Determination of 40 PFAS in Tilapia Tissue Following EPA 1633 Method Guidance

Importance of the Topic

The environmental persistence and bioaccumulation potential of per- and polyfluoroalkyl substances (PFAS) present a growing concern for food safety and ecosystem health. Analysis of PFAS in fish tissue is critical for monitoring human and wildlife exposure, guiding regulatory limits, and supporting remediation efforts. A robust and efficient analytical workflow for quantitating multiple PFAS in complex food matrices such as tilapia fillet helps laboratories respond to regulatory demands under the U.S. EPA Method 1633 guidance and similar international standards.

Objectives and Overview

This study aimed to develop and validate a multiresidue analytical protocol for the determination of 40 PFAS in tilapia tissue. Key goals included:

• Adapting QuEChERS extraction paired with Agilent Captiva EMR PFAS Food II mixed-mode passthrough cleanup.
• Deploying sensitive LC/MS/MS detection on an Agilent 6495D triple quadrupole with Jet Stream ionization.
• Meeting or exceeding EPA 1633 method quality control criteria (MDL, LOQ, recovery, precision).
• Comparing performance, solvent use, time, and cost against traditional weak anion exchange (WAX) SPE and a modified carbon/WAX SPE protocol.

Methodology and Instrumentation

Sample Preparation Workflow:

• Homogenize 5 g of tilapia fillet, spike with native PFAS and extracted internal standards (EIS) before extraction.
• Perform QuEChERS extraction using acetonitrile with 1% acetic acid and extraction salts; shake and centrifuge.
• Dilute crude extract with 10% water to facilitate mixed-mode cleanup.
• Apply passthrough cleanup on Agilent Captiva EMR PFAS Food II cartridges; collect filtrate without further drying.
• Spike post-extraction with nonextracted internal standards (NIS) and transfer directly to autosampler vials.

Instrumental Analysis:

• Liquid chromatography on an Agilent 1290 Infinity II UHPLC with Agilent ZORBAX RRHD Eclipse Plus C18 column (2.1 × 100 mm, 1.8 µm).
• Mobile phases: 5 mM ammonium acetate in water (A) and acetonitrile (B); gradient from 10% B to 100% B in 13 min.
• Detection on Agilent 6495D triple quadrupole MS in negative electrospray mode with Jet Stream ionization; MRM transitions from Agilent PFAS MRM database.
• Calibration using neat standards in acetonitrile with 1% acetic acid, diluted 1:1 with water to match the 10% water content of samples.

Main Results and Discussion

Chromatographic Separation and Sensitivity:
The optimized gradient and mobile phase composition produced baseline separation of critical PFAS isomers (e.g., PFOA, PFOS and branched forms) and eliminated co-eluting bile acid interferences. Representative chromatograms demonstrated clear peak shapes and retention stability across all 40 target compounds, EIS, and NIS.
Method Detection Limits (MDL) and Limits of Quantitation (LOQ):

• MDLs ranged from 0.002 to 0.046 µg/kg, consistently below EPA 1633 values (0.069–1.77 µg/kg).
• LOQs spanned 0.05 to 0.5 µg/kg, meeting or surpassing regulatory criteria.

Recovery and Precision:

• Mean recoveries for 24 extracted internal standards were 79–128%, within EPA acceptance limits (40–160%).
• Nonextracted internal standards averaged 92–101%, confirming minimal matrix bias after cleanup.
• Quantitation accuracy for 40 PFAS in QC samples at LOQ, 4× LOQ, and 40× LOQ levels ranged from 75–125% recovery.
• Intra-batch precision (RSD) was below 20% for all analytes across spiking levels.

Comparison with Traditional SPE Methods:
The QuEChERS-EMR protocol reduced sample preparation time by over 80% (from ~22 h to <2 h per sample), cut solvent usage by ~80%, and lowered consumable costs by >50% compared to Solvent-ext-Carbon/WAX and Solvent-ext-Carbon dSPE-WAX SPE methods. EIS recoveries under traditional SPE were broad (5–169%), whereas the new method showed tighter, more reliable recoveries (79–128%).

Benefits and Practical Applications

• High throughput: Simplified workflow supports rapid turnaround in regulatory and commercial labs.
• Cost efficiency: Significant reductions in solvent, consumables, and labor resources.
• Regulatory compliance: MDLs and LOQs meet or exceed EPA 1633 criteria for tissue matrices.
• Robust quantitation: Stable isotope-dilution strategy corrects for matrix effects, ensuring data reliability across complex food tissues.
• Versatility: Protocol can be adapted to other animal-origin food matrices and extended PFAS lists.

Future Trends and Applications

Emerging opportunities include automating QuEChERS-EMR cleanup on robotic platforms, expanding targeted PFAS panels with high-resolution MS, and integrating machine learning for automated data review. Novel sorbent chemistries may further enhance matrix removal for fatty or pigment-rich tissues. Standardization of EMR-based workflows across laboratories will support global PFAS monitoring in food and environmental samples.

Conclusion

The presented QuEChERS extraction with Agilent Captiva EMR PFAS Food II cleanup and LC/MS/MS detection offers a rapid, sensitive, and reliable method for quantifying 40 PFAS in tilapia tissue following EPA 1633 guidance. Compared to conventional SPE approaches, this protocol delivers superior analytical performance, reduced resource consumption, and streamlined operation, making it an attractive option for routine PFAS surveillance in food safety and environmental laboratories.

Reference

• U.S. EPA Method 1633, Revision A: Analysis of Per- and Polyfluoroalkyl Substances in Aqueous, Solid, Biosolid, and Tissue Samples by LC-MS/MS (EPA-820-R-24-007).
• U.S. EPA Method 537: Determination of Selected Perfluorinated Alkyl Acids in Drinking Water by SPE and LC/MS/MS (EPA/600/R-08/092).
• U.S. EPA Method 533: Determination of Per- and Polyfluoroalkyl Substances in Drinking Water by Isotope Dilution and SPE (EPA-815-B-19-020).
• U.S. EPA Multi-Industry PFAS Study – 2021 Preliminary Report (EPA-821-R-21-004).
• Genualdi et al. Anal. Bioanal. Chem. 2024, 416, 627–633.
• Hwang et al. Food Chem. 2024, 445, 138687.
• Zhao et al. Agilent Application Note 5994-7368EN (2024).
• Zhao et al. Agilent Application Note 5994-7370EN (2024).
• AOAC SMPR 2023.003: PFAS in Produce, Beverages, Dairy, Eggs, Seafood, Meat, and Feed.
• U.S. EPA Definition and Procedure for the Determination of the Method Detection Limit, Revision 2 (EPA-821-R-16-006).