injection of 0.25–5 DPM of radioactive materials (Brown et al., 2005, 2006).
For example, metabolite profiles of^14 C-R115777, a farnesyl transferase
inhibitor, in human urine, faces, and plasma have been determined by HPLC–
AMS after 50 mg^14 C-R115777 (687 nCi/mg) was dosed to subjects (Brown
et al., 2005, 2006). HPLC–AMS is especially useful for human ADME studies
when it is necessary to administer a very low radioisotope dose (either due to
safety considerations or because the total dose of the drug is low, i.e.,<1 mg)
or for the determination of long-term kinetics and metabolism in humans.
HPLC–AMS could be envisioned as a tool to allow metabolite profiles to be
determined very early in clinical development. However, the use of HPLC–
AMS in drug metabolism has not been widespread due to the relatively high
cost of AMS analysis and the challenge as inherent in sample collection and
preparation for HPLC–AMS analysis.
10.4 Radiochromatography in Conjunction with Mass Spectrometry
The use of radiolabeled drugs is not only crucial for the quantification of
unknown metabolites but has also long played a critical role in metabolite
identification studies. The utility of radiolabeled substrates include: (1) their
use as tracers of drug-related components during sample clean-up, concentra-
tion, profiling, and isolation from complex biological matrixes; and (2)
facilitation of LC-MS/MS detection of radiolabeled metabolites based on their
HPLC retention times, peak shapes, and in some cases, isotopic ratios.
10.4.1 LC-RFD-MS
General configurations of LC–RFD–MS are shown in Figs. 10.1a and 10.1b.
The configuration (A) is designed for conventional HPLC at flow rates of
approximately 1 mL/min. After splitting, a small portion (20 %) of the
HPLC effluent is introduced into a mass spectrometer, and the rest of the
effluent goes to the liquid detection cell of the RFD. This setup provides flow
rates suitable for both mass spectrometry and RFD and a large sample loading
capacity suitable forin vivosample analysis (Egnash and Ramanathan, 2002;
Hyllbrant et al., 1999; Mullen et al., 2002, 2003). Configuration (B) (Fig. 10.1b)
is more comparable with liquid radiochromatographic analysis at a flow rate of
approximately 0.2 mL/min. This setup is more sensitive in MS analysis, since
there is no splitting of HPLC effluent. Use of solid radioactivity detection cell
greatly reduces volume of radioactivity waste; however, the solid cell has lower
counting efficiency. Also, in some cases, radiolabeled drug or metabolites
adhere to the solid cell, resulting in peak broadening. LC–RFD–MS is the most
popular technique for simultaneous radioactivity profiling and metabolite
characterization because LC–RFD can provide results in the time frame
comparable to LC/MS (Mullen et al., 2002, 2003). In addition, metabolite
302 APPLICATIONS OF LIQUID RADIOCHROMATOGRAPHY TECHNIQUES