the metabolism of the drug, and the kinetics of the metabolic reactions (Ilett
et al., 2002; Lautala et al., 1999; Zhang et al., 2004, 2007a; Zhu et al., 2005a);
and (6) investigative drug metabolism studies to address issues related to drug
metabolism and disposition (Evans et al., 2004; Li et al., 2006; Meneses-
Lorente et al., 2006; Zhang et al., 2003). All these radiolabeled studies, except
for the tissue distribution study, require metabolite profiling to determine the
number and relative concentrations of individual metabolites in various
biological matrices using a radiochromatographic technique.
Applications of radioisotopes to studies of ADME have been comprehen-
sively reviewed recently (Dalvie, 2000; Marathe et al., 2004; Veltkamp, 1990).
This chapter focuses on the recent advance in liquid radiochromatographic
techniques and their application to metabolite detection and quantification in
drug metabolism studies. In addition, the use of liquid radiochromatography
coupled with mass spectrometry in metabolite profiling and structural
elucidation is described.
10.2 TRADITIONAL RADIOCHROMATOGRAPHY TECHNIQUES
Planar radiochromatography was the first widely used technique for separation
and detection of radiolabeled metabolites (Veltkamp, 1990). The technique is
very sensitive, fast, and has relatively low equipment and operating costs,
however, due to low separation resolution and poor precision in quantification,
it has largely been replaced by HPLC-based radiochromatographic techniques
in the pharmaceutical industry. Online radio flow detection (RFD) and off-line
liquid scintillation counting (LSC) have been routinely used for analysis of
radiolabeled metabolites for the last 15 years (Veltkamp, 1990).
10.2.1 HPLC-RFD
Figure 10.1a and b illustrate general setups of HPLC–RFD for radioactivity
profiling, in which a mass spectrometric detection can be coupled for
simultaneous metabolite characterization with or without splitting of the
HPLC effluent (Egnash and Ramanathan, 2002; Heath et al., 1997; Hyllbrant
et al., 1999; Vlasakova et al., 1998). Online HPLC–RFD has several
advantages including rapid analysis, high separation resolution (Fig. 10.2c),
and good accuracy and precision for radioactivity measurement. Currently,
HPLC–RFD is the most common liquid radiochromatographic technique used
in drug metabolism studies, and is especially suitable for radiochromatographic
method development, analysis of a large of number of samples necessary for
completing studies such as enzyme kinetic determination, and metabolite
characterization through coupling with a mass spectrometer (Morovjan et al.,
2002). The single major disadvantage of HPLC–RFD is its relatively poor
sensitivity due to short residence times (5–15 s) of the radioactive peaks in the
RFD detection cell (Zhu et al., 2005b). As listed in Table 10.1, the detection of
290 APPLICATIONS OF LIQUID RADIOCHROMATOGRAPHY TECHNIQUES