Different classes of GSH-conjugates appear to behave differently upon
collision-activated dissociation; not all afford a neutral loss of 129 Da as the
primary fragmentation pathway. For example, aliphatic thioether conjugates
may eliminate a glutathione molecule (307 Da) as a neutral fragment and/or
yield protonated glutathione (m/z 308) as the product ion; thioester
conjugates, on the other hand, typically fragment by a neutral loss of
147 Da (loss of pyroglutamic acid followed by a loss of H 2 O) (Baillie and
Davis, 1993; Dieckhaus et al., 2005). Hence, there is a need for a more broadly
applicable MS/MS survey scan for the detection of GSH-conjugates of
different structural classes. Dieckhaus et al. demonstrated that negative ion
MS/MS showed promise in overcoming this limitation since MS/MS spectra
of the deprotonated molecular ions [MH]of glutathione and major classes
of GSH-conjugates afforded a common fragment anion at m/z 272
(deprotonated g-glutamyl-dehydroalanyl-glycine), derived by loss of H 2 S
from the glutathionyl moiety. Therefore, precursor ion scan ofm/z272 in
negative ion mode could provide a generally applicable technique for the
detection of GSH-adducts (Dieckhaus et al., 2005). The utility of this
approach was demonstrated in a variety of compounds that are known to form
reactive metabolites. It should be noted that the MS/MS spectra of
deprotonated molecular ions of GSH-conjugates are dominated by fragment
ions from the tri-peptide moiety of glutathione, and few structurally
informative ions from the xenobiotics. Therefore, an approach that could
provide more information would be a combination of precursor ion scan of
m/z272 in negative ion mode as a survey scan for unambiguous detection of
GSH-adducts, with polarity switching to positive ion mode to acquire full scan
product ion spectra of MH+of the conjugates for structural elucidation
(Dieckhaus et al., 2005).
11.9 Conclusions and Future Directions
Metabolism studies play a pivotal role in drug discovery and development and
LC/MS has become an indispensable tool to elucidate metabolite structures
and metabolism pathways. Characterization of metabolic ‘‘hot spots’’ as well
as reactive and pharmacologically active metabolites is critical to designing
new drug candidates with improved metabolic stability, toxicological profile,
and efficacy. Metabolite identification in the preclinical species used for safety
evaluation is required in order to determine whether human metabolites
have been adequately tested during nonclinical safety assessment. High
performance liquid chromatography coupled with tandem mass spectrometry
has emerged as a cardinal apparatus for the identification of metabolites in
biological matrices. Additional techniques, such as chemical derivatization,
H/D exchange, stable isotope labeling, accurate mass measurements, and
software-assisted data acquisition and processing methods, have proved to be
useful for improving metabolite detection and identification.
CONCLUSIONS AND FUTURE DIRECTIONS 357