Automating Sample Extraction and Cleanup of Per- and Polyfluoroalkyl Substances (PFAS) in Fish Tissues Following EPA 1633 Guidance

Significance of the Topic

Per- and polyfluoroalkyl substances (PFAS) pose significant environmental and health risks due to their persistence and bioaccumulation. Monitoring PFAS in fish tissues is critical for assessing human exposure through diet, guiding regulatory actions, and ensuring ecosystem safety. Automating the extraction and cleanup workflow can enhance throughput while reducing contamination risk.

Objectives and Study Overview

This study demonstrates a fully automated sample preparation workflow for PFAS analysis in fish tissues following US EPA Method 1633. By integrating pressurized fluid extraction and automated solid phase extraction (SPE), the protocol reduces a traditional 2-day manual process to an 8-hour shift. Performance was benchmarked against established manual procedures across various fish matrices.

Methodology and Instrumentation

• Sample Preparation: Homogenization of salmon, tuna, and shrimp with dry ice; inclusion of a certified reference material (FAPAS CRM) and internal standards.
• Pressurized Fluid Extraction: CEM EDGE PFAS System, Q-Cup cells with S1 Q-Discs, Q-Matrix Hydra, spiking of extracted internal standards, extraction program per EPA 1633 guidelines, dilution to 250 mL, pH adjustment to <6.
• Automated SPE Cleanup: PromoChrom SPE-03 Gen 4, inline filtration, Oasis GCB/WAX cartridges, elution and addition of acetic acid plus non-extracted internal standard.
• LC-MS/MS Analysis: Waters ACQUITY Premier UPLC with BEH C18 analytical column and BEH C18 AX isolator, Xevo TQ Absolute MS in ESI- mode, waters_connect software for quantitation.

Main Results and Discussion

• Time Savings: Extraction and cleanup of 12 samples in series achieved within ~2 hours and hands-off cleanup in 2 hours, reducing total prep time to one workday.
• System Blanks: Method blanks confirmed no significant PFAS contamination from instrumentation; trace 6:2 FTS attributed to solvent.
• Recovery Performance: Extracted internal standards and fortified native PFAS in salmon, tuna, and shrimp exceeded EPA 1633 minimum recoveries and matched manual workflow results.
• CRM Accuracy: Quantified concentrations of PFOA, PFNA, PFHxS, and PFOS in the certified reference material aligned with certified values, supporting method accuracy.

Benefits and Practical Applications of the Method

• Improved Laboratory Efficiency: Single-shift sample preparation increases throughput.
• Reduced Analyst Involvement: Minimal hands-on steps lower labor demands and contamination risk.
• Regulatory Compliance: Fully meets EPA 1633 extraction and analysis criteria for tissue matrices.
• Matrix Versatility: Validated across fish tissues with diverse fat and protein contents.

Future Trends and Opportunities

• Expansion to Emerging PFAS: Automated workflows can incorporate additional analytes and matrices.
• Integration with Digital Labs: Connectivity and robotics promise seamless, traceable sample handling.
• High-Throughput Platforms: Miniaturized SPE and parallel extraction systems will further accelerate analysis.
• Advanced Data Analytics: Real-time monitoring and AI-driven quality control will enhance reliability.

Conclusion

The automated pressurized fluid extraction and SPE cleanup workflow for fish tissues under EPA Method 1633 delivers equivalent performance to manual procedures while condensing sample preparation into a single 8-hour shift. This approach ensures high data quality, reduces contamination risk, and increases laboratory throughput, making it a robust solution for PFAS monitoring in environmental and food safety laboratories.

Reference

1. US Environmental Protection Agency. EPA 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 3: Analysis of Soil and Tissue. Waters Application Note 720008230. 2024.