Sensitive and accurate determination of 32 PFAS in human serum using online SPE-UHPLC-HRMS

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.