depending on the extent various information are sought. Early in the drug
discovery process, metabolism studies are often aimed at understanding the
metabolic stability of a compound or a structural series. The major goal is to
locate the metabolically vulnerable spots in the structure that may increase the
rate of intrinsic metabolic clearance resulting in short half-life and low oral
bioavailability. Identification of the metabolic soft spots is vital in guiding
synthetic chemistry efforts to design new lead compounds with optimal
pharmacokinetic profiles (Ma et al., 2006).
Metabolic stability studies are performed by estimating the rate of
disappearance of the parent drug by measuring the concentration before and
following incubation in microsomal systems for a predetermined period
(Chovan et al., 2004). Modern LC/MS instruments, such as linear ion traps
(Hopfgartner et al., 2004; Xia et al., 2003) offer very high scan speed such that
multiple MS/MS experiments on the parent drug, as well as putative
metabolites, can be performed simultaneously not only to detect them at
high sensitivity but also to extract additional information on the site of
modification (Hopfgartner et al., 2003).
In vivometabolic stability, as well as preliminary metabolite characteriza-
tion, is usually performed when a compound is determined to be optimally
stable inin vitrosystems. Since radiolabeled compounds are rarely available in
the early stages of drug discovery, the detection of molecular ions of drug-
related components is mainly achieved from the analysis of the test and control
samples in parallel followed by the comparison of the extracted ion
chromatograms of potential metabolites according to predicted gains and
losses in their molecular mass. The common biotransformations and
correspondingm/zchanges are listed in Table 11.1 (Anari et al., 2004). The
structures of these putative metabolites are then determined from product ion
MS/MS and/or MSnexperiments (Tozuka et al., 2003). The tandem mass
spectrometry approach to revealing the molecular ions of unexpected
metabolites has focused on the use of PIS and CNLS. These two experiments
are particularly useful since they do not require previous knowledge of the
molecular weight of metabolites. The PIS allows detection of all molecular
entities that form a common product ion and signifies a structural relationship
between the administered drug and its metabolites. Typically, several product
ions of the parent drug are used to detect precursor ions of the metabolites.
These experiments take advantage of the fact that of any one metabolite, large
parts of the parent molecule do not undergo metabolic changes. The product
ion representing an unchanged substructure is used as a structure-specific
detection signal to detect the metabolites. As more than one substructure-
specific ion is used, one can detect metabolites that have modifications on
various portions of the molecule.
If a drug and its metabolites have a common structure that is lost in a MS/
MS experiments as neutral species, the mass spectrometer can be set to detect
the metabolite using CNLS, irrespective of its molecular weight. Several
conjugated metabolites fragment to lose a distinct neutral group. This distinct
330 APPLICATION OF LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY