Analysis of low level radioactive metabolites in biological fluids using high-performance liquid chromatography with microplate scintillation counting: method validation and application.

TopCount, a microplate scintillation counter (MSC), has been recently employed as an off-line liquid radiochromatographic detector for radioactive metabolite profile analysis. The present study was undertaken to validate TopCount for metabolite profiling with respect to sensitivity, accuracy, precision and radioactivity recovery. Matrix effects of various human samples on TopCount performance and capability of MSC for volatile metabolite analysis were also investigated. TopCount had a limit of detection (LOD) of 5 DPM and a limit of quantification (LOQ) of 15 DPM for [(14)C]-labeled compounds at a 10min counting time. It was two-fold more sensitive than a liquid scintillation counter (LSC), and 50-100-fold more sensitive than a radioactivity flow detector (RFD). TopCount had comparable accuracy and precision to RFD, and comparable precision to LSC for determining relative abundance of metabolites. Human liver microsome incubation (up to 1 mL), plasma (up to 1 mL), urine (up to 2 mL) and feces (up to 50mg) had no significant quenching effects on TopCount performance. Benzoic acid, a volatile metabolite, was detected by TopCount, but not by Microbeta counter after microplates were dried under vacuum. Radioactivity recovery in HPLC-MSC analysis was reliably determined using an LSC-based method. Examples of using HPLC-MSC for analysis of low levels of radioactive metabolites are presented, including determination of plasma metabolite profile, in vitro reactive metabolites trapped by [(3)H]glutathione, and metabolite concentrations in an enzyme kinetic experiment. The data from this study strongly suggest that HPLC in combination with TopCount is a viable alternative analytical tool for detection and quantification of low levels of radioactive metabolites in biological fluids.