Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Fish Fillet with LC-MS/MS

Significance of the Topic

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants that accumulate in wildlife and human food chains. Their stable fluorinated structure makes them resistant to degradation and leads to bioaccumulation. Analytical methods for measuring PFAS levels in fish are critical for assessing dietary exposure risks and guiding regulatory limits.

Study Objectives and Overview

This work aimed to develop a rapid, sensitive LC-MS/MS method for the simultaneous quantification of thirty PFAS compounds in fish fillet. Key goals included optimizing sample preprocessing, achieving reliable recovery rates across multiple concentration levels, and generating robust calibration curves within a fifteen-minute analysis window.

Methodology and Instrumentation

Sample Preparation and Extraction

• Homogenize 10 g frozen fish fillet using a freeze grinder
• Extract with 10 mL acetonitrile and QuEChERS salt packet
• Vigorously shake, centrifuge at 4 000 rpm, collect acetonitrile layer
• Dilute extract fivefold with water

Solid-Phase Purification

• Use Biotage EVOLUTE PFAS SPE cartridge (150 mg/6 mL)
• Condition with ammonia solution in methanol and formic acid in water
• Load 40 mL extract (equivalent to 8 g fish), wash, elute with ammonia/methanol/water mixture
• Add formic acid and inject 5 µL into the LC-MS/MS

Chromatography and Mass Spectrometry

• UHPLC: Shim-pack Scepter C18 column, gradient elution with acetonitrile/water plus ammonium acetate
• Flow switching to delay column prevents carry-over
• Detection: Triple quadrupole LCMS-8060NX, electrospray negative ion mode

Main Results and Discussion

All thirty PFAS eluted within eight minutes with baseline separation from bile acids. Calibration curves in the range 0.05–5 µg/kg exhibited r2 values ≥0.99 for most analytes. Recovery tests at 0.1, 1, and 5 µg/kg spiking levels demonstrated satisfactory performance:

• PFOS, PFOA, PFNA, PFHxS: limit of quantification (LOQ) 0.1 µg/kg, recoveries 80–120%, repeatability <20%
• Other PFAS: LOQ 1.0 µg/kg, recoveries 65–135%, repeatability <25%

Benefits and Practical Applications

This method enables high-throughput monitoring of a broad PFAS panel in fish within a short analysis time. It supports food safety laboratories, environmental monitoring programs, and regulatory compliance testing by offering:

• Rapid sample turnaround
• Quantitative accuracy across low-level contamination
• Minimal matrix effects through targeted SPE cleanup

Future Trends and Potential Uses

Advances may include automation of QuEChERS and SPE steps, expansion to additional food matrices, and integration with high-resolution mass spectrometry for suspect and non-target PFAS screening. Combining this workflow with isotope dilution standards could further enhance quantitation accuracy.

Conclusion

A validated LC-MS/MS method for thirty PFAS in fish fillet was established, delivering fast separation, strong linearity, and robust recovery. It addresses growing demands for comprehensive PFAS surveillance in dietary sources.

Used Instrumentation

• Shimadzu Nexera X3 UHPLC system
• Shim-pack Scepter C18 and delay columns
• Shimadzu LCMS-8060NX triple quadrupole mass spectrometer
• Biotage EVOLUTE PFAS SPE cartridges and PRESSURE+ manifold

References

1. AOAC SMPR 2023.003 Performance Requirements for Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Food Matrix