Simplifying the Analysis of PFAS in Aqueous Samples for EPA Method 1633 by Reducing Sample Volume

Significance of the Topic

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous environmental contaminants with high persistence and potential health risks. US EPA Method 1633 provides a standardized LC-MS/MS workflow for quantifying 40 PFAS in various aqueous and solid matrices. However, traditional workflows rely on large sample volumes (500 mL), leading to extended preparation times and logistical burdens. Reducing sample volume while maintaining method performance addresses practical challenges in field sampling, laboratory throughput, and cost efficiency.

Objectives and Overview of the Study

This application note aimed to simplify PFAS analysis in environmental waters by:

• Reducing aqueous sample volume from 500 mL (or 250 mL) to 50 mL.
• Implementing a fully automated SPE workflow to halve sample preparation time.
• Validating method sensitivity, recovery, and accuracy against EPA 1633 quality criteria.
• Demonstrating applicability to complex matrices, certified reference materials, and authentic water samples.

Instrumentation

The analytical workflow utilized:

• PromoChrom SPE-03 Gen 4 Automated SPE System with Oasis WAX/GCB bilayer cartridges for sample extraction and clean-up.
• ACQUITY™ Premier UPLC with Binary Solvent Management and Flow-Through Needle for chromatographic separation.
• ACQUITY Premier BEH C18 analytical column (2.1 × 50 mm, 1.7 µm) and Atlantis Premier BEH C18 AX isolator column.
• Xevo TQ Absolute Triple Quadrupole Mass Spectrometer operating in ESI- mode for sensitive MRM detection.
• waters_connect™ for Quantitation Software for data processing and reporting.

Methodology

Surface water, influent and effluent wastewater samples were collected directly into 50 mL polypropylene tubes, frozen until analysis, then weighed to verify volumes. Samples were spiked with extracted and non-extracted internal standards (25 ng/L), and loaded onto Oasis WAX/GCB SPE cartridges equipped with glass wool and inline filters to prevent clogging. The automated SPE method followed adapted EPA 1633 protocols with a 50 mL load, sequential conditioning, washing, and elution steps. Post-extraction, eluates were evaporated and reconstituted for LC-MS/MS. Chromatography employed a 11-minute gradient at 0.3 mL/min, 10 µL injection, with mobile phases of 2 mM ammonium acetate in water and acetonitrile. MS parameters included a 0.5 kV capillary voltage, 350 °C desolvation, and compound-specific MRM transitions.

Main Results and Discussion

Detection limits (MDLs) for all 40 PFAS using 50 mL samples were on average three times lower than EPA 1633 pooled MDLs from 500 mL extractions, with individual improvements ranging from 1.5× to 6.2×. Increasing injection volume to 10 µL compensated for the fivefold reduction in enrichment factor. Extracted internal standard recoveries across surface water, influent, and effluent matrices averaged 76–77% (RSD < 9%), surpassing EPA 1633 acceptance criteria. Native PFAS spike recoveries in surface water averaged 99% (RSD 7.1%). Analysis of a certified PFAS wastewater CRM in both 250 mL and 50 mL formats yielded mean trueness of 103–104%, confirming accuracy. Authentic field samples processed at 50 mL reliably detected trace PFAS at ng/L levels.

Benefits and Practical Applications

• Halved sample preparation time to ~1 hour per eight-sample batch.
• Reduced field collection, storage, and transport costs with smaller volumes.
• Lower risk of SPE cartridge clogging and enhanced throughput.
• Maintained or improved sensitivity and method performance against EPA quality criteria.
• Streamlined automated workflow suitable for high-volume environmental monitoring.

Future Trends and Opportunities

Further miniaturization and automation of PFAS workflows may integrate microflow UPLC and novel sorbent chemistries to enhance sensitivity while reducing solvent usage. Expansion to emerging PFAS analogs and high-resolution accurate-mass detection could broaden analyte coverage. Coupling on-site sample pre-treatment modules with remote data connectivity will facilitate real-time monitoring in diverse environmental settings. Development of standardized low-volume protocols may also support community-level water quality programs and regulatory compliance.

Conclusion

A fully automated SPE-LC-MS/MS workflow for EPA Method 1633 using only 50 mL aqueous samples delivers equivalent or improved sensitivity, recovery, and accuracy compared to larger volumes. The approach significantly reduces preparation time, logistical complexity, and cost, while ensuring compliance with EPA quality guidelines. This streamlined method enhances laboratory efficiency and supports robust environmental PFAS surveillance.

References

1. US Environmental Protection Agency. EPA Method 1633A: Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid, Biosolids, and Tissue Samples by LC-MS/MS; December 2024.
2. Organtini K., Rosnack K., Plummer C., Hancock P., Burt O. Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Accordance with EPA 1633 Part 2: Analysis of Aqueous Matrices. Waters Application Note 720008143; 2023.
3. Organtini K., Rosnack K., Burt O., Wan I. Automating the Sample Preparation Workflow for Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous Samples Following EPA Method 1633. Waters Application Note 720008825; 2025.
4. Code of Federal Regulations. Guidelines Establishing Test Procedures for the Analysis of Pollutants, 40 CFR Part 136, Appendix B.