Higher throughput bioanalysis by automation of a protein precipitation assay using a 96-well format with detection by LC-MS/MS

Generic methodology for the automated preparation and analysis of drug leve ls in plasma samples within a drug discovery environment was achieved throu gh the redesign of a protein precipitation assay to a microtiter (96-well) plate format and the application of robotic liquid handling for performance of all transfer and pipetting steps. Validation studies revealed that the application of robotics to sample preparation, in general, maintained the a nalytical accuracy and precision compared with preparing samples manually. The use of rapid gradient LC-MS/MS for analysis coupled with flow diversion of the solvent front allowed the introduction of protein-precipitated samp les into the mass spectrometer without the necessity for source cleaning. T he problem inherent in automatically pipetting plasma, caused by fibrinogen clots, was overcome by storing samples at -80 degrees C and thus precludin g clot formation. The resulting methodology allowed sample preparation for a 96-well plate designed to accommodate 54 unknowns, duplicate 12-point cal ibration curves, and 6 sets of quality controls at three levels in approxim ately 2 h, This approach allowed an increase in throughput of sample prepar ation and analysis to >400 samples per day per LC-MS/MS instrument with min imal manual intervention. Overall, substantial time savings were realized, demonstrating that automation is an increasingly essential tool in a drug d iscovery bioanalytical environment.