Sensitive Analysis for Per- and Polyfluoroalkyl Substances (PFAS) in Whole Blood

Importance of the Topic

Per- and polyfluoroalkyl substances (PFAS) are a widespread class of persistent environmental contaminants linked to adverse health outcomes. Accurate measurement of PFAS in whole blood is critical for clinical research, epidemiological studies, and regulatory monitoring. Achieving sensitive and reproducible analysis at low concentration levels helps improve risk assessment and supports public health decisions.

Study Objectives and Overview

This study aimed to develop and validate a streamlined workflow for the extraction and quantification of 40 PFAS compounds in whole blood. The approach combined offline protein precipitation with Agilent Captiva EMR–Lipid cleanup, followed by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS), to achieve low detection limits and robust performance.

Methodology

The sample preparation protocol consisted of:

• Prerinsing of a 1 mL Captiva EMR–Lipid cartridge (p/n 5190-1002) with 2 × 500 µL of 80:20 acetonitrile:water and drying under positive pressure.
• Aliquoting 150 µL of whole blood into an Eppendorf tube, spiking with 5 µL of internal standard mix.
• Protein precipitation by adding 450 µL of precooled 95:5 acetonitrile:methanol, vortexing for 3 minutes and centrifuging at 5 000 rpm for 3 minutes.
• Transferring the supernatant to the prerinsed cartridge and eluting under 2–5 psi into LC vials, followed by a brief drying step at ~9 psi.
• Adding performance internal standards to the eluate and vortexing prior to analysis.

Instrumentation

• Agilent 1290 Infinity II LC system with binary pump and autosampler (10 °C).
• Agilent 6495C triple quadrupole LC/MS equipped with PFC delay column (4.6 × 30 mm) to reduce background.
• Agilent InfinityLab Poroshell 120 EC-C18 analytical column (2.1 × 100 mm, 2.7 µm) at 50 °C.
• Mobile phases: A) 2 mM ammonium acetate in water; B) 95:5 acetonitrile:water, 0.4 mL/min gradient.

Results and Discussion

Matrix cleanup with Captiva EMR–Lipid removed over 99 % of phospholipids, significantly reducing matrix effects observed in protein precipitation (PPT)-only samples. Calibration curves from solvent standards overlaid those from post-extracted spiked matrix, confirming minimal bias. An optimized injection program—sandwiching 5 µL extract between 10 µL water plugs—improved peak shape for early eluters. Method performance demonstrated reproducible quantitation (RSD < 15 % for most analytes) across spiking levels of 0.03 to 0.67 ng/mL. The workflow efficiently handled small sample volumes and maintained sensitivity and linearity.

Benefits and Practical Applications

• Low blood volume requirement (150 µL) suitable for clinical and epidemiological studies.
• Comprehensive coverage of 40 PFAS compounds with high reproducibility at sub-ng/mL levels.
• Effective lipid removal minimizes matrix interference and extends column lifetime.
• Compatible with high-throughput laboratories and routine QA/QC workflows.

Future Trends and Opportunities

Advances may include automation of the protein precipitation and cartridge cleanup to increase throughput, expansion of target lists to emerging PFAS, integration with high-resolution mass spectrometry for suspect screening, and application in longitudinal exposure studies. Data analytics and informatics tools will further enhance interpretation of PFAS profiles in human biomonitoring.

Conclusion

The developed method provides a sensitive, reproducible, and practical solution for quantifying 40 PFAS in whole blood. By combining offline protein precipitation with Captiva EMR–Lipid cleanup and optimized LC-MS/MS conditions, the workflow achieves low detection limits and robust performance, supporting its use in clinical research and regulatory monitoring.

References

• Department of Health and Human Services, ATSDR. Toxicological Profile for Perfluoroalkyls, May 2021.
• Szabo D., Marchiandi J., Green M. P., Mulder R. A., Clarke B. O. Evaluation and Validation of Methodologies for the Extraction of PFAS in Serum of Birds and Mammals. Anal. Bioanal. Chem. 2022, 414(9):3017–3032.
• US EPA. Draft Method 1633: Analysis of PFAS in Aqueous, Solid, Biosolids, and Tissue Samples by LC-MS/MS, June 2022.
• Zhao L., Juck M. Protein Precipitation for Biological Fluid Samples Using Captiva EMR–Lipid 96-Well Plates. Agilent Technol. 2018, 5991-9222EN.
• Anumol T., Stevens J., Xu X. Analysis of PFAS in Biological Fluid Using a Novel Lipid Removing Sorbent and LC-MS/MS. Agilent Technol. 2017, 5991-8656EN.