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

Significance of the topic

Per- and polyfluoroalkyl substances (PFAS) are persistent, bioaccumulative anthropogenic chemicals with widespread use since the mid-20th century. Their environmental persistence and association with adverse health effects make sensitive, selective and high-throughput analytical methods essential for food safety monitoring and regulatory compliance. Coffee, as a frequently consumed beverage, can be a vector for low-level PFAS exposure through contaminated water, processing equipment or packaging, so validated methods for detecting multiple PFAS at sub-ppb levels in brewed coffee are important for public health surveillance and industry quality control.

Objectives and study overview

This single-laboratory study validated a targeted LC–MS/MS method for simultaneous determination of 30 PFAS in brewed coffee. The method used a rapid extraction with WAX solid-phase cleanup and quantitative analysis by a Shimadzu Nexera UHPLC coupled to a Shimadzu LCMS-8060NX triple quadrupole mass spectrometer operated in negative heated electrospray mode. Performance was evaluated against AOAC SMPR 2023.003 acceptance criteria using matrix-matched, isotope-dilution calibration and replicate spikes at four concentration levels.

Instrumental setup

• UHPLC: Shimadzu Nexera series (fast UHPLC separation, ~9 min run time).
• Mass spectrometer: Shimadzu LCMS-8060NX triple quadrupole with heated electrospray ionization (negative mode) and optimized ion focus for improved signal at higher gas flows/temperatures.
• Sample cleanup: Weak anion exchange (WAX) SPE cartridges with basic methanol elution.
• Quantitation approach: Multiple reaction monitoring (MRM) transitions for each PFAS with isotope‑labeled internal standards (exact analogs where available); matrix-matched isotopic-dilution calibration.
• Method development: Extensive optimization (1984 instrument settings and six column/gradient combinations) to maximize sensitivity and chromatographic resolution, especially for PFOA, PFHxS, PFNA and PFOS.

Methodology

• Sample matrix: Commercial brewed organic coffee. Test portions of 10 g were used.
• Sample preparation: Samples were spiked with native PFAS and 16 isotopically labeled internal standards, 10 mL acetonitrile was added, samples were shaken 1 min and centrifuged 5 min (4000 rpm). The acetonitrile layer was diluted fivefold with PFAS-free water, passed through WAX SPE, eluted with basic methanol, concentrated to dryness and reconstituted in 0.4 mL methanol–water prior to injection.
• Calibration and quantitation: Matrix-matched calibration standards covering 0.001–10 ng/g were prepared and processed identically to samples. Quantitation was performed at spike levels of 0.0055, 0.055, 0.55 and 5.5 ng/g using isotopic-dilution linear fits not forced through zero; residuals for curve points were within ±25%.
• Chromatography: Method achieved baseline separation of all targets in nine minutes, including resolution of branched versus linear isomers and a two-minute separation between PFOS and cholic-acid interferences.
• LOQ and acceptance criteria: LOQs were set as the lowest concentration meeting SMPR requirements for accuracy, precision (repeatability RSD), retention time, qualifier ion signal-to-noise (>3) and ion ratio within ±30% (some analytes required S/N >10 to meet recovery/precision limits).

Main results and discussion

• Analytical scope: Thirty PFAS were included (perfluoroalkyl carboxylates, sulfonates, sulfonamides, ether-acids and fluorotelomer sulfonates). Experimentally determined LOQs varied by analyte, with many long- and mid-chain PFAS achieving LOQs of 0.0055 ng/g (ppb) and some compounds at 0.055–0.55 ng/g, reflecting chemical- and matrix-dependent sensitivity.
• Chromatographic performance: Complete separation of all analytes within a nine-minute window; successful baseline resolution of PFOS isomers and separation from cholic acid interferences, which is critical to avoid false positives in food matrices.
• Accuracy and precision: Recoveries generally fell in the typical 80–110% range across spike levels; repeatability (RSD) was low for mid- to high-level spikes and acceptable near LOQ. Reported average recoveries and RSDs met the AOAC SMPR 2023.003 acceptance criteria for all analytes tested.
• Sensitivity improvements: Instrument and method optimization provided increased signal-to-noise for PFOA, PFHxS, PFNA and PFOS, improving LOQs and quantitation confidence for these regulatory‑relevant analytes.
• Isotope-dilution: Exact labeled analogs were used wherever possible to correct matrix effects; in a few cases alternative labeled standards were substituted to avoid matrix interferences.

Practical benefits and applications

• Regulatory readiness: The validated method meets AOAC SMPR 2023.003 criteria and is suitable for routine monitoring of PFAS in brewed coffee for compliance and surveillance programs.
• Throughput and robustness: Nine‑minute chromatographic runs combined with a rapid extraction and WAX cleanup enable relatively high sample throughput with reliable quantitation.
• Quantitative reliability: Matrix-matched isotopic-dilution calibration minimizes matrix bias and improves result traceability for laboratories performing regulatory testing or internal quality control.
• Adaptability: The sample preparation and instrument platform are adaptable to other aqueous food matrices after appropriate validation steps.

Future trends and potential applications

• Panel expansion and lower detection limits: As regulatory limits tighten, methods will need to extend to larger PFAS panels and push LOQs lower; continued instrument sensitivity improvements and optimized sample-concentration workflows will be important.
• High-resolution and non-target screening: Complementary high-resolution mass spectrometry (HRMS) workflows can enable suspect and non-target screening to detect novel or precursor PFAS not covered by targeted panels.
• Automation and green sample prep: Automated SPE and miniaturized extraction approaches can increase throughput and lower solvent use; adoption of greener solvents and reduced-volume workflows is likely to grow.
• Isomer-specific quantitation: Greater emphasis on distinguishing branched vs linear isomers for certain PFAS will require more labeled standards and refined chromatographic methods.
• Interlaboratory and method harmonization: Broader multi-laboratory validations and standardization will be needed to harmonize PFAS testing and support regulatory enforcement globally.

Conclusion

The described LC–MS/MS method using a Shimadzu Nexera UHPLC and LCMS-8060NX triple quadrupole, combined with WAX SPE cleanup and isotope-dilution calibration, provides fast, sensitive and robust quantitation of 30 PFAS in brewed coffee. Chromatographic optimization delivered complete separation, isomer resolution and mitigation of cholic-acid interference. Recoveries, repeatability and LOQs met AOAC SMPR 2023.003 criteria, supporting the method’s suitability for routine food-safety testing and regulatory monitoring.

Reference

1. AOAC INTERNATIONAL. SMPR 2023.003. Performance Requirements for the Determination of PFAS in Food Matrices. 2023.
2. Shimadzu Scientific Instruments. Application note: Determination of 30 PFAS in Coffee by LC–MS/MS using Nexera and LCMS‑8060NX. Shimadzu SSI, First Edition September 2024.