ring in RTCOOH. Similarly, the detection of doublet peaks with anm/z
difference of 2 Th at 113 and 115 Th in the mass spectrum suggested the
presence of TCONH 2 (M2). In the absence of a diagnostic isotopic pattern, the
presence of RTCOOH (M1) could not be confirmed and its MH+ion could
not be distinguished from interfering ions in the mass spectrum. This
customized stable isotope-labeling technique provided unambiguous identifica-
tion and characterization of ribavirin metabolites by the detection of diagnostic
doublet ions in the LC/MS spectra.
Stable isotope labeling is usually not necessary when the compound contains
Cl or Br, each of which has a unique natural isotopic abundance that can be
easily recognized in the mass spectra. Assuming that these halogens remain intact
during biotransformation, their unique isotope pattern can be used to search for
metabolites in a complex biological matrix. Several software packages have been
developed to analyze the MS data based on the isotopic pattern recognition,
which significantly improve throughput in metabolite identification.
11.4.3 Hydrogen/deuterium (H/D) Exchange MS
The exchange of labile hydrogen with deuterium in small organic molecules
has been widely used in structural characterization by mass spectrometry.
This technique now finds widespread use because it can be utilized under
LC/MS conditions. Biotransformation of xenobiotics usually involves
introduction of polar functional groups (such as OH,SH, N(R)H,
NH 2 , COOH) and leads to changes in the number of exchangeable
hydrogens. Determination of the number of exchangeable hydrogens in
metabolites can provide additional information to facilitate structural
elucidation (Olsen et al., 2000). This approach was applied to differentiate
sulfoxide and sulfone metabolites from the isomeric mono and dihydroxy-
lated metabolites, respectively (Liu et al., 2001). Ohashi et al. used the H/D
exchange method for drug metabolism studies of denopamine and
promethazine, in which they were able to successfully differentiateN-orS-
oxide from the hydroxylated metabolites (Ohashi et al., 1998). Miao et al.
demonstrated the use of H/D exchange LC/MS and LC/MS/MS in
characterization of a novel metabolite of ziprasidone (Miao et al., 2005). A
major metabolite, designated as M11, was detected from the incubation of
ziprasidone and hepatic cytosolic fractions of rat, dog, and human liver. Full
scan MS of M11 revealed a protonated molecular ion atm/z415; 2 Th higher
than the parent drug. MS/MS analysis suggested that the addition of two
mass units had occurred in the benzisothiazole moiety (Scheme 11.3). H/D
exchange LC/MS analysis of M11 showed a shift for the protonated
molecular ion fromm/z415 to 419, which suggested that M11 contains three
exchangeable hydrogen atoms. Comparison of the product ion mass spectra
of M11 atm/z415 to that ofm/z419 in the D 2 O mobile phase (Fig. 11.3)
indicated that the addition of two exchangeable hydrogen atoms had
occurred at the benzisothiazole moiety, potentially due to reductive cleavage
TECHNIQUES FOR IMPROVING METABOLITE DETECTION 341