Higher Throughput Bioanalysis by Automation of a Protein Precipitation Assay Using a 96-Well Format with Detection by LC-MS/MS
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- 作者：Alan P. Watt ; Denise Morrison ; Karen L. Locker ; and David C. Evans
- 刊名：Analytical Chemistry
- 出版年：2000
- 出版时间：March 1, 2000
- 年：2000
- 卷：72
- 期：5
- 页码：979 - 984
- 全文大小：179K
- 年卷期：v.72,no.5(March 1, 2000)
- ISSN：1520-6882
文摘
Generic methodology for the automated preparation andanalysis of drug levels in plasma samples within a drugdiscovery environment was achieved through the redesignof a protein precipitation assay to a microtiter (96-well)plate format and the application of robotic liquid handlingfor performance of all transfer and pipetting steps. Validation studies revealed that the application of robotics tosample preparation, in general, maintained the analyticalaccuracy and precision compared with preparing samplesmanually. The use of rapid gradient LC-MS/MS foranalysis coupled with flow diversion of the solvent frontallowed the introduction of protein-precipitated samplesinto the mass spectrometer without the necessity forsource cleaning. The problem inherent in automaticallypipetting plasma, caused by fibrinogen clots, was overcome by storing samples at -80 
C and thus precludingclot formation. The resulting methodology allowed samplepreparation for a 96-well plate designed to accommodate54 unknowns, duplicate 12-point calibration curves, and6 sets of quality controls at three levels in approximately2 h. This approach allowed an increase in throughput ofsample preparation and analysis to >400 samples per dayper LC-MS/MS instrument with minimal manual intervention. Overall, substantial time savings were realized,demonstrating that automation is an increasingly essentialtool in a drug discovery bioanalytical environment.
C and thus precludingclot formation. The resulting methodology allowed samplepreparation for a 96-well plate designed to accommodate54 unknowns, duplicate 12-point calibration curves, and6 sets of quality controls at three levels in approximately2 h. This approach allowed an increase in throughput ofsample preparation and analysis to >400 samples per dayper LC-MS/MS instrument with minimal manual intervention. Overall, substantial time savings were realized,demonstrating that automation is an increasingly essentialtool in a drug discovery bioanalytical environment.