Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Accordance With EPA 1633 Part 1: Establishing and Assessing the Method

Importance of the Topic

Per- and polyfluoroalkyl substances (PFAS) are widely recognized as persistent and bioaccumulative contaminants that pose risks to human health and ecosystems. Regulatory agencies such as the US EPA have established Method 1633 to standardize PFAS analysis across diverse matrices, including non-potable water, soils, biosolids, and tissues. Reliable quantitation of 40 target PFAS with stringent quality criteria is essential for environmental monitoring, remediation efforts, and compliance with emerging regulatory limits.

Objectives and Study Overview

This application note describes the first in a series of studies validating a comprehensive workflow for EPA Method 1633. The primary goals were to establish an 11-minute LC-MS/MS protocol on the ACQUITY Premier BSM FTN LC System coupled to the Xevo TQ Absolute Tandem Quadrupole Mass Spectrometer and to assess method performance using waters_connect for Quantitation Software. Key evaluation metrics included calibration linearity, retention time and ion ratio stability, sensitivity, and data processing efficiency.

Methodology

Sample preparation followed the draft EPA 1633 protocol using Oasis WAX solid phase extraction with graphitized carbon black cleanup. For aqueous samples, a reduced volume of 250 mL (50× concentration) was adopted to leverage the high sensitivity of the Xevo TQ Absolute and to improve throughput. Isotope-dilution calibration was employed with 31 labelled internal standards. Data review criteria from Method 1633 draft 4 included:

• Retention times within ±0.4 min of reference
• Elution within ±0.1 min of corresponding internal standard
• Resolution of bile salts at least 1 min from PFOS
• Minimum signal-to-noise of 3:1 for quantitation ions
• %RSD ≤ 20 % for response factors
• Calibration verification within 70–130 % of expected concentrations
• Ion ratio deviations within ±50 % of midpoint reference

Used Instrumentation

• ACQUITY Premier BSM FTN LC System with ACQUITY Premier BEH C18 column (2.1 × 50 mm, 1.7 µm)
• Atlantis Premier BEH C18 AX isolator column (2.1 × 50 mm, 5 µm)
• Xevo TQ Absolute Tandem Quadrupole MS operating in negative ESI mode
• waters_connect for Quantitation Software

Main Results and Discussion

Using acetonitrile as the organic mobile phase, the method achieved an 11-minute gradient that separated PFOS isomers from bile salt interferences by over 1 min. Calibration curves, extended 20× lower than the original method range, showed signal-to-noise ≥3 at the minimum quantitation level and %RSD of relative response factors below 15 %. Calibration verification injections over eight days remained within ±30 % deviation for virtually all analytes, demonstrating robust instrument stability. Retention time deviations peaked at 0.02 min (2.7 %) and all ion ratio measurements stayed within ±50 % of the reference, confirming compliance with EPA 1633 criteria. Isomer peak integration and automated summation were efficiently handled by waters_connect.

Benefits and Practical Applications

• Rapid 11-minute analysis for 40 PFAS compounds
• High method sensitivity enabling smaller sample volumes
• Robust retention time and ion ratio stability for routine monitoring
• Streamlined data processing and reporting with integrated software

Future Trends and Potential Applications

• Expansion of PFAS panels to include emerging analogues
• Field-deployable extraction and portable MS systems for on-site screening
• Integration of high-resolution MS and machine learning for non-targeted PFAS profiling
• Cross-laboratory validation and harmonization for broader regulatory adoption
• Automated sample preparation to increase throughput and reproducibility

Conclusion

The established LC-MS/MS workflow on the ACQUITY Premier BSM FTN and Xevo TQ Absolute fully meets EPA Method 1633 requirements, offering fast, sensitive, and robust PFAS analysis. Reduced sample volumes, stable calibration, and efficient software-driven data processing make this solution well-suited for environmental monitoring and compliance testing across various matrices.

References

1. US Environmental Protection Agency. Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid, Biosolids, and Tissue Samples by LC-MS/MS, Draft 4. July 2023.
2. Organtini KL, Rosnack KJ, Stevens D, Ross E. Analysis of Legacy and Emerging Perfluorinated Alkyl Substances in Environmental Water Samples Using SPE and LC-MS/MS. Waters Application Note 720006471; Revised November 2022.
3. Organtini K, Foddy H, Dreolin N, Adams S, Rosnack K, Hancock P. Ultra-trace Detection of PFAS in Drinking Water to Meet New US EPA Interim Health Advisory Levels. Waters Application Note 720007855; February 2023.