Liquid Chromatography-Accurate Radioisotope Counting and Microplate Scintillation Counter Technologies in Drug Metabolism Studies

Abstract

The present study involves an analysis of the performance of liquid chromatography (LC)-accurate radioisotope counting (ARC) and microplate scintillation counter (TopCount) technologies in drug metabolism studies. For the purpose of evaluating these systems, biological samples resulting from the metabolism of a radiolabeled [14C] compound, known as compound B, are analyzed using LC-ARC and TopCount under similar high-performance LC conditions. Counting efficiency is 83% for LC-ARC, 77% for TopCount, and linearity is R2 of 0.9998 versus 0.9984, respectively. The limit of detection for LC-ARC is 12 disintegrations per minute (dpm) with 1-min/fraction counting, yet for TopCount it is 8.7 dpm with 5-min/fraction counting. Under optimal conditions for each, the total run time of LC-ARC is approximately half that of TopCount. These results indicate that there is no significant difference between these two systems in terms of efficiency, linearity, and limit of detection. However, the LC-ARC system does not involve any manual operations, yet TopCount requires manual sample transfer and data import. This study shows that impressive progress has been made in the technology of radioisotope counting in drug metabolism using LC-ARC. This system enhances the resolution of radiochromatograms and is able to measure volatile metabolites that TopCount cannot detect at all. The ability to acquire mass spectra online is also a major advancement. The overall results suggest that the combination of LC-ARC with radioactivity detection and mass spectrometry has great potential as a powerful tool for radioisotope measurement in metabolite identification studies during drug discovery and development.

Reaction Phenotyping

“…HPLC -MSC provides excellent sensitivity, but has relatively low throughput. Stop -fl ow HPLC -RFD (Nassar et al, 2003(Nassar et al, , 2004 technique has the fl exibility to be operated in different modes (by -fraction, by -level, nonstop), the technique greatly extended the capacity of RFD by improving its sensitivity. It can be used in an automated fashion and has been used successfully in enzyme kinetics studies of muraglitazar glucuronidation (Zhao et al, 2008 ).…”

Radioactivity Detectors for High-Performance Liquid Chromatography in Drug Metabolism Studies

“…A general equation for the limit of detection (LOD) is: where T is the counting time in minutes, E is the counting efficiency as a percentage and B is the background radioactivity in dpm. [26] Obviously, changes in one of the three parameters B, E, or T effect the LOD. In most drug metabolism studies, radioactivity concentrations may be low, and with a low number of disintegrations N, the relative uncertainty becomes large.…”

Analytical Tools and Approaches for Metabolite Identification in Early Drug Discovery

“…Concentrations of metabolites in plasma are often quite low. When dealing with radioactivity studies, these low levels can be measured using stopflow or fraction collection techniques (55). LC/UV/MS approaches include column switching (56) to increase sample injection size for these relatively clean samples, Bon the fly^dynamic background correction on a quadrupole linear ion trap to greatly lower detection limits for metabolites (51) or fraction collection followed by nanospray infusion (57,58).…”

“…Alternatively all metabolites are quantified by incorporation of a 14 C or 3 H label into the structure followed by radiochemical detection of HPLC separated metabolites. StopYflow and microplate scintillation counting are recently developed improvements in low-level radiometabolite quantification (55). In many early metabolism studies neither of these classical approaches is practical.…”