Analysis of Per- and Polyfluoroalkyl Substances (PFASs) in Biological Fluid Using a Novel Lipid Removing Sorbent and LC-MS/MS

Importance of the Topic

Per- and polyfluoroalkyl substances are persistent industrial pollutants that accumulate in animal tissues and pose potential health risks such as liver, kidney, immune effects, and developmental problems. Reliable quantification of PFAS in plasma is crucial for environmental and toxicological studies.

Objectives and Study Overview

This application note presents a streamlined workflow for the extraction and quantification of 22 PFAS in human plasma. The approach combines in-well protein precipitation with targeted lipid removal using an Agilent Captiva EMR–Lipid cartridge followed by LC-MS/MS analysis.

Methodology and Used Instrumentation

• Sample Preparation: In-well precipitation using acetonitrile with 1% formic acid at a plasma to solvent ratio of approximately 1 to 4, followed by controlled passage through a 1 mL Captiva EMR–Lipid cartridge under low vacuum.
• Target Analytes: Twenty-two PFAS compounds including PFOA, PFOS, PFHxS, PFNA, and longer chain perfluorocarboxylic acids; matched 13C-labeled internal standards.
• Liquid Chromatography: Agilent 1290 Infinity II system with a ZORBAX Eclipse Plus 95Å C18 delay column and an InfinityLab Poroshell 120 EC-C18 analytical column at 50 °C; flow rate 0.5 mL/min; gradient elution from 30% to 100% acetonitrile.
• Mass Spectrometry: Agilent 6495 Triple Quadrupole with iFunnel technology in negative electrospray mode; dynamic MRM transitions optimized for each PFAS and a common m/z 184 fragment for phospholipid monitoring.

Main Results and Discussion

• Lipid Removal Performance: Captiva EMR–Lipid achieved more than 99% removal of phospholipids compared to protein precipitation alone, dramatically reducing matrix effects and potential instrument contamination.
• Chromatographic Quality: Clean, symmetric peaks were obtained at 5 ng/mL spiking levels with high signal-to-noise ratios, enabling accurate integration and minimal carryover.
• Linearity and Sensitivity: Calibration curves from 0.1 to 50 ng/mL were linear with coefficients of determination above 0.992, supporting reliable quantification down to 0.1 ng/mL.
• Recovery and Precision: Method recoveries ranged from 75% to 125% for all PFAS, with typical values around 90–110%. PFOA and PFOS recoveries averaged 92–93% with relative standard deviations of less than 14% at both 5 and 20 ng/mL levels.

Benefits and Practical Applications

The described protocol offers reduced sample handling, minimal analyte loss, and compatibility with high-throughput and automated workflows. Cleaner extracts lead to lower maintenance requirements and more robust long-term performance of LC-MS/MS systems.

Future Trends and Opportunities

Emerging sorbent materials and microfluidic integration may further enhance cleanup selectivity and throughput. Expanded target lists to include novel PFAS analogues and coupling with alternative ionization methods could broaden environmental and clinical applications.

Conclusion

Combining in-well protein precipitation with EMR–Lipid lipid removal delivers a rapid, reliable, and high-throughput method for multiresidue PFAS analysis in plasma. The workflow attains excellent sensitivity, accuracy, and instrument performance, making it well suited for environmental and toxicological research.

References

• US EPA Research on PFAS, 2017.
• Matuszewski BK et al. Strategies for assessment of matrix effects in HPLC-MS/MS bioanalysis. Anal Chem 2003;75(13):3019–3030.
• Chambers E et al. Systematic strategy for reducing matrix effects in LC/MS/MS analyses. J Chromatogr B 2007;852:22–34.