formation rates of the secondary metabolites in the incubations of the
individual primary metabolites as determined by radiochromatographic
analysis. The sequential metabolites identified in the metabolite incubation
approach were further used as metabolite standards for successfully determin-
ing the identities of buspirone metabolites in rat plasma. Results from this
example illustrate that this in vitro approach, using a combination of
metabolite incubation and LC–MSC–MS, is a simple and effective way to
determine the formation pathways and structures of sequential metabolites,
such as dihydroxy metabolites. A similar approach was applied to the
quantitative determination of formation pathways of capravirine in humans
using stop-flow HPLC–RFD analysis (Bu et al., 2005). The approach includes
three steps: (1) 30-min primary incubation of [^14 C]capravirine; (2) isolation of
radiolabeled metabolites from the primary incubation; and (3) 30-min
reincubation of the isolated metabolites supplemented with an ongoing (30-
min) microsomal incubation with nonlabeled capravirine. The formation
pathways of sequential metabolites were assigned based on the extent of
disappearance of the isolated primary metabolites and the formation of
sequential metabolites.
10.5.4 Enzyme Kinetic Studies
Enzymology studies in drug metabolism include the determination of kinetic
parameters, such asKmandVmax, and reaction phenotyping of enzymes that
catalyze formation of metabolites. These studies require quantification of
metabolites formed in a large number of incubation samples. Two analytical
approaches are often employed for these studies: (1) LC–MS/MS using
synthetic standards of metabolites; and (2) liquid radiochromatography using
radiolabeled substrates. Typically in the pharmaceutical industry, radiolabeled
drugs are available when a single compound is selected for development. The
online RFD method is the primary radiochromatographic technique used in
enzyme kinetic studies since it provides fast results and high separation
resolution (Zhu et al., 2005a). In some cases, metabolite quantitation is
challenging because the experiment is conducted with a drug at a low specific
activity or low substrate concentrations, or with slow turnover. Thus, use of
more sensitive liquid radiochromatographic techniques such as HPLC–MSC or
stop-flow RFD is required. Although HPLC–MSC provides excellent
sensitivity, due to relatively slow throughput (usually 6–8 HPLC injections
per day) and manual operation, it is not well suited for enzyme kinetic studies.
Stop-flow HPLC–RFD appears more attractive for enzyme kinetic experi-
ments since it can be performed in an automated fashion (Zhao, 2004). In
addition, the stop-flow RFD instrument has the conventional RFD function
suitable for high level radioactivity and the stop-flow function for analysis of
relatively low levels of radiolabeled metabolites, which is especially useful in
the determination of enzyme kinetic parameters.
310 APPLICATIONS OF LIQUID RADIOCHROMATOGRAPHY TECHNIQUES