Résumé
If very-high-pressure liquid chromatography (VHPLC) is to replace conventional HPLC as the ultimate separation tool for metabolism studies in development, coupling it efficiently with online radioactivity detection (RAD) is needed. We describe the successful combination of VHPLC/RAD, facilitated by improvements in online radioactivity detection, as well as in column loading and peak capacity. The sensitivity of 14C detection was improved by the use of a variable scintillation flow achieved via a simple modification to the classical online radiochemical detection set-up. A modification of the flow-through cell design in which internal diameter of the tubing was reduced further increased the sensitivity and resolution by decreasing peak tailing. The injection of relatively large injection volumes was made possible by the use of columns packed at ultra-high pressure with 2.2μm particles. Because of the reduced back pressure generated using these larger particle sizes, two 150mm×3mm columns could be coupled, allowing 4-fold larger injection volumes and a 50% increase in theoretical plate number at a similar back pressure compared to a standard 150mm×2.1mm Waters UPLC column. The value of the methodology described was demonstrated by the analysis of in vitro and in vivo metabolism samples of 3H- and 14C-labeled compounds and compared with conventional radio-HPLC. We have shown that metabolite separation can be achieved with increased efficiency while maintaining a sensitivity comparable to that of conventional HPLC/RAD.