rates with substrate concentrations was obtained by the stop-flow HPLC–RFD
method (Fig. 10.12a), which was very similar to that determined using off-line
HPLC–MSC (Fig. 10.12b). The results suggest that both stop-flow LC–RFD
and off-line LC–MSC can provide high quality data in the analysis of low level
radioactivity, however, stop-flow LC–RFD is 3–4 fold faster than HPLC–
MSC.
10.6 Summary
Metabolite profiling by liquid radiochromatographic techniques determines the
number and relative concentrations (% distribution of radioactivity) of
metabolites presented in a biological sample. Conventional online HPLC–
RFD is currently the primary liquid radiochromatographic tool employed in
drug metabolism laboratories for profiling radiolabeled metabolites, enzyme
kinetic determination of radiolabeled substrates, and metabolite structural
characterization by coupling with mass spectrometry. The recently introduced
stop-flow and dynamic flow techniques have significantly improved the
sensitivity of RFD. Ideally any new RFD devices would include nonstop,
stop-flow and dynamic flow functions that are completely interchangeable. Off-
line HPLC–MSC is a new and important tool for analysis of low level
radioactivity, which is especially useful in radiolabeledin vivoADME studies
that require very high radiodetection sensitivity for analysis of plasma
metabolites, and validated procedures for supporting drug development and
registration. In addition, the combination of HPLC–MSC and mass spectro-
metry allows for the detection and structural characterization of minor
radiolabeled metabolites that may not be detected by LC–RFD–MS. HPLC
coupled with AMS is an ultrasensitive liquid radiochromatographic technique
that has been applied in a few clinical studies to meet special needs. However,
the cost of the instrument and operation, and the difficulty in dealing with
radioactivity contamination limit its application in drug metabolism studies.
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