Introduction High clearance due to extensive metabolism by cytochrome P450 is a common ADME liability of discovery-stage compounds. To provide structural information to facilitate ADME optimization, we have established a higher-throughput in vitro soft spot identification assay using HRMS/MS, sample pooling and software-assisted structure elucidation, as reported previously1. In this work, we present an additional improvement to the assay by integrating the assessment of glutathione (GSH) adduct formation, which could indicate bioactivation to reactive metabolites, in an enhanced analysis and data-review workflow. Methods A dual-concentration (30 and 0.5 µM) incubation approach was used, with liver microsomes that were supplemented by both NADPH and GSH. A Shimadzu Nexera HPLC system and a Thermo Q Exactive tandem mass spectrometer were used for LC-MS analysis. A 10-min gradient was used with an Agilent Eclipse+ C18 column and a mobile phase of 0.2% formic acid in water and acetonitrile. During analysis of the 30 µM samples, the mass spectrometer was operated with alternating full scans and data dependent scans with an inclusion list generated automatically by Mass-MetaSite. For the 0.5 µM samples, only full scan data was collected. Software-assisted soft spot ID was performed using Mass-MetaSite, and semi-quantitation of metabolites was performed using GMSU/QC software. Preliminary Data The use of an inclusion list of both singly and doubly charged masses generated by Mass-MetaSite for the data dependent acquisition ensured the collection of important MS/MS spectra for potential metabolites and GSH adducts. In addition, all-ion fragmentation data was acquired following a separate injection in case an unexpected metabolite failed to trigger MS/MS spectrum acquisition. All data from the 30 µM samples was processed by Mass-MetaSite software, which compared chromatograms to identify metabolites and then assigned structures by comparing their theoretical and experimental MS/MS spectra in an unattended fashion. After major metabolites were assigned by Mass-MetaSite, their accurate masses were imported into GMSU/QC software for automatic peak extraction and integration, and %-remaining vs. time plots of metabolites detected in the 0.5 µM samples were generated automatically. Using this workflow, we established the feasibility of a higher-throughput assay for soft-spot identification with integrated assessment of GSH adduct formation, at a per batch capacity of 8 individual compounds in liver microsomes from 2 species. The results obtained from this workflow were consistent with those previously reported. The detailed assay incubation, data acquisition and data processing workflow, as well as results from literature compounds will be presented. 1 Paiva A, Klakouski C, Zvyaga T, Johnson B, Josephs J, Humphreys WG, Weller H and Shou WZ, “Optimization of a High-throughput Metabolic Soft Spot Assay with Pooled Sample Analysis and Software-assisted Structure Elucidation,” presented at the 61st Annual Conference of American Society for Mass Spectrometry (ASMS) on Mass Spectrometry and Allied Topics, Minneapolis, Minnesota, 2013. Novel Aspect The integrated assessment of glutathione adduct formation in a high-throughput metabolic soft spot assay.