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

Importance of the topic

Per- and polyfluoroalkyl substances (PFAS) are persistent, bioaccumulative anthropogenic contaminants of growing regulatory and public-health concern. Reliable, sensitive methods to detect low ng/g (ppb and sub-ppb) concentrations in complex food matrices such as protein powders are essential for exposure assessment, product safety, and compliance with emerging limits. Robust, validated analytical workflows allow food laboratories to deliver defensible data for surveillance, supplier control, and regulatory enforcement.

Objectives and overview of the study

This single‑laboratory validation aimed to demonstrate a rapid, quantitative LC–MS/MS method for 30 PFAS in a plant‑based protein powder matrix that meets AOAC SMPR 2023.003 acceptance criteria. Key goals included: matrix‑matched isotope dilution quantitation, determination of method limits of quantitation (LOQs), evaluation of recovery and precision across multiple spike levels, and chromatographic/ MS optimization to resolve isomers and interferences.

Methodology

Sample preparation and extraction

- Test portions: 10 g protein powder (commercial plant‑based product).
- Spiking: native PFAS (30 analytes) and 16 isotopically labeled internal standards; calibration standards and QC spikes prepared in matrix and carried through extraction.
- Extraction: add 10 mL water and 10 mL acetonitrile, shake 1 min; add QuEChERS packet, shake 1 min; centrifuge 5 min at 4000 rpm.
- Cleanup: transfer acetonitrile layer, dilute 13× with PFAS‑free water, apply to weak anion exchange (WAX) SPE cartridge; elute with basic methanol.
- Concentration: evaporate to dryness for sensitivity, reconstitute in 0.4 mL methanol:water (final extract concentrated prior to UHPLC injection).

Calibration and quantitation

- Calibration range: matrix‑matched spikes at 0.001, 0.01, 0.1, 1.0 and 10.0 ng/g used to build isotope‑dilution curves (linear model, not forced through zero).
- Quantitation spike levels: 0.0055, 0.055, 0.55 and 5.5 ng/g (triplicate).
- LOQ definition: lowest spike meeting SMPR retention time, accuracy/precision, ion ratio (±30%), and qualifier S/N (>3; some analytes required S/N>10).

Used instrumentation

- Shimadzu Nexera UHPLC (rapid separation, all analytes separated in ~9 minutes).
- Shimadzu LCMS‑8060NX triple quadrupole mass spectrometer with heated electrospray ionization (negative mode), operating multiple reaction monitoring (MRM).
- Method development included systematic evaluation (~1984 instrument parameter sets and 6 column/gradient combinations) to optimize peak shape, resolution and sensitivity for critical PFAS (notably PFOA, PFHxS, PFNA, PFOS).

MRM transitions and isotopic internal standards were assigned for each analyte; exact labeled analogs were used when available (alternative non‑interfering isotopes used where necessary). Chromatography was adjusted to separate PFOS from cholic‑acid interferences and to provide baseline resolution of branched vs linear isomers.

Main results and discussion

- Scope: 30 PFAS compounds including perfluoroalkyl carboxylic acids (C4–C14), sulfonates, fluorotelomer sulfonates, HFPO‑DA and other emerging PFAS.
- Chromatography: complete separation of all target peaks achieved within a nine‑minute run; co‑eluting background peaks observed in one region but did not interfere with targets.
- Sensitivity: LOQs experimentally determined for each analyte; many analytes showed LOQs at 0.055 ng/g, with a subset (including PFOA, PFHxS, PFNA, PFOS and several others) achieving lower LOQs of ~0.0055 ng/g; a few long‑chain or less responsive analytes had higher LOQs (e.g., some at 0.55 ng/g).
- Accuracy and precision: Recoveries generally ranged near 85–115% across spike levels for most analytes; repeatability (RSD) values were typically low (often single‑digit % at higher spikes, with higher variability near LOQ but within SMPR limits). Examples shown for PFOA, PFHxS, PFNA and PFOS calibration points demonstrated accuracies close to 90–110% over the calibration range.
- Specific improvements: Instrument and chromatographic optimization increased signal‑to‑noise for PFOA, PFHxS, PFNA and PFOS—critical analytes for regulatory monitoring and human exposure risk assessment.
- Validation outcome: All tested recovery, precision and LOQ criteria met the AOAC SMPR 2023.003 acceptance requirements in this single‑laboratory study.

Key practical takeaways and benefits

- Rapid workflow: QuEChERS extraction with WAX SPE cleanup and a 9‑minute UHPLC run provide a high‑throughput route for routine screening of protein powder samples.
- Robust quantitation: Matrix‑matched isotope dilution reduces matrix effects and improves accuracy; the method supports low ng/g and sub‑ng/g quantitation for many PFAS.
- Isomer resolution and interference management: Baseline separation of branched/linear isomers and deliberate separation of PFOS from cholic acids reduce false positives and improve confidence in identifications.
- Flexibility: The approach covers legacy PFAS and several emerging/shorter‑chain analogs, enabling broader surveillance of PFAS mixtures in food matrices.

Future trends and possible applications

- Wider matrix validation: Adapting and validating the workflow for other food matrices (dairy powders, flours, infant formula) to expand monitoring programs.
- Non‑target and suspect screening: Complementary high‑resolution mass spectrometry (HRMS) workflows to identify novel or transformation PFAS not included among targeted 30 analytes.
- Automation and miniaturization: Automated SPE and online cleanup to increase throughput and reduce analyst variability; microextraction approaches to reduce solvent consumption and cost.
- Harmonization and inter‑laboratory studies: Multi‑laboratory validations and ring trials to standardize LOQ definitions, isotope standards usage and reporting practices across regulatory laboratories.
- Regulatory alignment: Method adaptation to anticipated regulatory limits and reporting requirements, including low‑level surveillance and enforcement thresholds.

Conclusion

This study demonstrates a validated, high‑throughput LC–MS/MS workflow capable of quantifying 30 PFAS in a plant‑based protein powder matrix with sensitivity, accuracy and precision that meet AOAC SMPR 2023.003 criteria. The combination of QuEChERS extraction, WAX SPE cleanup, matrix‑matched isotope dilution, and an optimized Nexera/LCMS‑8060NX platform yields reliable sub‑ng/g quantitation for most target PFAS and effective separation of isomers and common interferences. The method is directly applicable to routine food safety testing and can be extended to additional matrices and regulatory use with further validation.

References

• AOAC SMPR 2023.003