Because of the pervasiveness, persistence, and toxicity of per-and polyfluoroalkyl substances (PFAS), there is growing concern over PFAS contamination, exposures, and health effects. The diversity of potential PFAS is astounding, with nearly 10,000 PFAS catalogued in databases to date (and growing). The ability to detect the thousands of known PFAS, and discover previously uncatalogued PFAS, is necessary to understand the scope of PFAS contamination and to identify appropriate remediation and regulatory solutions. Current non-targeted methods for PFAS analysis require manual curation and are time-consuming, prone to error, and not comprehensive. FluoroMatch Flow 2.0 is the first software to cover all steps of data processing for PFAS discovery in liquid chromatography-high-resolution tandem mass spectrometry samples. These steps include feature detection, feature blank filtering, exact mass matching to catalogued PFAS, mass defect filtering, homologous series detection, retention time pattern analysis, class-based MS/MS screening, fragment screening, and predicted MS/MS from SMILES structures. In addition, a comprehensive confidence level criterion is implemented to help users understand annotation certainty and integrate various layers of evidence to reduce overreporting. Applying the software to aqueous film forming foam analysis, we discovered over one thousand likely PFAS including previously unreported species. Furthermore, we were able to filter out 96% of features which were likely not PFAS. FluoroMatch Flow 2 increased coverage of likely PFAS by over tenfold compared to the previous release. This software will enable researchers to better characterize PFAS in the environment and in biological systems.

Thousands of per-and polyfluoroalkyl substances (PFAS) exist in the environment and pose a potential health hazard. Suspect and non-target screening with liquid chromatography (LC) high-resolution tandem mass spectrometry (HRMS/MS) can be used for comprehensive characterization of PFAS. To date no automated open source PFAS data analysis software exists to mine these extensive datasets. We introduce FluoroMatch, which automates file conversion, chromatographic peak picking, blank feature filtering, PFAS annotation based on precursor and fragment masses, and annotation ranking. The software library currently contains ~7,000 PFAS fragmentation patterns based on rules derived from standards and literature and the software This document is the postprint version of an article published in

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Omics-based technologies have enabled comprehensive characterization of our exposure to environmental chemicals (chemical exposome) as well as assessment of the corresponding biological responses at the molecular level. Systematically measuring exposures and linking these stimuli to biological perturbations can determine specific chemical exposures of concern, mechanisms and biomarkers of toxicity, and interventions. However, advancement of exposomics is limited by a lack of harmonization of gas chromatography high-resolution mass spectrometry (GC-HRMS) methods used for data acquisition and approaches for assigning confidence to chemical annotations. Here we discuss the major pieces of evidence provided by GC-HRMS workflows, including retention time and retention index, electron ionization, positive chemical ionization, electron capture negative ionization, and atmospheric pressure chemical ionization spectral matching, molecular ion, accurate mass, isotopic patterns, database occurrence, and occurrence in blanks. We then provide a qualitative framework for incorporating these various lines of evidence for communicating confidence in GC-HRMS data by adapting the Schymanski scoring schema developed for reporting confidence levels by liquid chromatography high-resolution mass spectrometry (LC-HRMS). Validation of our framework is presented using standards spiked in plasma, and confident annotations in outdoor and indoor air samples, showing a false positive rate of 12% for suspect screening for chemical identifications assigned as Level 2 (when structurally similar isomers are not considered false positives). This framework is easily adaptable to various workflows and provides a concise means to communicate confidence in annotations. Further validation, refinements, and adoption of this framework will ideally lead to harmonization across the field, helping to improve the quality and interpretability of compound annotations obtained in GC-HRMS.

There are thousands of different per- and polyfluoroalkyl substances (PFAS) in everyday products and in the environment. Discerning the abundance and diversity of PFAS is essential for understanding sources, fate, exposure routes, and the associated health impacts of PFAS. While comprehensive detection of PFAS requires use of non-targeted mass spectrometry, data-processing is time intensive and prone to error. While automated approaches can compile all mass spectrometric evidence (e.g., retention time, isotopic pattern, fragmentation, and accurate mass) and provide ranking or scoring metrics for annotations, confident assignment of structure often still requires extensive manual review of the data. To aid this process, we present FluoroMatch Visualizer which was developed to provide interactive visualizations which include normalized mass defect plots, retention time versus accurate mass plots, MS/MS fragmentation spectra, and tables of annotations and meta-data. All graphs and tables are interactive and have cross-filtering such that when a user selects a feature, all other visuals highlight the feature of interest. Several filtering options have been integrated into this novel data visualization tool, specifically with the capability to filter by PFAS chemical series, fragment(s), assignment confidence, and MS/MS file(s). FluoroMatch Visualizer is part of FluoroMatch Suite, which consists of FluoroMatch Modular, FluoroMatch Flow, and FluoroMatch Generator. FluoroMatch Visualizer enables annotations to be extensively validated, increasing annotation confidence. The resulting visualizations and datasets can be shared online in an interactive format for community based PFAS discovery. FluoroMatch visualizer holds potential to promote harmonization of non-targeted data-processing and interpretation throughout the PFAS scientific community.