Assessing Relative Response of Four European-Regulated PFAS in Human Serum Using Cyclic Ion Mobility MS

Significance of the topic

Human exposure to per- and polyfluoroalkyl substances (PFAS) is an active public health and regulatory concern because many PFAS are persistent, bioaccumulative and associated with adverse developmental, hepatic and other toxic effects. Reliable measurement of PFAS at trace levels in human serum is therefore critical for exposure assessment, biomonitoring and for informing regulatory action. Advanced separation strategies that combine liquid chromatography with ion mobility and high-resolution mass spectrometry (LC-Cyclic-IMS-MS) can increase confidence in identification and help distinguish structurally related PFAS in complex biological matrices, supporting both targeted and non-targeted surveillance.

Objectives and study overview

This work evaluated the relative analytical response and occurrence of four European-regulated PFAS (perfluorooctane sulfonate PFOS, perfluorohexane sulfonate PFHxS, perfluorooctanoic acid PFOA, and perfluorononanoic acid PFNA) in human serum collected from Ghanaian firefighters and e-waste handlers. The main aims were to demonstrate the performance of LC-Cyclic-IMS-MS for confident trace-level identification in serum, to report measured concentration ranges in occupationally exposed cohorts, and to illustrate the utility of collision cross section (CCS) and arrival time distribution (ATD) information for feature discrimination.

Methodology

• Sample cohorts: Human serum from two occupational groups (firefighters and e-waste handlers) in Ghana, analyzed alongside NIST reference standards for method validation and comparability.
• Sample preparation: High-throughput solid-phase extraction (SPE) using 96-well µElution plates with polymeric reversed-phase/weak anion exchange sorbent to enrich PFAS and reduce matrix interferences.
• Chromatography: Reversed-phase UPLC with a 22-minute gradient using aqueous (95:5 water:methanol) and methanolic mobile phases containing 2 mM ammonium acetate; ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.8 µm) at 35 °C and 10 µL injection volume.
• Separation specificity: Use of PFAS-minimized system components (PFAS-free conversion kit and an isolator column) to limit background contamination.
• Mass spectrometry and ion mobility: Quadrupole–Cyclic ion mobility time-of-flight (Q-Cyclic IMS-TOF) instrument with cyclic IMS resolution approximately R ~65–145, allowing measurement of CCS and ATD for enhanced feature discrimination.
• Quantification and identification: Isotopically labelled internal standards, accurate mass, retention time matching and CCS agreement with library values were combined to achieve high-confidence identifications at trace concentrations.

Instrumentation

• Mass spectrometer: Quadrupole-Cyclic IMS time-of-flight mass spectrometer (SELECT SERIES Cyclic IMS platform).
• LC system: Modified ACQUITY i-Class UPLC with PFAS-free conversion kit; columns used included ACQUITY UPLC BEH C18 (100 × 2.1 mm, 1.8 µm) and Atlantis Premier BEH C18 AX isolator column (2.1 × 50 mm, 5 µm).
• Consumables and kits: PFAS Analysis Kit for ACQUITY UPLC Systems and SPE 96-well µElution polymeric RP/WAX plates.

Main results and discussion

• Detection frequency: Combined detection for the four regulated PFAS (PFOS, PFHxS, PFOA, PFNA) was approximately 83% across serum samples and reference materials, indicating the method’s sensitivity and applicability to human biomonitoring.
• Concentration ranges: Measured concentration ranges (reported per sample cohort) showed higher levels among firefighters relative to e-waste handlers. Representative ranges reported were:
  • E-waste handlers: PFOS ~0.008–0.064 ng/mL; PFHxS ~0.001–0.012 ng/mL; PFOA ~0.005–0.04 ng/mL; PFNA ~0.004–1.513 ng/mL.
  • Firefighters: PFOS ~0.006–2.979 ng/mL; PFHxS ~0.001–0.088 ng/mL; PFOA ~0.002–0.324 ng/mL; PFNA ~0.003–5.713 ng/mL.
• Ion mobility benefits: CCS and ATD measurements agreed well with library values and provided an orthogonal descriptor to retention time and accurate mass, improving specificity. Ion mobility-resolved spectra successfully separated PFAS signals from co-eluting matrix background, enabling confident discrimination of native analytes and isotopically labelled standards at low ng/mL to sub-ng/mL levels.
• Fragmentation and ion chemistry: Under the soft ionisation conditions used, perfluoroalkyl carboxylic acids (PFCAs) often produced dominant [M–CO2H]− fragment ions rather than simple deprotonated [M–H]− species because of labile neutral losses. These fragment ions retained characteristic ATD features that were reproducible and useful for PFCA classification when combined with CCS, retention time and internal standards.
• Analytical confidence: Combining isotopically labelled internal standards, accurate mass, retention time and CCS led to robust identification at trace levels; the study demonstrates the approach’s value for both targeted and non-targeted PFAS surveillance in complex biological matrices.

