facilitate high throughput screening for reactive metabolites of molecules in the
discovery stage programs.
Recently, Castro-Perez et al. (2005b) reported an LC/MS/MS method for
screening glutathione conjugates using exact mass neutral loss of 129.0426 Da
(corresponds to the exact mass of pyroglutamic acid) in a hybrid quadrupole
time-of-flight mass spectrometer to eliminate false positives. The neutral losses
of endogenous compounds in biological matrices may have the same nominal
mass of 129 Da but less likely to have the same exact mass; therefore, exact
mass measurements can play a pivotal role in excluding false positives. The
instrument acquired survey mass spectra sequentially at low and high energy
by switching the collision energy from 5 to 20 eV. The data system was set to
examine these mass spectra in real-time to look for ions with a mass difference
of 129.0426 Th within a narrow mass tolerance window in the high energy MS
survey scan. Whenever an exact neutral loss was detected, the instrument was
automatically switched to MS/MS mode to acquire a full scan MS/MS
spectrum that was then used to elucidate the structures of detected GSH
adducts. This approach allowed a selective detection and identification of
GSH-adducts. In addition, a single injection provided accurate mass MS and
MS/MS data on each detected GSH-conjugate.
FIGURE 11.8 Collision induced product ion spectra of natural (a,m/z457) and
isotope-labeled GSH adduct (b, m/z 460). Reprinted from Yan et al. (2004) with
permission of the American Chemical Society.
356 APPLICATION OF LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY