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

Significance of the Topic

The reliable determination of per- and polyfluoroalkyl substances (PFAS) in food matrices is a high priority for public health laboratories, food producers, and regulatory agencies because PFAS are persistent, bioaccumulative, and associated with adverse health effects. Eggs are a relevant animal-derived food matrix prone to contamination through environmental exposure, feed, processing, or packaging; therefore robust, sensitive, and high-throughput analytical methods are required to support monitoring, compliance and risk assessment.

Objectives and Overview of the Study

This single-laboratory validation evaluated a workflow for the quantitative measurement of 30 PFAS in whole egg using a rapid QuEChERS-based extraction followed by UHPLC separation and triple quadrupole LC–MS/MS detection (Shimadzu Nexera + LCMS-8060NX). Key aims were to: demonstrate method performance against AOAC SMPR 2023.003 criteria; establish Limits of Quantitation (LOQs); evaluate recovery and precision across multiple spike levels; and optimize chromatographic and MS conditions to resolve interferences and PFAS isomers.

Methodology

Sample preparation and extraction

• Whole eggs were homogenized using dry ice and ground material subsampled (10 g test portions).
• Samples were spiked (for validation) with native PFAS (30 targets) and 16 isotopically labeled internal standards.
• Extraction: 10 mL acetonitrile added to 10 g sample, vortex 1 min, QuEChERS salt packet addition, shake 1 min, centrifuge 5 min at 4000 rpm.
• Cleanup: aliquot of acetonitrile layer diluted 5× with PFAS-free water, passed through a weak anion exchange (WAX) SPE cartridge, eluted with basic methanol, concentrated to dryness and reconstituted in 0.4 mL methanol:water for analysis.

Calibration, quantification and QA criteria

• Matrix-matched calibration curves were prepared with test portions spiked at 0.001, 0.01, 0.1, 1, and 10 ng/g (isotope dilution, linear model not forced through zero).
• Quantitation spikes on additional whole egg samples were performed in triplicate at 0.0055, 0.055, 0.55 and 5.5 ng/g.
• LOQ determination used SMPR criteria including recovery, repeatability (RSD), retention time, ion ratio tolerance (±30%), and S/N >3 for qualifier ion (with some analytes requiring S/N >10 for robust LOQ).

Instrumentation Used

The study used a Shimadzu Nexera UHPLC system coupled to a Shimadzu LCMS-8060NX triple quadrupole mass spectrometer with heated electrospray ionization (HESI) operated in negative ion mode. Method optimization included evaluation of approximately 1,984 instrument parameter combinations and six column/gradient configurations to optimize peak shape, inter-analyte separation, and signal-to-noise for critical analytes (notably PFOA, PFHxS, PFNA, PFOS). Isotopically labeled analogs (exact matches where possible) were used for isotope-dilution calibration; alternative non-interfering isotopes were substituted in a few cases where matrix effects were detected.

Main Results and Discussion

Scope and targets

• Thirty PFAS were targeted across perfluoroalkyl carboxylates, sulfonates, sulfonamides and emerging PFAS (including HFPO-DA and several FTS homologues).
• Sixteen isotopically labeled internal standards supported isotope-dilution quantitation.

Chromatography and separation

• All targeted PFAS were separated within a nine-minute UHPLC runtime, enabling high sample throughput.
• Chromatographic conditions were tuned to resolve PFOA from potential cholic-acid interferences and to achieve baseline separation between branched and linear isomers (notably PFOS isomers).

Sensitivity, LOQs and calibration performance

• Experimentally determined method LOQs met AOAC SMPR 2023.003 acceptance criteria for all analytes. Many analytes had LOQs at 0.0055 ng/g (ppb) or 0.055 ng/g depending on chemical class and response.
• Calibration employed a linear isotope-dilution approach with residuals within ±25% across the calibration range (0.001–10 ng/g).
• Sensitivity improvements were particularly notable for PFOA, PFHxS, PFNA and PFOS following extensive parameter optimization.

Recovery and precision

• Average recoveries for most analytes fell within the typical acceptable range (~85–115%), and repeatability (RSD) values across spike levels met the SMPR criteria in this single-lab validation.
• Where matrix interferences affected a labeled standard (13C2 in a few cases), alternative labeled standards were used to maintain quantitation integrity.

Quality attributes

• Method met SMPR requirements for retention-time tolerance, ion-ratio agreement (±30%), qualifier S/N and repeatability for the tested egg matrix.

Benefits and Practical Applications

This workflow delivers several practical advantages for routine PFAS monitoring in eggs and similar fatty biological matrices:

• High throughput: complete chromatographic separation of 30 PFAS in a nine-minute run supports larger sample loads.
• Sensitivity: low LOQs enable reliable detection at levels relevant to current regulatory and monitoring needs.
• Robustness: QuEChERS extraction combined with WAX SPE cleanup and isotope-dilution quantitation yields reproducible recoveries and RSDs within SMPR limits.
• Practical implementation: the method uses common laboratory consumables and established cleanup strategies, facilitating adoption in food testing and environmental labs.

Future Trends and Potential Uses

Potential directions to extend and leverage this work include:

• Matrix expansion: adaptation and validation across other food matrices (meat, dairy, seafood, produce) to build comprehensive monitoring programs.
• Method automation and miniaturization: integration of automated SPE and online cleanup to increase throughput and reduce manual handling and contamination risk.
• Broader analyte panels and non-target screening: coupling targeted triple quad workflows with high-resolution MS for suspect and non-target PFAS discovery.
• Standardization and regulatory uptake: adoption of validated approaches into standardized methods and inter-laboratory studies to harmonize reporting and compliance testing.
• Emerging cleanup strategies: evaluation of alternative sorbents and microextraction techniques to improve matrix removal for very low-level PFAS analyses.

Conclusion

The Shimadzu Nexera UHPLC combined with the LCMS-8060NX triple quadrupole mass spectrometer, paired with a QuEChERS extraction and WAX SPE cleanup, provides a validated, high-throughput method for quantifying 30 PFAS in whole egg. The method met AOAC SMPR 2023.003 criteria in a single-laboratory validation, delivering low LOQs, acceptable recoveries, good precision, and robust chromatographic separation including resolution of isomeric PFAS and biological interferences. This protocol is well suited for routine monitoring and can be adapted further for broader surveillance efforts and regulatory testing.

References

• AOAC SMPR 2023.003