile, and can readily react with certain
hard electrophiles, such as iminium ions. It is applicable to use a mixture of
KCN=K^13 C^15 N (1:1) in trapping studies, and the isotopic patterns of the
corresponding cyanide adducts might be observed in LC/MS/MS analysis
(Evans et al., 2004). Inhibitory effects of trapping agents on the metabolism of
compounds may need to be evaluated before conducting trapping studies. It
was reported that higher concentration of sodium cyanide inhibited
metabolism and adduct formation (Ho and Castagnoli, 1980; Ward et al.,
1982). However, sodium cyanide at 1 mM or less did not significantly inhibit
cytochrome P450 activity in liver microsomes. In addition, investigators may
need to pay attentions to some cases, where the formation of trapped adducts
was due to artifacts in trapping studies (Gorrod and Sai, 1997).
Even though radiolabeled compounds are not required for trapping studies,
because investigators may use LC/MS/MS and NMR techniques to
qualitatively characterize the trapped adducts, from which the structures of
reactive intermediates may be inferred, the use of the^3 H-or^14 C-labeled
compounds provides an easy means for identifying, characterizing, and
quantifying potential adducts in trapping studies. In addition, dansyl
glutathione (dGSH, Fig. 14.1), a fluorescence labeled GSH, can also be used
as a trapping agent for both qualitative and quantitative analysis of thiol
adducts of unlabeled compounds (Gan et al., 2005). Fluorescence detection
after HPLC separation allows universal quantitation of adducts without a need
of synthetic standards or radiolabels. In addition, online HPLC/MS/UV
detection provides another means of semiquantitative analysis of trapped
adducts of unlabeled compounds.
It was reported that some GSH adducts formed in trapping studies are
highly unstable, and readily undergo degradation or rearrangement (Alt
and Eyer, 1998; Doss et al., 2005). Thus, the constant neutral loss scanning of
m/z129 with triple quadrupole mass spectrometry to screen GSH adducts
may yield false negative results. Investigators may consider other alternative
thiol-containing trapping agents (e.g., NAc, GSH–EE). It was reported that a
mixture of GSH and GSH–EE was effective in trapping studies for detecting
reactive intermediates (Ge et al., 2006). Two separate GSH and GSH–EE
adducts at a mass difference ofm/z28 were detected in LC–MS analysis. This
approach may increase the probability for detection of trapped adducts. On
the contrary, compounds which are known to form reactive intermediates
GLUTATHIONE,N-ACETYLCYSTEINE, AND POTASSIUM CYANIDE 451