retention times and peak shapes in a radiochromatogram and its corresponding
ion chromatograms recorded from LC–RFD–MS analysis are the same, which
can greatly enhance the identification of metabolite peaks. However, the
relatively poorer radioactivity detection of RFD limits the use of LC–RFD–MS
in the analysis of the samples with low levels of radioactivity.
10.4.2 Stop-Flow and Dynamic Flow LC–RFD–MS
Stop-flow LC–RFD coupled with a mass spectrometer has been applied to
radioactivity profiling and metabolite characterization (Nassar et al., 2003).
The configuration of this system was similar to that displayed in Fig. 10.1a. In
the analysis, the HPLC flow was stopped for both radioactivity counting by
RFD and mass spectral data acquisition by the mass spectrometer. Although
mass spectral data of metabolites were obtained from these studies, no ion
chromatographs from mass spectrometric analysis were reported. When the
HPLC flow is completely stopped, the HPLC effluent is not introduced into the
mass spectrometer. As a result, the retention times and shapes of radiolabeled
metabolite parks in reconstituted ion chromatograms may be different than
those in the corresponding radiochromatograms. Another disadvantage of the
stop-flow HPLC–RFD–MS is that it takes a much longer run time (up to 4 h,
Fig. 10.5). The newly developed dynamic flow LC–RFD would be an improved
online radiodetection technology regarding coupling with a mass spectrometer,
since the HPLC flow is not stopped or interrupted in the dynamic flow analysis
(Lee, 2006).
10.4.3 LC-MSC-MS
The combination of HPLC–MSC with mass spectrometry (LC–MSC–MS) is
often employed for metabolite analysis and is especially useful for metabolites
with a low level of radioactivity (Zhang., et al 2007a and 2007b). Two
configurations of LC–MSC–MS are shown in Fig. 10.3. In configuration (A)
the HPLC effluent is split so that a small portion flows to the mass
spectrometer and the remaining portion flows to 96-well microplates.
Radioactivity in the 96-well plates is counted by MSC after either removing
solvent under vacuum or direct addition of scintillation cocktail. Conventional
HPLC columns (3.9–4.6 mm ID) are typically employed in LC–MSC–MS
analyses because a large column provides consistent retention times and high
separation resolution of analytes in complex biological samples. It also
provides better MSC and mass spectrometric sensitivity because larger sample
volumes can be injected without sacrificing chromatographic performance. In
general, MSC provides a level of detection comparable to or better than LC–
MS/MS when 40–100mCi radioactivity is dosed to each animal or human
subject in ADME studies. Figure 10.7 shows an example of using LC–MSC–
MS for quantification and structure elucidation of metabolites in a rat bile
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