A Method for the Extraction and Analysis of PFAS from Human Serum Utilizing Weak Anion Exchange (WAX) Chemistry and Xevo TQ-S micro

Importance of the Topic

The widespread use and environmental persistence of per- and polyfluoroalkyl substances (PFAS) have led to their accumulation in human tissues. Monitoring PFAS in serum provides critical insight into exposure pathways, bioaccumulation trends, and potential health risks. A reliable extraction and analysis protocol for multiple PFAS is essential for occupational health, environmental exposure assessment, and epidemiological studies.

Objectives and Study Overview

This study aimed to develop and validate a robust, high-throughput solid phase extraction (SPE) method coupled with LC-MS/MS for quantifying 30 PFAS in human serum. The method leverages weak anion exchange (WAX) chemistry for cleanup and a Xevo TQ-S micro tandem quadrupole mass spectrometer to achieve sensitive, selective detection. Method performance was assessed using pooled human serum lots and a NIST standard reference material (SRM 1957).

Methodology

Sample preparation employed Oasis WAX µElution 96-well plates. Human serum or PFAS-free fetal bovine serum (for calibration) underwent protein precipitation (1:3 serum:acetonitrile) and acid dilution to disrupt PFAS–protein binding. After loading, plates were washed to remove interferences and eluted for analysis.

Used Instrumentation

• UPLC system: ACQUITY UPLC I-Class PLUS modified with PFAS kit
• Column: ACQUITY UPLC HSS T3, 2.1 × 100 mm, 1.8 µm, 35 °C
• Mass spectrometer: Xevo TQ-S micro, ESI negative mode
• Software: MassLynx 4.2 with TargetLynx XS for data processing

Main Results and Discussion

Optimization of sample pretreatment demonstrated that 1:3 acetonitrile protein precipitation minimized PFAS breakthrough compared to direct acid dilution. Column selection was critical: the HSS T3 phase resolved PFAS from coeluting steroid sulfates better than a BEH C18 column, improving quantitation accuracy. Calibration curves in FBS ranged from 0.05 to 20 ng/mL with R² ≥ 0.996 (except 6:2 FTS at 0.992). Residuals were ≤15% at LOQ and ≤10% elsewhere. Recoveries across six pooled serum lots were 85–120% for 29 PFAS; FBSA showed ~20% recovery due to its neutral chemistry. Analysis of NIST SRM 1957 (eight replicates) yielded values within ±10% of certified concentrations with RSD <4%, confirming accuracy and robustness. Application to six human serum pools revealed variable total PFAS levels and isomer ratios, enabling potential exposure fingerprinting.

Benefits and Practical Applications

• High-throughput, accurate PFAS quantitation in complex serum matrices
• Reduced instrument maintenance through efficient cleanup
• Applicability to occupational health, environmental biomonitoring, and toxicology
• Capability to distinguish isomeric profiles for source identification

Future Trends and Potential Applications

Advancements may include expansion to non-target PFAS screening, integration of high-resolution mass spectrometry for unknowns, automation of sample preparation, and correlation of PFAS fingerprints with exposure sources in large-scale epidemiological studies. Emerging extraction chemistries may improve recovery of neutral or low-molecular-weight PFAS.

Conclusion

The presented SPE-UPLC-MS/MS method offers a reliable, sensitive, and reproducible approach for quantifying a broad range of PFAS in human serum. Validation with multiple serum sources and certified reference material demonstrates its suitability for routine biomonitoring and research applications.

Reference

1. Post GB, Cohn PD, Cooper KR. Perfluorooctanoic Acid (PFOA), An Emerging Drinking Water Contaminant: A Critical Review of Recent Literature. Environ Res. 2012;116:93–117.
2. Zeng Z, et al. Assessing the Human Health Risks of Perfluorooctane Sulfonate by In Vivo and In Vitro Studies. Environ Int. 2019;126:598–610.
3. United States Environmental Protection Agency. Health Effects Support Document for Perfluorooctane Sulfonate (PFOS). EPA 822-R-16-002; 2016.
4. United States Environmental Protection Agency. Health Effects Support Document for Perfluorooctanoic Acid (PFOA). EPA 822-R-16-003; 2016.
5. Sunderland EM, et al. A Review of the Pathways of Human Exposure to Poly- and Perfluoroalkyl Substances (PFAS) and Present Understanding of Health Effects. J Expo Sci Environ Epidemiol. 2019;29(2):131–147.
6. Huber S, Brox J. An Automated High-Throughput SPE Micro-Elution Method for Perfluoroalkyl Substances in Human Serum. Anal Bioanal Chem. 2015;407(13):3751–3761.
7. Chan E, et al. Endogenous High-Performance Liquid Chromatography/Tandem Mass Spectrometry Interferences and the Case of Perfluorohexane Sulfonate (PFHxS) in Human Serum. Rapid Commun Mass Spectrom. 2009;23(10):1405–1410.