be significantly reduced or eliminated by the dual stages of chromatographic
separation. (3) Higher sensitivity of mass spectrometric analysis is achieved by
the use of very low flow rates. (4) Use of the second HPLC allows LC–MS
conditions to be optimized for the analysis of each metabolite. The method
using an infusion nanoelectrospray mass spectrometer (Nanomate) to analyze
radiolabeled metabolites recovered from microplates (Fig. 10.3b) is also
employed for metabolite profiling. The approach increases acquisition time up
to 30–60 min for each recovered radioactivity peak so that multiple MS
experiments such as MSnanalyses can be conducted with improved sensitivity
(Staack et al., 2007).
10.4.4 An Integrated Radiochromatography–Mass Spectrometry Approach
Recently, an integrated approach using a combination of RFD, MSC and mass
spectrometers has been developed (Fig. 10.8) for quantification and structural
characterization of both high and low level radiolabeled metabolites (Zhao,
2006). In that study, a sample from a rat hepatocyte incubation of
[^14 C]nefazodone (NEF) was injected onto an HPLC (4.6 250 mm). A portion
(20 %) of the HPLC effluent was directed into a linear ion-trap MS (LTQ,
Thermo) and the remainder was passed through a solid scintillation cell and
then collected into 96-well plates. Radioactive peaks of interest were recovered
and infused using chip-based nanoelectrospray (Nanomate, Advion) into a
high resolution MS (LTQ FT) with its mass resolution set to 25,000 for
FIGURE 10.8 A set up of integrated approach using a combination of RFD, MSC,
and mass spectrometers for quantification and identification of both high and low levels
of radiolabeled metabolites.
RADIOCHROMATOGRAPHY IN CONJUNCTION 305