approximately 500 DPM of radioactivity or more are required for detection of
minor metabolites (10 DPM, 10 min counting time) corresponding to 2 % of
the total radioactivity on column. For the detection of the same minor
radioactive components, approximately 25,000–50,000 DPM of radioactivity is
required in the LC–RFD analysis. MSC is also applicable in the detection and
profiling of in vitro metabolites when the specific radioactivity of the
radiolabeled drug is low, metabolic turnover is low, or quantification of minor
radiolabeled metabolites is required.
10.5.2 Analysis of Metabolites of Nonradiolabeled Drugs Using Radiolabeled
Cofactors or Trapping Agents
Another application of liquid radiochromatographic techniques in drug
metabolism is the detection and quantification of conjugated metabolites or
reactive metabolites of nonradiolabeled drugs formed in incubations with
radiolabeled cofactors such as [^14 C]uridine diphosphate glucuroinic acid
(UDPGA) (Ethell et al., 1998) or reactive metabolite trapping agents such as
[^3 H]- or [^35 S]-glutathione (GSH) (Thompson et al., 1995; Zhu et al., 2005b).
For analyzing a large number of samples in enzyme kinetic experiments or
screening reactive metabolites, the use of online radiochromatographic
techniques such as HPLC–RFD and stop-flow LC–RFD should be the first
choice of the methods. HPLC–MSC is an alternative tool for sensitive analysis
of a limited number of samples. Figure 10.10 shows radioactivity profiles of a
nonradiolabeled drug incubated with human liver microsomes in the presence
of 1 mM [^3 H]GSH with and without the addition of NADPH. GSH trapped
reactive metabolites M1 and M2 were detected and quantified by HPLC–MSC
(Fig. 10.10a). These same conjugates were not detected in the incubation in
which NADPH was omitted (Fig. 10.10b). The sensitivity of MSC allows for
detection of low quantities of GSH adducts even when the specific activity and
total concentration of [^3 H]GSH in the incubations were kept low. If the same
experiment was conducted using a radio-flow detector, 50–100-fold more
[^3 H]GSH would be required to achieve the same level of detection.
10.5.3 Determination of Structures and Formation Pathways
of Sequential Metabolites
Many drugs, such as buspirone, undergo multiple oxidative biotransformation
reactions. In cases such as buspirone the secondary metabolites, which are
minor metabolitesin vitro, are major metabolites in the circulation or excreta in
humans and animals because primary metabolites are rapidly converted to
secondary metabolites. To determine the formation pathways and structures of
secondary or sequential metabolites, a method using ‘‘metabolite incubation’’
and HPLC–MSC–MS analysis was developed and demonstrated using
buspirone as an example (Zhu, 2002). [^14 C]buspirone was incubated with
HLM, and metabolic profiling was carried out using HPLC–MSC. The
308 APPLICATIONS OF LIQUID RADIOCHROMATOGRAPHY TECHNIQUES