A simplified protein precipitation-based LC–MS/MS workflow for legacy and emerging PFAS determination in serum and whole blood

Reliable determination of per- and polyfluoroalkyl substances (PFAS) in biological matrices is essential due to their persistence, bioaccumulation and strong binding to blood proteins. In this study, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method based on protein precipitation (PPT) and extract dilution was developed and validated for the simultaneous determination of 20 legacy and emerging PFAS in serum and whole blood. This simplified workflow was suitable for both protein-rich matrices, minimizing sample handling while avoiding SPE or additional clean-up steps. The method demonstrated excellent analytical performance in both matrices, with matrix-matched calibration curves showing coefficients of determination (R 2 ) ≥ 0.99 for all analytes. Recoveries were within the acceptance criteria, and robust analytical sensitivity was achieved, with limits of detection below 0.1 ng mL -1 for nearly all target PFAS. Limits of quantification ranged from 0.035 to 0.70 ng mL -1 , with most analytes validated at 0.35 ng mL -1 or lower. The applicability of the method was successfully demonstrated through the analysis of 40 human serum and 40 whole blood samples. Eleven PFAS were quantified, and all analyzed samples contained between 6 and 11 PFAS simultaneously. The combination of a straightforward sample preparation procedure, high analytical sensitivity, applicability to both serum and whole blood, and simultaneous determination of legacy and emerging PFAS makes the proposed method a practical tool for routine human biomonitoring and PFAS exposure assessment studies, particularly given the lack of simplified PPT-only workflows validated for liquid whole blood and the limited availability of harmonized methods for both matrices.