In vitro metabolic stability assays are used to screen compounds for stability in the presence of various drug metabolizing enzymes, usually cytochrome P450 in liver preparations (e.g., liver microsomes). High-throughput metabolic stability assays using pooling methods have been developed to keep pace with screening requirements at the lead ADME optimization stage. In our laboratory, we have improved the metabolic stability assay using the cassette analysis method, column switching, and incorporated time saving techniques in method development to yield a robust method which reduces data turnaround time, increases compound throughput, and maximizes mass spectrometer usage. This method can determine metabolic stability using microsomes or hepatocytes from any species. We describe our findings following incubation of 40 different compounds with human liver microsomes and analysis by the cassette and discrete analysis methods. Similar metabolic stability results were obtained using the cassette analysis and discrete analysis method. An overall 70% time savings was achieved by pooling four new compounds into one sample for method development/MS optimization, cassetting four samples into one sample to minimize the number of injections on LC/MS/MS analysis, and using a column switching system to analyze the samples, which results in a two-fold decrease in the LC/MS/MS analysis time.
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