acquisition of accurate mass spectral data. The online RFD and linear ion-trap
MS analyses detected multiple oxidative and conjugated metabolites with high
levels of radioactivity (>250 DPM). The off-line MSC radiochromatogram
from the same HPLC run displayed nine additional metabolites with low level
of radioactivity. Nanoelectrospray FTMS analyses of a number of the
metabolites recovered from microplates were carried out under optimized
analytical conditions with continuous infusion for up to 30 min. As a result,
high quality accurate mass full scan MS and MSnspectra were obtained. In
some cases, metabolite ions could not be recognized in the full scan MS spectra
due to interference from coeluting biological components and background
ions. Mass defect fiter (Zhu et al., 2006) removed the majority of these intense
interfering ions so that the metabolite ions became predominant and were
easily recognizable. As a result, a single LC/RFD/MS run followed by MSC
radioactivity profiling and nanoelectrospray FTMS analysis enabled the
detection, quantitative determination, and structural characterization of
more than 20 NEF-related components, including several very minor
metabolites. This example demonstrates that the integrated approach can
provide both quantitative analysis and structural characterization of radi-
olabeled metabolites at both high and low levels.
10.5 APPLICATION OF NEW RADIOCHROMATOGRAPHY
TECHNIQUES IN DRUG METABOLISM STUDIES
10.5.1 Profiling of Radiolabeled Metabolites in Plasma
One of important objectives of drug metabolism studies in drug development is
to assess whether human circulating metabolites are present in plasma of
toxicology species at appropriate levels, which is usually accomplished by
profiling plasma metabolites in radiolabeled human and animal ADME
studies. However, radioactivity analysis of plasma metabolites is a challenging
task because concentrations of metabolites in plasma are much lower
compared to those in urine, bile or feces, and volumes of plasma sample
obtained from ADME studies are very limited, especially when multiple
plasma samples were collected for AUC determination. In most cases, HPLC–
RFD is not sensitive enough for this application. In addition, high throughput
is not required in plasma metabolite profiling since a limited of number of
pooled plasma samples are subjected to analysis in ADME studies (Zhang
et al., 2007b). Therefore, off-line LC–MSC technique well suits for analysis of
plasma and other samples from ADME studies. Furthermore, unlike stop-flow
or dynamic flow LC–RFD methods, the use of LC–MSC technique for
metabolite profiling has been fully validated with respect to accuracy,
precision, sensitivity, matrix effect and radioactivity recovery (Bruin et al.,
2006; Zhu et al., 2005b). Thus, high quality results of metabolite profiling by
MSC can be obtained as long as proper procedures are followed.
306 APPLICATIONS OF LIQUID RADIOCHROMATOGRAPHY TECHNIQUES