seems to have no problem in maintaining the consistency of the analyte
retention times since the total HPLC run time and the HPLC flow rate in the
dynamic flow analysis are the same as those in conventional HPLC–RFD
analysis (Fig. 10.6) (Lee, 2006). Although further evaluation and detailed
validation of the use of dynamic flow LC–RFD for metabolite profiling are
required, the preliminary data have shown that the new radiochromatographic
technique enables a significant increase in the sensitivity of RFD without a stop
or interruption of the HPLC flow.
10.3.4 UPLC-Radiodetection
Ultra-performance liquid chromatography (UPLC) coupled with UV and MS
has been applied to metabolite profiling with improved sensitivity and shorter
HPLC run times (Johnson and Plumb, 2005; Wang et al., 2006a, 2006b). The
width of metabolite peaks in UPLC is usually 2–6 s, equivalent to peak
volumes of approximately 10–20mL. Even with low volume detection cells the
residence times of peaks in such a UPLC–RFD setting would result in a very
significant decrease in radiodetection sensitivity. Therefore, RFD is not
practically suitable for UPLC. Alternatively, MSC has been applied as an off-
line radioactivity detector of UPLC for radioactive metabolite profiling (Dear
et al., 2006). The configuration of UPLC–MSC is the same as that of HPLC–
MSC (Fig. 10.3a). In the analysis, the UPLC effluent was collected into 96-well
plates at a rate of 2 s/well using a modified rapid fraction collection system, and
then the 96-well plates were counted on TopCount. Compared to HPLC–
MSC, UPLC–MSC provided better radiodetection sensitivity (2–5 fold), faster
analysis, and comparable separation resolution (Dear et al., 2006).
Additionally, since smaller volume fractions are collected in the UPLC–MSC
analysis, liquid scintillation cocktail can be directly added into 96-well plates
without a drying process, which eliminates the possibility of the loss of volatile
metabolites and reduces the matrix effect. Ultra-performance liquid chroma-
tography coupled with MSC provides an alternative tool for high sensitivity
radiolabeled metabolite profiling.
10.3.5 HPLC-AMS
Accelerator mass spectrometry (AMS) is an ultrasensitive analytical method
for radioactivity analysis. AMS offers 10^3 –10^9 -fold increases in sensitivity over
LSC or other decay counting methods so that levels as low as 0.0001 DPM can
be detected (Brown et al., 2005, 2006). AMS has been applied to mass balance
determination, pharmacokinetic studies of total radioactivity, and measure-
ment of chemically modified DNA and proteins in humans after the
administration of a low radioisotope dose (approximately 10 nCi/person for
mass balance and drug metabolism studies) (Buchholz et al., 1999; Garner,
2000; Garner et al., 2002; Liberman et al., 2004; White and Brown, 2004). In
addition, off-line HPLC–AMS has been explored for metabolite profiling after
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