Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Wastewater

Importance of the topic

The rising regulatory and public-health attention to per- and polyfluoroalkyl substances (PFAS) has driven demand for validated, high‑throughput analytical workflows able to quantify dozens of PFAS across diverse water matrices. EPA Method 1633A provides a comprehensive, standardized approach for multi‑matrix PFAS analysis; practical, low‑contamination sample‑preparation systems that reliably implement this method are essential for routine monitoring, compliance testing, and research in wastewater, surface water, groundwater and treated drinking water.

Objectives and study overview

This application study evaluated the Fluid Management Systems (FMS) EZPFC semi‑automated solid phase extraction (SPE) workflow for implementing EPA Method 1633A on aqueous matrices. Goals included demonstrating method performance for 40 targeted PFAS (accuracy, precision, method detection limits), confirming low system‑derived contamination, verifying robustness in particulate‑rich samples (no cartridge clogging), and assessing throughput and practical usability for environmental laboratories.

Methodology and instrumentation

Key analytical approach:

• Sample volume: 500 mL aqueous samples spiked with native PFAS and extracted internal standards as required by Method 1633.
• SPE: Agilent Bond Elut PFAS WAX 150 mg/6 mL cartridges were used on the FMS EZPFC semi‑automated SPE platform; sample loading rates were ≈5–10 mL/min under vacuum (~8 inHg).
• Sample processing: a three‑stage extraction sequence based on Method 1633 aqueous procedures — cartridge conditioning (1% methanolic ammonium hydroxide then 0.3 M formic acid), sample loading and sequential rinses (reagent water and acid:methanol), short cartridge drying, followed by a basic methanolic elution step with collection of 5 mL eluates.
• Cleanup and preparation for LC/MS/MS: small volumes of acetic acid and loose carbon were added to eluates for matrix removal, samples were shaken, centrifuged, transferred to clean polypropylene tubes with addition of non‑extracted internal standards, filtered through nylon syringe filters and analyzed directly (no final concentration step required).
• MDL determination: method detection limits were calculated following the 40 CFR Part 136 Appendix B procedure using multiple low‑level spikes.

Used instrumentation

• FMS EZPFC Semi‑Automated SPE System with compatible vacuum pump and stainless steel bottle filters / cartridge filtration wool to mitigate particulate plugging.
• Agilent 1290 Infinity II LC System modified with the Agilent InfinityLab PFAS Analysis HPLC Conversion kit.
• Agilent 6475 triple quadrupole LC/MS operated in electrospray negative ion mode with dynamic MRM acquisition.
• Analytical column: Agilent ZORBAX Eclipse Plus C18, 3.0 × 50 mm, 1.8 µm.
• Consumables and reagents: Agilent Bond Elut PFAS WAX cartridges (5610‑2150), ultrapure DI water, pesticide‑grade methanol, ammonium hydroxide, formic acid, loose carbon, nylon syringe filters, polypropylene tubes, extracted internal standards (EIS) and non‑extracted internal standards (NIS).

Main results and discussion

Performance summary:

• Accuracy and precision: Four replicate synthetic wastewater samples spiked across 40 PFAS targets produced spike recoveries within EPA Method 1633A acceptance windows. Relative standard deviations (RSDs) for target compounds were consistently below 10%, demonstrating excellent repeatability.
• EIS performance: Recoveries for the set of 24 extracted internal standards met the acceptance criteria for aqueous matrices, supporting quantitative integrity across the extraction and analysis workflow.
• Method detection limits: MDLs determined from multiple low‑level spikes yielded sub‑ng/L sensitivity for the majority of analytes, consistent with the detection needs of environmental monitoring programs.
• System contamination: Process (reagent water) blanks contained PFAS levels well below calculated MDLs, indicating effective contamination control in the EZPFC workflow and associated consumables.
• Matrix application and robustness: Triplicate analyses of well, river and tap water showed good reproducibility. Short‑chain PFAS (for example, PFBA, PFBS, PFHxA) and legacy analytes (PFOA, PFOS) were detected at environmentally relevant concentrations (typically low ng/L to sub‑ng/L levels depending on matrix). No cartridge clogging or flow issues were observed, attributed to the integrated bottle filters and cartridge wool.
• Throughput and workflow advantages: The semi‑automated EZPFC processed 6–12 samples in under 70 minutes without a final concentration step, reducing labor and turnaround time relative to fully manual workflows.

Benefits and practical applications

The validated EZPFC + Agilent LC/MS/MS workflow provides several practical advantages for PFAS laboratories:

• High analytical throughput with minimal hands‑on time, enabling expanded monitoring capacity.
• Robust contamination control that preserves low‑level detection capability, a critical requirement for PFAS analysis.
• Reproducible quantitative performance across 40 target PFAS, supporting compliance testing and large‑scale surveys using EPA Method 1633A.
• Compatibility with particulate‑laden matrices (wastewater, groundwater, surface water) through built‑in particulate mitigation, reducing sample loss and repeat analyses.
• Cost and operational efficiency: semi‑automation reduces operator variability and lowers per‑sample labor costs compared with fully manual SPE processing.

Future trends and potential applications

Anticipated developments and ways the described workflow can be extended:

• Further automation and integration with laboratory information management systems (LIMS) to streamline chain‑of‑custody and reporting for regulatory monitoring programs.
• Expansion of targeted lists and hybrid workflows that combine the validated targeted Method 1633A approach with high‑resolution mass spectrometry for non‑target and suspect screening to capture emerging PFAS chemistries.
• Method miniaturization and greener solvent usage to lower waste and operational costs while preserving sensitivity.
• Adaptation to solid and biological matrices (soil, biosolids, tissues) consistent with Method 1633 scope, with matrix‑specific cleanup optimizations.
• Wider adoption in municipal and industrial labs to support source tracking, treatment performance evaluation, and regulatory compliance as PFAS standards evolve.

Conclusion

The FMS EZPFC semi‑automated SPE system coupled with Agilent LC/MS/MS reagents and instrumentation reproducibly implements EPA Method 1633A for aqueous PFAS analysis. The workflow delivered validated accuracy and precision for 40 target PFAS, sub‑ng/L MDLs, minimal system contamination, reliable performance with particulate‑rich matrices, and high sample throughput without an evaporation/concentration step. These attributes make the approach well suited for routine environmental monitoring, regulatory testing, and research applications requiring robust, low‑contamination PFAS extraction.

References

1. U.S. Environmental Protection Agency. Method 1633A: Analysis of Per‑ and Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid, Biosolids, and Tissue Samples by LC‑MS/MS; EPA 820‑R‑24‑007; Office of Water, Engineering and Analysis Division: Washington, DC, December 2024.
2. U.S. Environmental Protection Agency. Definition and Procedure for the Determination of the Method Detection Limit—Revision 2; Appendix B to Part 136, Title 40 Code of Federal Regulations; U.S. Government Printing Office: Washington, DC, 2025.