Game of Unknowns: Non-Target Analysis Strategies for Identifying Per- and Polyfluoroalkyl Substances (PFAS) in Water

Importance of the Topic

Per- and polyfluoroalkyl substances (PFAS) are widely used for their unique chemical and physical properties. Their persistence, bioaccumulation and potential health impacts make them priority environmental contaminants. Conventional targeted analysis covers only a small fraction of PFAS present, leaving many unknown or emerging compounds undetected. Developing robust non-target screening strategies is essential for comprehensive monitoring, risk assessment and informed remediation decisions.

Objectives and Study Overview

This study demonstrates advanced workflows on an LC-Q/TOF platform to screen, identify and quantify known and unknown PFAS in water. Key goals include improving sensitivity for targeted PFAS, expanding the scope to biotransformation products and homologous series, enhancing confidence with retention time prediction and in silico tools, and reducing reliance on analytical standards.

Methodology and Instrumentation

Environmental water samples were mixed 1:1 with methanol, acidified, filtered and centrifuged. A 30 μL injection of the 50/50 methanol/water extract was analyzed by high-resolution accurate mass LC-Q/TOF. Screening strategies employed proprietary databases, predictive software and mass defect analysis without requiring standards for every compound.

• Sample preparation: dilution, acidification, filtration, centrifugation
• Chromatography: gradient LC on Agilent 1290 Infinity II
• Detection: Agilent 6546 Q-TOF in full scan and MS/MS modes
• Data analysis: MassHunter Quantitative, Mass Profiler Professional, Biotransformation and Molecular Structure Correlator modules

Used Instrumentation

• Agilent 1290 Infinity II LC system
• Agilent 6546 Q-TOF mass spectrometer
• Agilent MassHunter Quantitative software
• Agilent MassHunter Mass Profiler Professional
• Agilent Biotransformation module
• Agilent Molecular Structure Correlator

Main Results and Discussion

Targeted quantification of 22 PFAS achieved detection limits down to 2–20 ng/L for PFOA, PFOS and PFBA, demonstrating improved sensitivity and linearity (20–2000 ng/L for PFBA; 2–2000 ng/L for PFOS/PFOA).

• Library-based screening: Personal compound databases (PCD) and PCD libraries (PCDL) provided accurate mass, formula and MS/MS spectra matching for known PFAS.
• Biotransformation products: In silico software predicted PFOS metabolites, enabling detection of degradation products not in standard lists.
• Retention time prediction: Models regressing measured RTs on LogP, LogS and –CF2– count improved identification confidence for suspects such as perfluoro(2-ethoxyethane) sulfonic acid.
• Kendrick mass defect analysis: Identification of homologous series by recurring –CF2– units streamlined detection of multiple PFAS congeners.
• In silico fragment prediction: Molecular Structure Correlator annotated unknown PFAS fragments, aiding structural elucidation without standards.

Benefits and Practical Applications

The integrated workflow enables comprehensive PFAS screening in water, from regulated compounds to novel precursors and transformation products. It supports environmental monitoring, remediation planning and regulatory compliance with minimal reliance on standards. High-throughput analysis and automated data processing enhance laboratory efficiency and data quality.

Future Trends and Opportunities

Continued expansion of PFAS libraries, integration of machine learning for retention time and fragmentation prediction, and development of real-time field-deployable HRMS platforms will further advance non-target analysis. Collaborative data sharing and open-access spectral databases are critical for identifying emerging PFAS worldwide.

Conclusion

This study presents a versatile LC-Q/TOF workflow combining targeted quantification and non-target screening strategies for PFAS in water. By leveraging HRMS accuracy, predictive software and innovative data analysis approaches, laboratories can detect a broader range of PFAS contaminants with high confidence and sensitivity.

Reference

• Anumol T, Tölgyesi L, Weil D, Pyke J, Clarke B. Game of Unknowns: Non-Target Analysis Strategies for Identifying Per- and Polyfluoroalkyl Substances in Water. SETAC SciCon 2020.