are recorded by collision-induced dissociation (CID) of molecular ion of the
test compound (MH+) species. Several approaches might be used to detect
molecular ions of potential adducts. First, by comparing UV spectra of the
samples in the presence and absence of trapping agents, the additional UV
peaks shown in the samples in the presence of trapping agents might be
corresponding to the trapped adducts. Subsequent MSnanalysis of the peaks of
interest should be performed to further confirm and characterize the structures
of the potential adducts. Secondly, different molecular ions of potential
adducts can be generally screened in MS and MSnanalysis. Them/zvalues
commonly used in screening are the molecular weights of the trapping agents
plus MH+, or plus molecular weights of modified test compounds (MH++14,
MH++16, MH++18, MH++32) (Note 3). Any potential adducts should be
further characterized in MSnanalysis. Thirdly, postrun processing of mass
spectra generated from data-dependent MSn acquisition for the neutral
fragment (loss) ofm/z129 or 27/29 can be performed in MS^2 analysis for the
detection of potential GSH/NAc adducts and cyano adducts, respectively.
Subsequent MSn(n= 3 and 4) analysis of the molecular ions of potential
adducts should be performed for further characterization. Two examples of
LC/MS/MS analyses of trapped adducts are shown below.
Figure 14.2 shows LC/MS/MS analysis of the bis-cyano adductBfrom
incubation of (2S,3R)-3-(4-hydroxyphenyl)-2-[4-(2-piperidin-1-ylethoxy)phenyl]-
2,3-dihydro-1,4-benzoxathiin-6-ol (A, Fig. 14.1) in human liver microsomes in
the presence ofKCN=K^13 C^15 N(Zhang et al., 2005). The MS spectrum extracted
in chromatography atm/z531 (MH+NH 3 ) in total ion chromatogram revealed
a significant peak at 26.1 min (Fig. 14.2a). The MS spectrum of this adduct
showed isotopic peaks atm/z531, 533, and 535 at an approximate ratio of 1:2:1
(Fig. 14.2b), suggesting the formation of a bis-cyano adduct. The MS^2 spectrum
of this adduct, obtained by CID of them/z531 species, exhibited product ions at
m/z514 (loss of NH 3 ) andm/z487 (loss of NH 3 and HCN) (Fig. 14.2c). Two
minor fragments atm/z347 and 391 associated with the right-hand side of the
dihydrobenzoxathiin moiety were also detected. The MS^3 spectrum, obtained by
CID ofm/z487, showed loss of a second HCN molecule (27 Da) to give an ion at
m/z460 (Fig. 14.2d). The other product ions are also shown in Fig. 14.2d. The
MS^2 CID spectrum of them/z533 species exhibited product ions atm/z516 (loss
of NH 3 ),m/z489 (loss of NH 3 and HCN), andm/z487 (loss of NH 3 andH^13 C^15 N)
(Fig. 14.2e). A minor fragment atm/z259 is associated with the loss of the 4-(2-
piperidin-1-ylethoxy)phenyl portion. Furthermore, the MS^3 CID spectrum of
m/z489 showed loss of theH^13 C^15 Nmoiety (29 Da) to give an ion atm/z 460
(Fig. 14.2f). Collectively, these LC/MS/MS data suggest the presence of two
cyano groups in the piperidine ring, the most logical positions beinga- to the ring
nitrogen in the adductB(Fig. 14.1). This structure was further confirmed by
NMR analysis (Zhang et al., 2005). These results suggest that compoundAis
metabolized by cytochrome P450 in human liver microsomes to form reactive
iminium ions, and the proposed mechanism for the formation of the bis–cyano
adductBis shown in Fig. 14.3a.
GLUTATHIONE,N-ACETYLCYSTEINE, AND POTASSIUM CYANIDE 453