radioactive peaks corresponding to the major buspirone metabolites 3^0 -OH,
5-OH and N-oxide were recovered from the 96-well microplates and
reincubated with HLM. Secondary metabolites derived from the incubations
of buspirone and these primary metabolites were analyzed by HPLC–MSC in
conjunction with ion-trap and accurate mass LC/MS.
Based on HPLC retention times, MSnspectra and diagnostic fragment ions
from accurate mass analysis, four secondary metabolites 5,3^0 -di-OH,N,3^0 -di-
OH, 5,6^0 -di-OH and N,6^0 -di-OH derivatives were identified. The major
precursors of these secondary metabolites were also elucidated based on
FIGURE 10.10 Analysis of [^3 H]GSH trapped reactive metabolites by HPLC with
TopCount (Zhu et al., 2005b). A nonlabeled drug (50 mM) was incubated with a
mixture of GSH (1 mM) and trace [^3 H]GSH (1–2mCi/mL) in human liver microsomes.
After precipitating proteins, the samples were analyzed by HPLC (1 mL/min) with
TopCount (4 wlls/min, 10-min counting time); (a) Radioactivity profile of the
incubation. M1 and M2 were GSH-trapped reactive metabolites; and (b)
Radioactivity profile of a control incubation sample (without NADPH).
APPLICATION OF NEW RADIOCHROMATOGRAPHY TECHNIQUES 309