Sensitive and Accurate Determination of 32 PFAS in Human Serum Using Online SPE- UHPLC-HRMS

Authors: Masho Hilawie Belay, Elisa Robotti, Arianna Ghignone, Alessia Fabbris, Jessica Brandi, Daniela Cecconi, Maria Angela Masini, Francesco Dondero and Emilio Marengo

Abstract: Per- and polyfluoroalkyl substances’ (PFAS) extreme persistence has been linked to many adverse effects on human health including increased risk of certain cancers. This study presents the development and validation of a new, highly sensitive method for the quantification of 32 PFAS in human serum using online solid-phase extraction (SPE) coupled with ultra-high performance liquid chromatography–high resolution mass spectrometry (UHPLC–HRMS). Legacy and emerging PFAS were targeted. Main steps of sample pretreatment include protein precipitation (PP), pellet rinsing, centrifugation, preconcentration through solvent evaporation, and online SPE using a weak anion-exchange polymeric sorbent. The PP and pellet-rinsing procedures were optimized through a comprehensive exploration of solvent combinations. Following this, a pretreatment that offers the best compromise for the targeted PFAS was identified using principal component analysis. The method demonstrated excellent linearity (R² = 0.977–0.997) with limits of quantification ranging from 8.9 to 27 ng/L, 5 to 15 times lower than previous methods. Precision (intraday 2.6–14.0% and interday 1.3–11.0% relative standard deviation) and accuracy (recoveries 72.7–106%) were robust. The method was validated in accordance with ISO/IEC 17025 and successfully applied to five human serum samples, confirming its suitability for high-throughput profiling of PFAS in biomonitoring studies. This method is the first to use online SPE for the simultaneous determination of a broad range of PFAS, including ether congeners such as perfluoro(2-ethoxyethane) sulfonic acid and Nafion byproduct 2. Furthermore, control charts were employed to assess instrument performance during routine analysis and implement necessary actions.

Keywords: PFAS, Method development, High resolution mass spectrometry, Human biomonitoring, Serum pretreatment