Refined SPE-LC-MS/MS method for simultaneous quantification of legacy PFAS in Korean population serum under...

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants whose accurate quantification in human serum demands sensitive, rigorously validated analytical methods. A refined solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) workflow was developed and validated for the simultaneous quantification of four legacy PFAS (PFOA, L-PFOS, PFNA, and PFHxS) in 100 μL of human serum. Chromatographic conditions were systematically optimized on a Kinetex® C18 column (2.6 μm, 2.1 × 50 mm) to achieve baseline resolution of linear and branched PFOS isomers within a 6-min total run time. Four critical SPE parameters (protein precipitation solvent, acid additive, evaporation temperature, and reconstitution solvent) were optimized to maximize extraction efficiency and signal reproducibility from the limited-volume matrix. Calibration linearity was established over 0.3-40 ng/mL (PFOA) and 0.2-40 ng/mL (L-PFOS, PFNA, PFHxS), with all correlation coefficients (r²) exceeding 0.99. Validation conducted according to current FDA and EMA guidelines demonstrated within- and between-batch accuracy (RE, -5.3% to +7.0%) and precision (RSD ≤8.0%), IS-normalized matrix effects within 95.6-111.4% (RSD <15.0%), and confirmed analyte stability under bench-top, long-term, freeze-thaw, and autosampler conditions. The validated method was applied to serum samples from 200 participants in a rural Korean cohort; all four PFAS were quantifiable in every sample, yielding mean (± SD) concentrations of 2.9 ± 1.3, 15.9 ± 10.9, 2.3 ± 1.4, and 2.9 ± 2.7 ng/mL for PFOA, L-PFOS, PFNA, and PFHxS, respectively (total PFOS: 21.5 ± 15.7 ng/mL, calculated as the sum of linear and branched fractions). Concentration ranges were consistent with Korean National Environmental Health Survey and National Health and Nutrition Examination Survey reference datasets. Stratified analyses confirmed the method's capability to resolve biologically meaningful sex- and age-dependent PFAS differences. By combining volume efficiency with rigorous chromatographic and bioanalytical performance, this method provides a practical platform for large-scale PFAS biomonitoring under sample-limited conditions.