Drug Metabolism in Drug Design and Development Basic Concepts and Practice

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10.2. Minor metabolite peaks M1, M2, M4, M10, M13 and M14 were detected
in all analyses with TopCount (Fig. 10.2a), but not detected by RFD even
when four times as much sample was injected (Fig. 10.2c). Most minor
metabolites were detected by LSC, but a few minor peaks detected by MSC
were not observed with LSC analysis. For example, M14 (15 CPM) was not
observed in the LSC radiochromatogram shown in Fig. 10.2b.


10.3.1.2 Precision and Accuracy The precision and accuracy of the
TopCount instrument for metabolite profiling were recently determined and
compared to those from RFD and/or LSC analysis (Bruin et al., 2006; Zhu
et al., 2005b). The precision for determining the relative radioactivity
abundance of buspirone metabolites by HPLC–TopCount (Fig. 10.2a) ranged
from 2 to 11 %, which was comparable to that determined by the LSC and
RFD methods (Table 10.2). The accuracy values obtained on a TopCount for
the buspirone metabolites (Fig. 10.2a) were1 % to 12 % of nominal values
obtained by LSC and were similar to those determined by radio-flow detection
(Table 10.2). Excellent accuracy for metabolite profiling by HPLC–MicroBeta
counter was also demonstrated in the analysis of a plasma sample (Wallace
et al., 2004).


TABLE 10.2 Precision of metabolite profiling by LSC, MSC, and RFD (Zhu et al.,
2005b).


Radioactivity peak Percentage radioactivitya


LSC RFD MSC
Meanb RSDc Mean RSD Mean RSD

M3 11.24 7.0 11.73 8.8 12.56 4.5
M5 5.96 10.8 5.40 12.7 6.00 3.1
M6 6.88d 10.0d 2.17 11.5 2.18 10.5
M7 4.77 11.4 4.92 4.9
M8 8.22 8.4 8.05 8.6 7.68 3.4
M9 13.20 5.0 13.42 3.5 12.92 4.2
M11 7.98 4.6 7.89 10.4 7.90 1.6
Buspirone 32.80 4.3 34.96 7.5 33.00 2.9
M12 7.26 3.7 6.84 8.7 6.60 2.1


aProfiles of buspirone metabolites were determined by HPLC with three radiodetection techniques


(see Fig. 10.2). Percent distribution of radioactive peaks was calculated by dividing the
radioactivity of a metabolite peak by the total radioactivity determined in the HPLC run.
(8000 DPM per injection for HPLC–LSC and HPLC–MSC, and32,000 DPM per an injection
for HPLC–RCD).
bMean of % radioactivity (n= 5), which was calculated based on five HPLC injections.
cRSD is the relative standard deviation.
dThe value represents the sum of M6 and M7 radioactivity since the two metabolites were not


separated in HPLC–LSC analysis.

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