FluoroMatch 3.0 – Automated PFAS Non-Targeted Analysis and Visualizations Applied to Mammalian Biofluids

Importance of the Topic

Per- and polyfluorinated substances (PFAS) are persistent environmental contaminants of growing concern due to their bioaccumulation and toxicity. Recent regulatory and research efforts have focused mainly on a limited number of PFAS, leaving a large fraction of unknown or emerging PFAS unmonitored. Non-targeted analysis is therefore crucial to fully assess PFAS exposure and potential health impacts.

Objectives and Study Overview

This work introduces FluoroMatch 3.0, an automated software platform designed to expand PFAS detection beyond targeted methods. The study applies FluoroMatch to dried blood spot samples to demonstrate its ability to identify both known and novel PFAS species in mammalian biofluids.

Methodology

• Sample preparation: Whole blood was dried on QIAcard blood spot cards and spiked with 20 native PFAS standards, with blanks analyzed for background correction.
• Chromatography and mass spectrometry: An Agilent 1290 Infinity II LC with Poroshell ECC18 and PFC Delay Column was coupled to an Agilent 6546 LC/Q-TOF instrument. Data were acquired in full-scan, data-dependent acquisition (DDA), and iterative exclusion DDA modes to maximize PFAS coverage.
• Data processing: Raw data were processed using MZMine for peak picking, followed by FluoroMatch Flow and Modular workflows for feature grouping, blank filtration, annotation, and visualization.

Used Instrumentation

• Agilent 1290 Infinity II LC system with Poroshell ECC18 column (2.1 × 100 mm, 2.7 µm) and PFC Delay Column.
• Agilent 6546 LC/Q-TOF mass spectrometer for high-resolution MS and MS/MS with DDA and iterative exclusion strategies.
• FluoroMatch software suite for automated library matching, fragmentation screening, and visual analytics.

Main Results and Discussion

• A total of 28 PFAS across five homologous series (FTS, PFCA, PFECA, PFSA, PFESA) were annotated in blood spot samples.
• The workflow detected 95 % of spiked PFAS standards, with a false negative rate below 5 % and a similar false positive rate.
• Five novel or rarely screened PFAS, including PFECA isomers and unsaturated PFECA, were identified, indicating human exposure beyond commonly monitored compounds.
• Fragment screening highlighted multiple ether-linked PFAS, expanding the scope of detectable PFAS in biofluids.

Benefits and Practical Applications

• Enhanced PFAS coverage, enabling comprehensive exposure profiling in environmental and biomedical studies.
• Automated annotation and visualization streamline non-targeted workflows, reducing manual review time.
• Customizable libraries and modular scripts allow integration with in-house analytical pipelines and regulatory monitoring programs.

Future Trends and Opportunities

Ongoing expansion of PFAS libraries through EPA collaboration and in silico biotransformation predictions will improve detection of emerging contaminants. High-throughput screening, integration with machine learning for fragmentation prediction, and cloud-based interactive dashboards represent promising directions for next-generation PFAS analysis.

Conclusion

FluoroMatch 3.0 offers a robust, automated solution for non-targeted PFAS analysis, achieving high annotation confidence, uncovering novel PFAS in human blood, and supporting comprehensive exposure assessment and regulatory applications.