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

Significance of the topic

Per- and polyfluoroalkyl substances (PFAS) are persistent anthropogenic contaminants with widespread use and potential adverse health effects. Accurate, sensitive, and robust analytical methods are required for monitoring PFAS in food matrices to support consumer safety, regulatory compliance, and contamination source assessment. Pet food is an important matrix for surveillance because PFAS can enter the food chain via raw materials, processing, packaging, or environmental contamination. The described method provides a validated approach for simultaneous determination of 30 PFAS at sub-ng/g levels, addressing needs for speed, sensitivity, and regulatory performance criteria (AOAC SMPR 2023.003).

Objectives and study overview

• Develop and validate a single-laboratory LC–MS/MS method for 30 PFAS in pet food using a rapid QuEChERS-based extraction and a UHPLC–triple quadrupole MS.
• Demonstrate method performance against AOAC SMPR 2023.003 acceptance criteria, including recovery, precision, and limit of quantitation (LOQ).
• Optimize chromatographic separation and mass spectrometer parameters to resolve isomers and interferences, and maximize sensitivity—especially for PFOA, PFHxS, PFNA, and PFOS.

Methodology and sample preparation

• Sample homogenization: Commercial dry dog food was ground with dry ice and stored frozen after sublimation of the dry ice.
• Test portion: 2.0 g aliquots were spiked (triplicate) at three levels (0.5, 5.0, 25 ng/g) plus blanks; calibration used matrix-matched extracted standards at 0.25–25 ng/g.
• Extraction: Add 10 mL water and 10 mL acetonitrile to 2 g sample, shake 1 min, add QuEChERS packet, shake 1 min, centrifuge 5 min at 4000 rpm.
• Cleanup: Transfer acetonitrile layer, dilute 5× with PFAS-free water, pass through a weak anion exchange (WAX) SPE cartridge; elute PFAS with basic methanol. A 0.5 mL aliquot of eluate was acidified with formic acid prior to analysis.
• Quantitation strategy: Isotope dilution using 16 isotopically labelled internal standards (exact labeled analogs where possible); in a few cases alternate non-interfering isotopes were substituted due to matrix effects.
• Calibration: Linear matrix-matched isotopic-dilution calibration not forced through zero; residuals for calibration points were within ±25%.

Instrumentation used

• UHPLC: Shimadzu Nexera series; chromatographic method providing separation of all targets in a nine-minute run and baseline resolution of linear and branched isomers and separation of PFOS from cholic acid interferences.
• Mass spectrometer: Shimadzu LCMS-8060NX triple quadrupole operated with heated electrospray ionization (HESI) in negative-ion mode; multiple reaction monitoring (MRM) transitions optimized per analyte.
• Optimization effort: Instrument and chromatography optimization included ~1,984 instrument setting permutations and six column/gradient combinations to maximize peak shape, resolution, and signal-to-noise for critical analytes.

Main results and discussion

• Scope: The method covers 30 PFAS including perfluoroalkyl carboxylic acids (C4–C14), sulfonic acids, sulfonamides, chlorinated ether sulfonic acids, HFPO-DA (GenX), DONA, and several fluorotelomer sulfonates.
• LOQ: Experimentally determined LOQ for all target analytes was 0.5 ng/g (ppb), defined as the lowest concentration meeting SMPR criteria (recovery, repeatability, ion ratio within ±30%, retention time, and qualifier ion S/N >3; for some compounds S/N >10 was required).
• Accuracy and calibration: Representative calibration accuracy for PFOA, PFHxS, PFNA and PFOS across 0.25–25 ng/g ranged approximately 92–106% with consistent performance at low-level points (0.25–0.5 ng/g), indicating good fit of the matrix-matched, isotope-dilution calibration model.
• Recovery and precision: Average recoveries for spiked levels (0.5, 5, 25 ng/g) were typically within approximately 90–110% with repeatability RSDs generally low (most RSDs <10%), meeting AOAC SMPR 2023.003 criteria across analytes and levels.
• Chromatographic separation: Baseline separation achieved between branched and linear isomers and a two-minute window separated PFOS from cholic acid interferences, reducing false signals and improving ion-ratio consistency.
• Sensitivity improvements: Targeted optimization increased sensitivity particularly for PFOA, PFHxS, PFNA, and PFOS, critical analytes for regulatory monitoring.

Benefits and practical applications

• Regulatory compliance: Method meets AOAC SMPR 2023.003 single-laboratory acceptance criteria, making it suitable for regulatory surveillance and method adoption by testing laboratories.
• High throughput: Nine-minute UHPLC runtime with robust cleanup (QuEChERS + WAX SPE) supports routine sample throughput for monitoring programs.
• Quantitative reliability: Use of matrix-matched isotope-dilution calibration and labeled internal standards yields accurate quantitation even at low-ng/g levels.
• Wide analyte coverage: Inclusion of 30 PFAS, including emerging compounds (e.g., HFPO-DA, DONA) and chlorinated ether sulfonates, provides comprehensive screening relevant to modern regulatory priorities.
• Practical workflow: Simple sample handling and a straightforward SPE cleanup make the method compatible with standard laboratory equipment and workflows.

Future trends and potential applications

• Panel expansion: Extend analyte lists to include additional novel PFAS and transformation products as industry and regulatory lists evolve.
• Interlaboratory validation: Adoption of multi-lab validation to support standardization and routine adoption by regulatory testing networks.
• High-resolution MS complements: Use HRMS (Q-TOF/Orbitrap) for non-target screening and identification of unknown PFAS in complex food matrices.
• Automation and miniaturization: Automating extraction/cleanup, on-line SPE, or microextraction to increase throughput and reduce solvent use.
• Lower LOQs: Continued instrumental and sample-prep refinements to push LOQs lower for increasingly stringent regulatory limits.

Conclusion

The validated LC–MS/MS method using QuEChERS extraction, WAX SPE cleanup, matrix-matched isotope-dilution calibration, Shimadzu Nexera UHPLC, and LCMS-8060NX triple quadrupole MS achieves robust quantitation of 30 PFAS in pet food at an LOQ of 0.5 ng/g. Chromatographic and MS optimization produced effective separation of isomers and common interferences, high sensitivity for key PFAS, and recoveries and precision within AOAC SMPR 2023.003 acceptance criteria. The approach is applicable for routine monitoring, regulatory compliance, and targeted surveillance of PFAS in food matrices.

Reference

• AOAC SMPR 2023.003