Large-Scale Characterization of ToxCast Compounds and Their Metabolites by High-Throughput Ion Mobility-Mass Spectrometry: Applications in Human Exposome Studies

The exposome refers to the complete array of non-genetic factors that an individual experiences during their lifetime. Accurately measuring the chemical exposome, which may provide novel insights into environmental determinants of chronic disease, presents significant analytical challenges. Complex interactions between co-exposures can occur but are difficult to quantitatively predict. These mixture effects are likely due to the heterogeneous nature of common environmental chemicals, which represent thousands of classes and span vast concentration ranges in human blood. Additionally, inter-individual variation in xenobiotic metabolism can lead to different toxicological outcomes within an exposed population. High-resolution mass spectrometry (HRMS) has emerged as a powerful technique for broadly profiling xenobiotics and their transformation products in complex matrices. Despite recent advances in HRMS-based exposome analysis, however, there is a paucity of MS reference data for common environmental chemicals and their metabolites. There also remains a crucial need to harmonize measurements of exposome molecules across different laboratories and instrumental platforms. To address these challenges, we propose the development of large- scale, multidimensional reference databases dedicated to common environmental chemicals in the ToxCast library. These databases, which are essential for MS-based compound identification workflows, leverage ion mobility-mass spectrometry (IM-MS) to enhance selectivity and provide additional structural information. Gas-phase analyte ions in IM-MS are rapidly separated by size and shape using a dynamic electric field (traveling wave IM; TWIM) with a neutral buffer gas (e.g., N2). Ions with smaller rotationally averaged surface area, or collision cross section (CCS), exhibit greater mobility within the drift tube than less structurally compact gas-phase ions. IM-MS consequently enables three-dimensional separation when coupled to polarity-based LC separation and mass-based MS. The high inter-day and inter-laboratory reproducibility of TWIMCCSN2 measurements across diverse analyte classes, complemented by its compatibility with high- throughput analytical workflows, makes IM-MS ideal for confidently identifying exposome molecules in complex mixtures. Therefore, Specific Aim 1 will utilize LC-IM-MS/MS to create a multidimensional reference database that incorporates accurate mass-to-charge (m/z), CCS, retention time, and MS/MS fragmentation spectra for the ~4500 ToxCast compounds. To expand the scope of human exposome research to xenobiotic transformation products, Specific Aim 2 will focus on elucidating the major phase I/II metabolic pathways of the ToxCast compounds by human liver. The proposed research will generate crucial reference data for confidently identifying exposome molecules and their metabolites in complex matrices. Project Number: 1F31ES037537-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Environmental Health Sciences (NIEHS) | Principal Investigator: Ryan Nguyen | Institution: UNIVERSITY OF WASHINGTON, SEATTLE, WA | Award Amount: $44,939 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F04-S (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11162749