PT - JOURNAL ARTICLE AU - Pranav Shah AU - Edward Kerns AU - Dac-Trung Nguyen AU - R. Scott Obach AU - Amy Q. Wang AU - Alexey Zakharov AU - John McKew AU - Anton Simeonov AU - Cornelis E. C. A. Hop AU - Xin Xu TI - An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software AID - 10.1124/dmd.116.072017 DP - 2016 Oct 01 TA - Drug Metabolism and Disposition PG - 1653--1661 VI - 44 IP - 10 4099 - http://dmd.aspetjournals.org/content/44/10/1653.short 4100 - http://dmd.aspetjournals.org/content/44/10/1653.full SO - Drug Metab Dispos2016 Oct 01; 44 AB - 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 (t½ < 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.