Benefits and practical applications

• Enhanced selectivity: The additional ion mobility dimension (CCS/ATD) improves discrimination of isobaric or co-eluting PFAS and reduces false positives from matrix interferences.
• Trace-level biomonitoring: The workflow supports reliable detection and quantification of regulated PFAS in human serum at sub-ng/mL concentrations, suitable for occupational and population exposure studies.
• Non-target screening capability: Cyclic IMS coupled with high-resolution MS enables discovery of emerging or unexpected PFAS in complex samples, informing exposure assessment and regulatory prioritization.
• Operational advantages: Use of PFAS-minimised LC components and automated 96-well SPE supports higher throughput while limiting background contamination—important for routine biomonitoring labs.

Limitations and considerations

• Requirement for standards: Confident quantification and CCS library matching depend on availability of isotopically labelled standards and reference CCS values for analytes of interest.
• Ion chemistry complexity: PFAS fragmentation patterns (e.g., [M–CO2H]− formation) can complicate interpretation; robust method validation and orthogonal criteria (RT, CCS, labelled standards) are needed for reliable identifications.
• Background control: Stringent PFAS control in consumables and LC systems is essential to avoid artefactual contamination that would compromise low-level measurements.

Future trends and potential applications

• Expanded CCS libraries: Development and community sharing of high-quality CCS databases for a wider range of PFAS and metabolites will increase confidence in non-target identifications.
• Standardization: Harmonized workflows, reference materials and inter-laboratory studies will be needed to support regulatory biomonitoring programs and cross-study comparisons.
• Integration with toxicology: Linking molecular-level exposure data with toxicogenomic and clinical endpoints can improve risk assessment and prioritize PFAS of concern.
• Automated data analytics: Machine learning and feature-processing pipelines that use ATD/CCS signatures alongside mass and chromatographic information will accelerate non-target discovery and reduce manual review time.
• High-throughput biomonitoring: Continued optimization of SPE automation and shortened LC gradients combined with IMS resolution improvements could permit larger population studies while maintaining specificity.

Conclusions

The study demonstrates that LC-Cyclic-IMS-MS is a powerful analytical approach for detecting and characterizing regulated PFAS in human serum at trace concentrations. Ion mobility-derived CCS and ATD descriptors provide valuable orthogonal specificity that, together with isotopically labelled standards, retention time and accurate mass, enable reliable identification in complex biological matrices. Application to occupational cohorts revealed widespread detection of PFOS, PFHxS, PFOA and PFNA, with generally higher levels observed in firefighters compared with e-waste handlers. The method is well suited to both targeted monitoring of regulated PFAS and broader non-target screening for emerging fluorinated contaminants.

Reference

1. U.S. Environmental Protection Agency. Method 1633 Final Report. 2024.
2. European Union Reference Laboratory for Persistent Organic Pollutants (EURL-POPs) working group resources on PFAS.
3. Drinking Water Inspectorate information letter on PFAS monitoring. 2021.
4. Maine Department of Environmental Protection. PFAS products information.
5. French National Assembly document on PFAS regulation proposals.
6. U.S. EPA. Body Weight Data for IRIS PFNA Assessment. 2024.
7. Conley JM, Lambright CS, Evans N, et al. Hexafluoropropylene oxide-dimer acid (GenX) alters maternal and fetal metabolism and causes neonatal toxicity in Sprague-Dawley rats. Environ Int. 2021;146:106204.
8. Waters Corporation. Enhanced Identification Confidence and Specificity for PFAS Analysis Using Cyclic Ion Mobility Mass Spectrometry Collision Cross Sections. Application Note. 2024.
9. Waters Corporation. PFAS Analysis Kit for ACQUITY UPLC Systems User Guide. 2024.
10. McCullagh M, Kass I, Lioupi A, Theodoridis G, Plumb R, Dowd S, Adams S. The utility of Cyclic ion mobility to improve selectivity and analysis efficiency of environmental PFAS contamination and exposure. Poster, ASMS 2024.