@article {Shah1653, author = {Pranav Shah and Edward Kerns and Dac-Trung Nguyen and R. Scott Obach and Amy Q. Wang and Alexey Zakharov and John McKew and Anton Simeonov and Cornelis E. C. A. Hop and Xin Xu}, title = {An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software}, volume = {44}, number = {10}, pages = {1653--1661}, year = {2016}, doi = {10.1124/dmd.116.072017}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {Advancement of in silico tools would be enabled by the availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure data set by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (P450) isozyme. To achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle a large volume of compound sets. The substrate depletion method [in vitro half-life (t1/2) method] was chosen to determine CLint. The assay (384-well format) consisted of three parts: 1) a robotic system for incubation and sample cleanup; 2) two different integrated, ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and 3) an automated data analysis system. The CYP3A4 assay was evaluated using two long t1/2 compounds, carbamazepine and antipyrine (t1/2 \> 30 minutes); one moderate t1/2 compound, ketoconazole (10 \< t1/2 \< 30 minutes); and two short t1/2 compounds, loperamide and buspirone (t{\textonehalf} \< 10 minutes). Interday and intraday precision and accuracy of the assay were within acceptable range (\~{}12\%) for the linear range observed. Using this assay, CYP3A4 CLint and t1/2 values for more than 3000 compounds were measured. This high-throughput, automated, and robust assay allows for rapid metabolic stability screening of large compound sets and enables advanced computational modeling for individual human P450 isozymes.}, issn = {0090-9556}, URL = {https://dmd.aspetjournals.org/content/44/10/1653}, eprint = {https://dmd.aspetjournals.org/content/44/10/1653.full.pdf}, journal = {Drug Metabolism and Disposition} }