A Fully Automated Workflow for PFAS Analysis in Seafood for Regulatory Screening

Significance of the Topic

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants that accumulate in aquatic ecosystems and marine organisms. Detecting trace levels of PFAS in seafood is critical for food safety and regulatory compliance. Traditional manual extraction methods are labor-intensive and prone to variability, motivating the development of a fully automated, high-throughput workflow.

Objectives and Study Overview

This study aims to establish and validate a fully automated protocol for quantitative PFAS analysis in seafood using a CTC PAL3 Series 2 RTC autosampler coupled to an Agilent 6495D triple quadrupole LC/MS system. The goals include:

• Automating calibration preparation, QuEChERS salting-out solvent extraction, micro-SPE cleanup, and LC/MS injection.
• Evaluating method performance (linearity, sensitivity, recovery, repeatability, reproducibility) for 73 PFAS analytes.
• Meeting or exceeding U.S. FDA, EU, EURL POPs, and AOAC regulatory criteria.

Methodology and Instrumentation

The workflow integrates automated sample preparation and analysis:

• Calibration: Robotic serial dilution of analyte, surrogate, and internal standard stock solutions to generate 12 calibration levels (1–50 000 ng/L), stored at 5 °C.
• Sample Preparation: 4 g shrimp powder undergoes QuEChERS salting-out extraction with ACN/H₂O, vortexing, centrifugation; micro-SPE cleanup in 2 mL vials; direct dilute-and-shoot with isotope-labeled internal standards.
• Online Sequence: MassHunter software orchestrates parallel sample preparation on PAL3 and LC/TQ data acquisition, enabling continuous throughput.

Used Instrumentation

• CTC PAL3 Series 2 RTC autosampler with liquid syringe, dilutor, micro-SPE, Fast Wash, solvent, and tray cooler modules.
• Agilent 1290 Infinity II UHPLC with PFC-free HPLC conversion kit and ZORBAX RRHD Eclipse Plus C18 column (2.1 × 100 mm, 1.8 µm).
• Agilent 6495D triple quadrupole LC/MS with Jet Stream ESI in negative mode.

Main Results and Discussion

• Linearity: R² ≥ 0.99 for all 73 PFAS, five or more calibration points.
• Sensitivity: Method detection limits (MDL₍cal₎) ≤ 10 ng/kg and limit of quantitation (LOQ₍cal₎) ≤ 50 ng/kg for ≥ 95% of targets; 28 mandatory PFAS met MDL ≤ 10 ng/kg and LOQ ≤ 35 ng/kg per U.S. FDA.
• Regulatory Compliance: LOQ₍vali₎ ≤ 0.3 µg/kg for PFOS, PFNA, PFHxS; ≤ 0.3–3 µg/kg for other PFAS per EU, EURL POPs, AOAC.
• Recovery: 65–135% for ≥ 89% of analytes at mid-level spike (0.3 µg/kg); critical PFAS recoveries of 80–120%.
• Repeatability: Intrabatch RSD ≤ 20% for 93% of analytes; critical PFAS RSD ≤ 12%.
• Reproducibility: Interbatch RSD ≤ 20% for 93% of analytes; regulated PFAS RSD ≤ 12%.

Benefits and Practical Applications

• Fully automated calibration and sample preparation reduce manual labor, human error, and variability.
• Parallel operation of PAL3 and LC/TQ maximizes throughput and instrument utilization.
• Robust performance against stringent regulatory standards ensures reliable PFAS monitoring in seafood and related matrices.

Future Trends and Opportunities

Automation of PFAS analysis is expected to expand into other complex food and environmental matrices (e.g., dairy, eggs, feed). Integration with advanced data-processing algorithms and cloud-based reporting will further streamline compliance workflows. Miniaturized sample preparation modules and AI-driven method optimization may enhance sensitivity and reduce solvent consumption.

Conclusion

The integrated CTC PAL3 Series 2 RTC autosampler and Agilent 6495D LC/TQ deliver a robust, fully automated workflow for PFAS quantitation in seafood. The method achieves excellent linearity, sensitivity, recovery, and reproducibility, meeting or surpassing U.S. FDA, EU, EURL POPs, and AOAC requirements. This approach improves laboratory efficiency and data quality, supporting regulatory screening and public health protection.

Reference

1. U.S. FDA, Foods Program Compendium of Analytical Laboratory Methods: Chemical Analytical Manual (CAM).
2. European Commission Regulation (EU) 2023/915 on PFAS in food.
3. European Commission Recommendation (EU) 2022/1431 on PFAS analytical limits.
4. EURL POPs Guidance Document v1.2 for PFAS determination in food and feed.
5. AOAC SMPR 2023.003 Standard Method Performance Requirements for PFAS.
6. EPA Method 1633 for PFAS analysis in various matrices.