Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Animal Products with an Enhanced Sensitivity LC-MS/MS Method using Fish Reference Materials as a Case Study

Importance of PFAS Analysis in Animal Products

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants that accumulate in food chains. Recent regulatory limits and growing dietary exposure concerns make reliable PFAS quantification in animal-derived foods critical to protect public health and comply with EU directives.

Objectives and Study Overview

This study aimed to optimize a formerly published alkaline digestion and WAX SPE extraction procedure for PFAS in fish, enhancing chromatographic speed and detection sensitivity. Fish reference materials from Fapas were used to assess method performance and regulatory compliance.

Methodology and Instrumentation

Samples of homogenized fish and quality control materials were spiked with isotopically labeled extraction and injection standards. Alkaline methanol digestion was followed by centrifugation and Oasis WAX SPE cleanup. Extracts were analyzed by UPLC–MS/MS using an ACQUITY Premier UPLC system modified with an isolator column and PFAS kit, coupled to a Xevo TQ Absolute tandem quadrupole MS. Ionization was evaluated by comparing conventional electrospray to the UniSpray source.

Instrumentation

• ACQUITY Premier UPLC with Atlantis Premier BEH C18 AX isolator column
• Xevo TQ Absolute Tandem Quadrupole Mass Spectrometer
• UniSpray negative ionization source
• Oasis WAX SPE cartridges (6 cc, 150 mg)
• waters_connect software for quantitation

Main Results and Discussion

The revised UPLC gradient reduced run time by 50% and achieved clear separation of cholic acid interferents. The isolator column effectively delayed mobile phase PFBA and PFOA contaminants by over 30 s. UniSpray ionization improved PFAS peak areas at least three- to fourfold and enhanced signal-to-noise ratios by 1.5–2 times compared to electrospray. Solvent-based calibration indicated quantification limits of 0.025 µg/kg for most PFAS (0.05 µg/kg for PFBA). Analysis of Fapas fish materials yielded recoveries of 86–118% for key PFAS (PFOA, PFOS, PFNA, PFHxS) with RSD ≤ 14%, meeting EURL POPs guidance. Internal standard recoveries fell within 80–120%, except for known neutral or matrix-affected compounds.

Practical Applications and Benefits

• Enhanced throughput through shortened LC runs and reduced injection volumes
• Greater analytical confidence via improved separation of interferences and contaminant isolation
• Lower detection limits supporting compliance with stringent EU PFAS maximum residue levels
• Robust performance on certified reference materials ensuring reproducibility in QA/QC laboratories

Future Trends and Potential Uses

Advances in ionization techniques like UniSpray will continue to push sensitivity boundaries, enabling routine determination of emerging PFAS at sub-ppt levels. Integration of high-resolution MS and automated sample prep could further accelerate multi-matrix monitoring. Data-driven workflows and standardized kits will support harmonized surveillance across the food sector.

Conclusion

The updated LC-MS/MS method delivers rapid, sensitive, and reliable determination of 30 PFAS in fish and other animal products. UniSpray ionization and a dedicated isolator column enhance signal quality and mitigate contamination, while method performance on reference materials aligns with EU guidance. This workflow supports regulatory testing and dietary exposure assessment.

References

1. EFSA CONTAM Panel, EFSA Journal 2020;18(9).
2. Commission Regulation (EU) 2022/2388.
3. Commission Recommendation (EU) 2022/1431.
4. Organtini et al., Waters Application Note, 720007482, 2022.
5. EURL POPs Guidance Document on PFAS, May 2022.