Drug Metabolism in Drug Design and Development Basic Concepts and Practice

(also see later in Section 11.7.2) alone is a challenging task because these analytes
exhibit the samem/zfor their protonated or deprotonated molecular ions, and
their product ion mass spectra are usually very similar. Chemical reduction of
N-oxides by TiCl 3 provides an efficient and selective way to differentiateN-oxides
from hydroxylated metabolites (Kulanthaivel et al., 2004; Seaton et al., 1984).
The reduction can be carried out in the presence of biological matrices,
sulfoxides, and other labile groups (Kulanthaivel et al., 2004).

11.4.2 Stable Isotope Labeling

Stable-isotope-labeled (^2 H,^13 C,^15 N,^18 O,^34 S, etc.) xenobiotics can facilitate
metabolite detection and identification by mass spectrometry, especially when
radiolabeled parent drug is not available. Custom-designed isotopic clusters
resulting from the mixture of natural and synthetically enriched isotopes can
greatly facilitate the detection and identification of metabolites (Mutlib and
Shockcor, 2003; Wienkers et al., 1995, 1996).
Chowdhury et al. utilized stable isotope-labeled drug to aid in the detection
and identification of ribavirin metabolites in rats (Chowdhury et al., 2005). A
mixture containing equal amounts of ribavirin (RTCONH 2 )and^13 C 3 -
RTCONH 2 and a relatively small mass of^14 C-RTCONH 2 was dosed to
male Sprague-Dawley rats (60 mg/kg) to facilitate metabolite identification.
Two major metabolites, RTCOOH and TCONH 2 (Scheme 11.2), were detected
in the pooled urine. The detection of a doublet peak with anm/zdifference of
3 Th at 246 and 249 Th in the mass spectrum suggested the presence of
RTCOOH (M1). Its identity was further confirmed by MS/MS of 246 and
249 Th. The respective major product ions at 114 and 116 Th were consistent
with the unlabeled and^13 C 2 -labeled carbons of carboxylic acid and triazole

N

O

N

OH

N

HO

HO

H 2 N

O

N

O

N

OH

N

HO

HO

HO

O

N

H

N

N

H 2 N

O

*

*

*

* *

*

*

*

Ribavirin (RTCONH 2 ) M1 (RTCOOH) M2 (TCONH 2 )

SCHEME 11.2 Chemical structures of ribavirin, metabolite M1, and metabolite M2

*denotes the position of (^13) Clabel.
340 APPLICATION OF LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY