Determination of 30 PFAS in Fish by Liquid Chromatography Triple Quadrupole Mass Spectrometry (LC-MS/MS)

Determination of 30 PFAS in Fish by LC-MS/MS — Summary

Importance of the topic

Per- and polyfluoroalkyl substances (PFAS) are persistent anthropogenic contaminants with documented human health risks and widespread environmental mobility. Monitoring PFAS in foodstuffs, particularly fish, is essential for consumer safety, regulatory compliance, and source attribution. Reliable, sensitive, and high-throughput analytical methods are therefore required to quantify multiple PFAS at low ng/g (ppb) levels in complex biological matrices such as fish tissue.

Objectives and overview of the study

The study describes a single-laboratory validation for the simultaneous measurement of 30 PFAS in fish using a rapid QuEChERS-based extraction followed by UHPLC separation on a Shimadzu Nexera system and detection by a Shimadzu LCMS-8060NX triple quadrupole mass spectrometer operating in negative heated electrospray ionization. The method was evaluated against AOAC SMPR 2023.003 criteria with emphasis on limits of quantitation (LOQ), recovery, precision (repeatability), chromatographic resolution (including branched vs linear isomers), and robustness for routine food testing.

Methodology

• Sample preparation: Edible portions of tuna were diced, ground with dry ice, frozen, and portioned (10 g). Samples were spiked at three concentrations (0.1, 1.0 and 5.0 ng/g) in triplicate and processed alongside matrix-matched calibration standards (0.05–5.0 ng/g).
• Extraction: 10 mL acetonitrile was added to 10 g test portions, shaken, and QuEChERS salt packets were applied. After centrifugation, the acetonitrile layer was diluted (5×) with PFAS-free water.
• Cleanup: Extracts were passed through a weak anion exchange (WAX) SPE cartridge and eluted with basic methanol; aliquots were acidified with formic acid prior to analysis.
• Calibration and quantitation: Isotope dilution was used with 16 isotopically labeled internal standards. Matrix-matched calibration (linear, not forced through zero) produced the best fit; residuals for calibration points were within ±25%.
• Chromatography and MS: UHPLC separation achieved baseline separation of all targets in nine minutes, including baseline resolution of branched and linear isomers and separation of PFOS from cholic acids. The LCMS-8060NX used heated ESI in negative mode with compound-specific MRM transitions for quantitation and qualifier ions for confirmation.
• Optimization: Instrument and LC conditions were extensively optimized (1984 instrument parameter permutations and testing of six column/gradient combinations) to maximize signal-to-noise for critical analytes (notably PFOA, PFHxS, PFNA, PFOS).

Used Instrumentation

• Shimadzu Nexera UHPLC (high-performance liquid chromatography).
• Shimadzu LCMS-8060NX triple quadrupole mass spectrometer with heated electrospray ionization (negative mode).
• Standard laboratory equipment for QuEChERS extraction (centrifuge, SPE manifold) and dry-ice grinding.

Main results and discussion

• Analyte panel: 30 PFAS covering perfluoroalkyl carboxylates, sulfonates, sulfonamides, ether-acids and fluorotelomer sulfonates were targeted. Method LOQs were typically 0.1 ppb (ng/g) for the majority of analytes, with some compounds set at the minimum concentration that met all performance criteria.
• Sensitivity and separation: Nine-minute chromatographic method provided full separation of the target panel, with particular attention to resolving PFOS from endogenous cholic acid interferences and discriminating branched vs linear isomers.
• Accuracy and precision: Recoveries across spike levels generally fell within acceptable ranges required by AOAC SMPR 2023.003, with mean recoveries near quantitative (~90–110% for most analytes). Repeatability (RSD) values were low for most compounds, typically well within SMPR limits; some individual analytes showed higher variability but remained compliant.
• LOQ determination: LOQs were assigned as the lowest spiking level meeting combined criteria including recovery, repeatability, retention time agreement, qualifier ion signal-to-noise (>3, in several cases >10), and ion ratio tolerance (±30%).
• Quantitation strategy: Isotope dilution with labeled analogs (exact labeled analogs used where available) improved accuracy and compensated for matrix effects. Alternative labeled standards were selected for a few analytes when specific labels showed matrix interferences.

Benefits and practical applications of the method

• High throughput: Short run time (≈9 minutes) supports larger sample loads in routine monitoring laboratories.
• Robust sensitivity: LOQs at or below 0.1 ng/g allow detection of PFAS at regulatory-relevant concentrations.
• Comprehensive panel: Simultaneous analysis of 30 PFAS classes enables broad surveillance and reduces need for multiple methods.
• Matrix control: Matrix-matched calibration and isotope dilution provide reliable quantitation in complex fish tissue matrices.
• Simplified workflow: QuEChERS extraction combined with WAX SPE cleanup offers a rapid, cost-effective sample preparation route adaptable to food-testing labs.

Future trends and applications

• Panel expansion and non-target screening: Laboratories will likely extend targeted suites to emerging PFAS and incorporate high-resolution MS for suspected unknowns.
• Automation and miniaturization: Automated sample preparation and reduced solvent protocols (microextraction) to increase throughput and sustainability.
• Standardization and interlaboratory validation: Wider multi-laboratory validation and harmonized reference materials will strengthen regulatory acceptance.
• Isomer-specific risk assessment: Improved chromatographic and MS methods to distinguish and quantify branched vs linear isomers for refined exposure assessment.
• Integration with environmental forensics: Combined food and environmental monitoring to trace PFAS sources and inform mitigation strategies.

Conclusion

The described QuEChERS—WAX SPE—UHPLC-MS/MS workflow using Shimadzu Nexera and LCMS-8060NX achieved reliable quantitation of 30 PFAS in fish with LOQs generally at 0.1 ng/g, acceptable recoveries, and repeatability in line with AOAC SMPR 2023.003. The method’s fast chromatographic cycle, robust isotope-dilution calibration, and effective cleanup make it suitable for routine food-safety laboratories tasked with PFAS surveillance in seafood.

Reference

• AOAC SMPR 2023.003