sample. MSC was able to detect and quantify both minor and major
metabolites (Fig. 10.7a), which is consistent with the metabolite profile
determined by high resolution LC/MS (Fig. 10.7c).
To enhance the sensitivity of mass spectrometry in the analysis of
radiolabeled metabolites, an alternative method using a combination of LC–
MSC and MS has been recently developed as shown in Fig. 10.3b (Gedamke,
2003; Zhu, 2002, 2003, 2005c). First, a radioactivity profile is determined by
MSC without splitting HPLC effluent. Based on the resultant metabolite
profile, radioactive metabolites of interest are then recovered from the
microplates and structurally characterized by capillary and nano LC–MS or
nanospray mass spectrometry via direct infusion. A combination of LC–MSC
and nano LC/MS for analyzing rat plasma metabolites showed several
advantages to this approach (Gedamke, 2003). (1) The superior radiodetection
sensitivity was maintained due to the high loading capacity of the large HPLC
column. (2) Suppression of metabolite ionization by coeluting components can
FIGURE 10.7 Profiles of radiolabeled metabolites in a rat bile sample determined by
HPLC-MSC-MS. A majority of HPLC effluent (1 mL/min) was collected into 96-well
microplates (four fractions per min) followed by radioactivity counting using
TopCount. The top panel is the radiochromatogram of this sample. A potion of the
effluent was analyzed by LTQ FTMS. The middle panel is the total ion chromatogram
(TIC) from accurate mass full scan MS analysis. The bottom panel is a mass defect filter
processed TIC (Zhu et al., 2006). The peak indicated is a GSH adduct that was not
presented in the unprocessed TIC.
304 APPLICATIONS OF LIQUID RADIOCHROMATOGRAPHY TECHNIQUES