PFAS Analysis in Human Plasma Using the Agilent Ultivo Triple Quadrupole LC/MS

Significance of the topic

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are persistent synthetic compounds widely used in industry and consumer products. Their stability leads to environmental accumulation and bioaccumulation in human tissues, where they have been associated with adverse health effects including metabolic, reproductive, and oncological outcomes. Sensitive quantification of PFAS in plasma is essential for biomonitoring, exposure assessment, and understanding molecular mechanisms of toxicity.

Objectives and study overview

This application note describes the validation of an LC/MS/MS method for targeted measurement of 14 PFAS in human plasma using the compact Agilent Ultivo Triple Quadrupole LC/MS. The method aims to deliver high sensitivity, robust performance, and streamlined sample preparation suitable for large-scale epidemiological studies.

Methodology and instrumentation used

• Sample preparation: Protein precipitation of 100 µL plasma with cold methanol, spiking of mass-labeled internal standards, centrifugation, and collection of supernatant.
• Chromatography: Agilent 1290 Infinity II LC with Poroshell 120 EC-C18 column (2.1×50 mm, 2.7 µm), mobile phases of 5 mM ammonium acetate in water (A) and methanol (B), 10-minute gradient at 0.5 mL/min, column at 50 °C.
• Mass spectrometry: Agilent Ultivo Triple Quadrupole in negative electrospray ionization, dynamic multiple reaction monitoring, MassHunter Acquisition and Quantitative Analysis software.
• Calibration: 13-point curve from 0.003 to 12.5 ng/mL, demonstrating linearity with R²>0.996; LODs defined at S/N ≥3:1 and LOQs at S/N ≥10:1.

Main results and discussion

• Baseline chromatographic separation of all 14 PFAS achieved within a 10-minute runtime.
• LOQs ranged from 0.009 to 0.195 ng/mL and LODs from 0.003 to 0.048 ng/mL.
• Carryover was <20% at the LOQ level for analytes and <5% for internal standards.
• Mean recoveries between 67–110% across low, medium, and high quality controls, meeting US EPA criteria.
• Precision (%CV) below 10% for both intra-day and inter-day reproducibility.
• Stability of PFAS in QC samples at room temperature and 4 °C maintained with %CV <15%.
• Application to 96 human plasma specimens detected 11 PFAS above LOQ with a total concentration of 6.85±3.1 ng/mL; significant gender differences observed for PFHxS and PFOA.

Benefits and practical applications of the method

The streamlined workflow requires minimal sample volume and simple protein precipitation, enabling high-throughput analysis. The method’s sensitivity, precision, and compliance with regulatory guidelines support its use in population biomonitoring, clinical studies, and combination with lipidomics to investigate PFAS-related metabolic disruptions.

Future trends and potential uses

• Expansion of PFAS panels and integration with multi-omics strategies for comprehensive exposome research.
• Automation of sample preparation to increase throughput for large cohorts.
• Adaptation of the method to additional biological matrices such as serum, urine, and tissues.
• Development of portable LC/TQ systems for point-of-care or field-based monitoring.

Conclusion

The validated Agilent Ultivo Triple Quadrupole LC/MS method offers a compact, robust, and sensitive approach for quantifying PFAS in human plasma. Its performance meets regulatory standards and facilitates large-scale studies and mechanistic research on PFAS exposure.

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