As the drug enters Phase I clinical trials the drug metabolism efforts focus
on identifying both circulating and excreted metabolites in humans. These
results are then compared to those observed in the animal species used for
safety assessment (Ma et al., 2006; Nassar and Talaat, 2004). All metabolism
studies in clinical drug development are conducted with great austerity to be
compliant with all regulatory guidelines required for marketing registration.
Mass balance studies are designed to collect most of the excreted drug-derived
material where the characterization of metabolites is more complete. Typically,
a single LC/MS method is used for all matrices and species so that metabolites
observed in humans and animals can be directly compared using their LC
elution time, single MS spectra, and MS/MS results (Ramanathan et al., 2005).
To assess human risk, qualitative and quantitative differences in metabolite
profiles are examined to establish exposure in a preclinical species relative to
humans. Attempts should be made as early as possible in the drug development
process to identify the differences in drug metabolism between animals used in
nonclinical safety assessments and humans (US Food and Drug Administration,
2005). It is important to identify unique metabolites only produced in humans or
formed to a much greater extent in humans compared to that in animals as early
as possible to allow for timely assessment of potential safety issues (Davis-Bruno
and Atrakchi, 2006; Humphreys and Unger, 2006; Smith and Obach, 2006).
Therefore, the primary objectives of metabolite profiling and identification in
drug development are to compare metabolism across species, to fully
characterize major metabolites, and to search for human-specific metabolites
or any metabolite(s) formed to a much greater extent in humans compared to
those in preclinical species used in safety testing.
The most widely used approach for metabolite profiling and identification in
drug development involves administration of radiolabeled drug (i.e.,^14 Cand
(^3) H). Following collection of excreta and blood, analysis of these matrices is
typically performed using LC coupled to both a mass spectrometer (MS) and
a flow scintillation analyzer (FSA). An LC/MS/FSA method allows for
simultaneous acquisition of mass spectra (and/or MS/MS spectra) and the
radiochromatographic response for each metabolite. The former provides the
identification of metabolites and the latter the relative amounts of each. Thus,
circulating levels of each detected metabolite and/or unchanged drug can be
estimated from the plasma metabolite profile. In addition, percentage of
administrated dose for each drug-derived entity excreted in urine and feces
can be calculated from total excretion data and the radiochromatographic
response for each metabolite in urine and feces. Recently, Ramanathan et al.
reported the characterization of more than 50 metabolites of loratadine in male
and female mice, rats, and monkeys using the approach described above and
provided a very comprehensive interspecies comparison (Ramanathan et al.,
2005). This robust approach to metabolite characterization across species not
only allows for identification and semiquantification of individual metabolites,
but also establishes whether all human metabolites have been adequately tested
in nonclinical safety assessments.
338 APPLICATION OF LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY