is especially useful in HPLC method development and rapid profiling of
metabolites with medium-to-low levels of radioactivity. Another attractive
application of stop-flow RFD is in the quantitative measurement of the
metabolite formation of radiolabeled substrates in enzyme kinetic studies such
as CYP reaction phenotyping and determination ofKmandVmaxvalues, since
these studies often deal with a large number ofin vitroincubation samples
(Zhao, 2004). One of the major limitations of stop-flow RFD is the significant
variability of the analyte retention times when different operation modes are
used. As showed in Fig. 10.5a, the major radioactivity peak eluted at 29.42 min
in the analysis using the nonstop mode, while the same peak eluted earlier in
the analyses using the by-level mode (28.93 min, Fig. 10.5c), and the by-fraction
mode (28.08 min, Fig. 10.5b). The shifts of metabolite HPLC retention times
seem to depend on the total HPLC run times: the longer total run time, the
shorter retention times of analytes. This observation is consistent with those
reported in the literature (Nassar et al., 2003, 2004). Most likely, the decrease of
the HPLC retention times in the stop by-fraction or stop by-level analyses is the
result of some diffusion of analytes when the HPLC flows are stopped. The
significant shifts in analyte retention times among different operation modes or
of different HPLC run times make the stop-flow HPLC–RFD very difficult to
determine identities of radioactive metabolites on the basis of their HPLC
retention times.
FIGURE 10.5 Analysis of two radioactive compounds using stop flow HPLC-RFD;
(a)nonstop mode; (b) stop by fraction mode; and (c) stop by level mode. Radioactivity
in stop by fraction and stop by level analyses were counted for 1 min each fraction.
NEW RADIOCHROMATOGRAPHY TECHNIQUES 299