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

Importance of the topic

Accurate, sensitive and robust methods for measuring per- and polyfluoroalkyl substances (PFAS) in food matrices are critical because PFAS are persistent, bioaccumulative and associated with adverse health effects. Offal (e.g., liver, kidney) can concentrate PFAS and represents an important food safety matrix for monitoring and regulatory compliance. A validated, rapid analytical workflow that meets consensus performance criteria (AOAC SMPR 2023.003) enables routine surveillance, risk assessment and supports regulatory decision-making.

Study objectives and overview

This single-laboratory study aimed to develop and validate a rapid LC–MS/MS method for the quantitative determination of 30 PFAS in beef kidney (offal). Primary goals were to achieve low limits of quantification (LOQs) consistent with AOAC SMPR 2023.003, high precision and recovery, clear chromatographic separation (including branched/linear isomers), and a practical extraction workflow suitable for routine laboratory implementation.

Methodology

• Matrix and test portions: Locally purchased beef kidney was sliced, frozen, ground with dry ice and stored frozen. Ten-gram test portions were used for extraction and calibration spike preparation.
• Spiking and calibration: Samples were spiked in triplicate at five levels. Matrix-matched calibration curves were prepared by spiking 10 g kidney portions at 0.1, 0.5, 1.5, 5 and 15 ng/g. Quantitation samples were spiked at 0.2, 0.4, 1, 4 and 10 ng/g. Isotope dilution was used with 16 isotopically labeled internal standards; exact labeled analogs were used where possible, with non-interfering alternatives substituted when necessary.
• Extraction: 10 g test portions were spiked, 10 mL acetonitrile added, samples vortexed (1 min) and centrifuged (5 min, 4000 rpm). The acetonitrile layer was diluted fivefold with PFAS-free water and cleaned using a weak anion exchange (WAX) SPE cartridge. PFAS were eluted with a basic methanol–water mixture.
• Chromatography: UHPLC separation on a Shimadzu Nexera system provided baseline separation of all target analytes within a nine-minute run. Chromatography was optimized to resolve PFOS from cholic acid interferences and to separate branched and linear isomers.
• Mass spectrometry: Targeted quantitation employed a Shimadzu LCMS-8060NX triple quadrupole with heated electrospray ionization in negative mode using multiple reaction monitoring (MRM). Instrument optimization included evaluation of ~1,984 settings and six column/gradient combinations; special effort focused on improving sensitivity for PFOA, PFHxS, PFNA and PFOS.
• Calibration model and QA criteria: A linear isotope-dilution calibration not forced through zero was used. LOQ determination followed AOAC SMPR 2023.003 requirements: correct retention time, acceptable ion-ratio (±30%), qualifier ion S/N > 3, and recovery/precision within SMPR limits. For PFBA, PFPeA and PFOSA LOQs were set at the lowest level meeting recovery/precision and S/N > 10.

Instrumentation used

• Shimadzu Nexera UHPLC system.
• Shimadzu LCMS-8060NX triple quadrupole mass spectrometer with heated electrospray ionization, negative mode, operating in MRM for targeted PFAS analysis.
• Weak anion exchange (WAX) SPE cartridges for extract clean-up.

Main results and discussion

• Analytes: 30 PFAS were targeted, covering short- to long-chain perfluoroalkyl carboxylates, sulfonates, sulfonamides and newer replacement chemistries (e.g., HFPO-DA).
• LOQs: The experimentally determined LOQs met AOAC SMPR 2023.003 for all compounds; most reported LOQs were 0.2 ng/g (ppb) for the majority of analytes.
• Recovery and precision: Recoveries across spike levels generally ranged from approximately 90% to 115% for most analytes, with repeatability (RSD) typically low (commonly single-digit percent RSD at routine spike levels). Detailed recovery and RSD data per analyte and spike level were generated and met SMPR acceptance criteria.
• Chromatographic performance: Complete separation of the 30 analytes was achieved within nine minutes. The method provided baseline resolution between branched and linear isomers for PFOS and sufficient separation of PFOS from cholic acid interferences, reducing false positives/quantitation bias.
• Method robustness: Extensive method optimization (nearly 2,000 instrument settings evaluated and multiple column/gradient trials) improved sensitivity particularly for PFOA, PFHxS, PFNA and PFOS. Use of matrix-matched, extracted calibration standards and isotope dilution minimized matrix bias.

Benefits and practical application

• Regulatory readiness: Method performance complies with AOAC SMPR 2023.003, making it applicable for monitoring programs and compliance testing in offal matrices.
• High throughput: Short nine-minute chromatographic run and a straightforward extraction/SPE workflow support efficient sample throughput for routine laboratories.
• Analytical reliability: Isotope dilution quantitation and matrix-matched calibration provide accurate results across low ng/g concentration ranges, with robust control for matrix effects.
• Applicability: The workflow is readily adaptable to other animal tissue matrices with appropriate validation and can be implemented in food safety, environmental monitoring and research laboratories.

Future trends and potential uses

• Extension to additional matrices: Validation across other foodstuffs (liver, muscle, dairy, eggs) and environmental samples will broaden surveillance capability.
• Automation and sample prep miniaturization: Automated SPE and robotic sample handling can increase throughput and reduce variability.
• Broader target panels and suspect screening: Integration with high-resolution mass spectrometry would enable non-target and suspect PFAS screening alongside targeted quantitation.
• Standardization and inter-laboratory validation: Multi-lab studies and ring trials would support formal standard method adoption and regulatory harmonization.
• Improved isotope label coverage: Expanding labeled internal standard availability for emerging PFAS will improve quantitation accuracy for substitution chemistries.

Conclusion

A rapid LC–MS/MS method using acetonitrile extraction, WAX SPE clean-up, a nine-minute UHPLC separation and triple-quadrupole MRM detection on the Shimadzu Nexera/LCMS-8060NX platform reliably quantified 30 PFAS in beef kidney. The single-laboratory validation met AOAC SMPR 2023.003 criteria for LOQ, recovery and precision, demonstrating a practical, high-throughput option for PFAS monitoring in offal with strong chromatographic separation and robust isotope-dilution quantitation.

Reference

• AOAC International. SMPR 2023.003: Standard Method Performance Requirements for the Determination of PFAS in Food Matrices.