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

Determination of 30 PFAS in Fish Oil by LC-MS/MS — Validated Single-Lab Method Using Shimadzu Nexera + LCMS-8060NX

Importance of the topic

Per- and polyfluoroalkyl substances (PFAS) are persistent anthropogenic contaminants with demonstrated human health risks. They can enter food chains via environmental contamination, processing, or packaging. Reliable analytical methods with low limits of quantitation (LOQs), robust precision, and accurate quantitation are essential for food safety monitoring, regulatory compliance, and risk assessment—particularly for lipid-rich matrices such as fish oil where matrix effects and interferences complicate analysis.

Objectives and study overview

- Validate a routine analytical workflow for 30 PFAS in fish oil using a single-laboratory study following AOAC SMPR 2023.003 acceptance criteria.
- Achieve low LOQs, good recoveries, and repeatability with a rapid sample preparation and short UHPLC runtime.
- Use matrix-matched calibration and isotope dilution quantitation to compensate for matrix effects.

Methodology

Sample collection and spike design

- Commercial fish oil soft gels were used. Test portions were prepared by expelling oil from capsules into tubes.
- Samples were spiked in triplicate at five concentrations to assess recovery and precision; calibration standards were prepared in matrix at 0.1, 0.5, 1.5, 5.0 and 15.0 ng/g.

Extraction and cleanup

- 10 g fish oil test portions were weighed, spiked with analytes and 16 isotopically labeled internal standards, and 10 mL acetonitrile was added.
- Samples were vortexed 1 minute and centrifuged 5 minutes at 4000 rpm. The acetonitrile layer was diluted 5× with PFAS-free water.
- Clean-up employed weak anion exchange (WAX) solid-phase extraction; PFAS were eluted with a basic methanol–water mixture.

Chromatography and mass spectrometry

- UHPLC separation on a Shimadzu Nexera system achieved adequate separation of 30 PFAS in a nine-minute run.
- Shimadzu optimized ~1984 instrument settings and tested six column/gradient combinations to maximize peak shape, resolution, and sensitivity—especially for PFOA, PFHxS, PFNA and PFOS.
- Detection used a Shimadzu LCMS-8060NX triple quadrupole with heated electrospray ionization in negative ion mode, performing MRM transitions for each analyte and associated labeled internal standard (isotope dilution quantitation).

Calibration and quantitation strategy

- Matrix-matched isotopic dilution calibration with a linear model (not forced through zero) provided best fit and recoveries; residuals of curve points were within ±25%.
- Quantitation spikes covered 0.25–10 ng/g; LOQs were assigned as the lowest concentration meeting SMPR criteria for recovery, repeatability, retention time, qualifier ion S/N (>3) and ion ratio tolerance (±30%). For PFBA, PFPeA and PFOSA, LOQs were set at the minimum concentration meeting recovery/repeatability with S/N >10.

Used instrumentation

• Shimadzu Nexera UHPLC system.
• Shimadzu LCMS-8060NX triple quadrupole mass spectrometer with heated electrospray ionization (negative mode).
• Weak anion exchange (WAX) SPE cartridges for cleanup.

Main results and discussion

Analytical scope and LOQs

- Thirty PFAS were included: a range of perfluoroalkyl carboxylic acids (C4–C14), perfluoroalkyl sulfonic acids (C4–C12, and additional complex sulfonates), PFOSA, chlorinated ether-sulfonates, HFPO-DA, DONA and FTS homologs (4:2 to 10:2).
- Experimentally determined LOQs were 0.25 ng/g for the large majority of analytes; 10:2 FTS had an LOQ of 0.5 ng/g. All LOQs met AOAC SMPR 2023.003 requirements.

Recovery and precision

- Recovery and repeatability were evaluated across five spike levels (0.25–10 ng/g). Average recoveries were generally within approximately 90–110% for most analytes across concentrations, with many values close to 100%.
- Relative standard deviations (RSDs) for repeatability were typically low (single-digit percentages across mid-to-high spikes); some higher RSDs occurred at the lowest spikes for a few analytes but still satisfied SMPR acceptance limits.
- Matrix-matched isotope dilution effectively compensated for matrix effects; exact labeled analogs were preferred, with substitutions used only when isotopic interferences were observed.

Chromatographic performance and selectivity

- Baseline resolution was achieved for linear vs. branched isomers (e.g., PFOS and PFHxS isomer separation), and a two-minute separation between PFOS and potential cholic acid interferences was established to avoid false positives from bile acid co-elution.
- Optimization efforts improved signal-to-noise for PFOA, PFHxS, PFNA and PFOS—critical analytes for regulatory monitoring.

Practical performance summary

- The validated workflow delivered low LOQs, good accuracy and precision, rapid throughput (nine-minute analysis per injection) and robust selectivity in a challenging lipid matrix.

Benefits and practical applications

- Routine monitoring of PFAS in fish oil and other high-lipid food matrices for regulatory compliance and quality control.
- The combination of fast UHPLC separation, targeted triple-quadrupole sensitivity, and isotope-dilution calibration supports reliable quantitation at trace ng/g levels.
- The method’s relatively simple extraction and SPE cleanup make it adaptable for high-throughput laboratory environments.

Limitations and considerations

- Single-laboratory validation demonstrates method fitness but inter-laboratory validation would strengthen method transferability and robustness across different instrument platforms and operators.
- Some low-level measurements showed larger variability; careful attention to blank controls, PFAS-free consumables, and internal standard selection is necessary to maintain data quality.

Future trends and potential uses

• Extension of analyte panels to include emerging and ultra-short-chain PFAS and transformation products, and use of HRMS for non-target screening to capture unknowns.
• Inter-laboratory collaborative validations to produce standardized methods for regulatory adoption and proficiency testing.
• Automation and miniaturization of extraction and SPE to increase throughput and reduce solvent use; greener solvent strategies may be explored.
• Lowering LOQs through further instrument and separation optimization, and advancing approaches to quantify PFAS in more complex or lower-mass matrices.
• Surveillance applications linking product monitoring with environmental and biomonitoring programs to better assess human exposure pathways.

Conclusion

The Shimadzu Nexera UHPLC coupled with the LCMS-8060NX triple quadrupole MS supports a validated single-laboratory method for determination of 30 PFAS in fish oil with rapid sample preparation, nine-minute UHPLC analysis, matrix-matched isotopic dilution calibration, and WAX SPE cleanup. The workflow achieved LOQs at or below AOAC SMPR 2023.003 requirements, robust recoveries and repeatability, and sufficient chromatographic selectivity to resolve isomers and remove cholic acid interferences. This method is suitable for routine analytical and regulatory testing of PFAS in high-lipid food matrices.

Reference

1. AOAC SMPR 2023.003. Standard Method Performance Requirements (SMPR) for the Determination of Per- and Polyfluoroalkyl Substances (PFAS) in Foods.
2. Lipps W., Gruszecka D., Imoto E., Matsubara T. Determination of 30 PFAS in Fish Oil by Liquid Chromatography Triple Quadrupole Mass Spectrometry (LC-MS/MS). Shimadzu Scientific Instruments Application Note; 2024.