11.8.2 Screening for Glutathione Conjugates
Glutathione (GSH, g-glutamylcysteinylglycine) is present virtually in all
mammalian tissues and therefore serves as a natural scavenger for chemically
reactive metabolites. A screen for the formation of glutathione conjugates
could potentially identify a significant portion of reactive metabolites formed
from a drug. Fragmentation of glutathione conjugates following collision-
induced dissociation (CID) is illustrated in Scheme 11.8 (Haroldsen et al.,
1988; Levsen et al., 2005; Murphy et al., 1992). Similar to CID of peptides, the
fragmentations of GSH conjugates mainly result from the cleavage of
the peptide backbone of the glutathione moiety. Even though the relative
abundances of different types of fragment ions are sometimes dependent on
the nature of the conjugated species, glutathione conjugates generally undergo
a neutral loss of 129 Da (pyroglutamic acid) to producee-type fragment ions
(Scheme 11.8). Therefore, glutathione conjugates can be easily detected by a
constant neutral loss (CNL) scan of 129 Da. One of the main disadvantages of
this technique is its poor selectivity, as endogenous compounds presented in
biological matrices may also give rise to a neutral loss of 129 Da. Therefore,
false positives are routinely detected. To overcome this drawback, Yan and
Caldwell developed a novel approach that demonstrated great selectivity and
reliability for high-throughput screening for reactive metabolites using a
stable-isotope-labeled trapping agent (Yan and Caldwell, 2004; Yan et al.,
SCHEME 11.8 Characteristic fragment ions of glutathione conjugates under collision-
induced dissociation. Adapted from Ma and Subramanian (2006) with permission of
John Wiley and Sons Limited.
354 APPLICATION OF LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY