Drug Metabolism in Drug Design and Development Basic Concepts and Practice

limit of RFD is 250–500 disintigrations per minutes (DPM) and the limit of
quantification of RFD ranges from 750 to 1500 DPM, which greatly limits its
use in the analysis of low levels of radiolabeled metabolites such as plasma
metabolites.

10.2.2 HPLC-LSC

Figure 10.1c illustrates a general setup of an HPLC coupled with an LSC
instrument for radioactivity profiling. Off-line LSC radioactivity counting is
at least 25-fold more sensitive than HPLC–RFD (Figs. 10.2b and 10.2c and
Table 10.1) because individually collected effluent fractions can be counted for
a longer time (usually 10 min or more). LSC has been traditionally employed
for quantification of low levels of radiolabeled metabolites (Chando et al.,
1998; Everett et al., 1991). Radioactivity analysis by off-line LSC consists of
four steps: HPLC separation, fraction collection into individual vessels,
mixing with scintillation cocktail, and radioactivity counting one fraction at a
time. The entire process is time consuming and relatively labor intensive. In
addition, due to the labor and reagent consumption of the process HPLC
fractions are often collected at timeintervals, which result in suboptimal peak
resolution compared to other techniques (Fig. 10.2b and 10.2c). Because of
these reasons, the use of HPLC–LSC for profiling of low level radiolabeled
metabolites has generally been replaced by HPLC–microplate scintillation

LC/UV

RFD

(b) (solid cell) MS

LC/UV

RFD

(liquid cell)

MS

Cocktail

(a)

LC/UV

Fraction

collector

MS

Cocktail

LSC

(c)

FIGURE 10.1 Configuration of HPLC with online RFD (aandb); and off-line of LSC
(c) with or without coupling with a mass spectrometer.

TRADITIONAL RADIOCHROMATOGRAPHY TECHNIQUES 291