Methods: Two different methods were evaluated for increasing the amount of materials fractionated into 384-well micoplate. The first method involved increase loading using larger bore column and with maximum amount diverted to fraction collector. The other method involved collection of multiple HPLC runs into same 384-well microplate (LumaPlate, Perkin-Elmer, Shelton, CT) at the rate of 5 or 6 s/well with a fraction collector (PAL, Leap Technologies, Carrboro, NC). HPLC solvents in microplate were evaporated using a speed vacuum system (SpeedVac SPD-2010, Thermo Savant, Holbrook, NY). Radioactivity (CPM value) of the fractions in 384-well plates was determined with TopCount (Perkin-Elmer, Shelton, CT). Up to 12 well were counted simultaneously with a 5 min counting time.
Results: Biological samples such as plasma, urine, and feces with low concentrations of radioactivity were analyzed using HPLC with on-line radioactivity flow detection and off-line TopCount detection with multiple injections. Comparing the on-line and off-line approaches, the radioactivity profile from TopCount showed much more detail and the sensitivity was improved since the multiple injections allowed the double or triple radioactivity accumulated in 384-well microplate.
Conclusion: The data from this study strongly suggest that HPLC with multiple injections in combination with TopCount is a viable alternative analytical tool for detection and quantification of low levels of radioactive metabolites in biological fluids.