Advancing PFAS biomonitoring with a validated high-throughput suspect screening workflow in a longitudinal birth cohort.

Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals of increasing global concern due to their environmental persistence, bioaccumulation, and associated health risks. Advances in high-resolution mass spectrometry and suspect/non-target screening (SNTS) have expanded PFAS detection, enabling a deeper understanding of this chemical group. In this study, a PFAS-specific workflow was developed and validated to identify exposome-related PFAS in human plasma from a cohort of children. Limits of identification (LOIs) and true positive rates (TPRs) were evaluated using standards and plasma spiked with 42 PFAS. LOIs of 1-5 ng/mL were achieved for 14% of compounds, 67% ranged from 5 to 10 ng/mL, and 19% were above 20 ng/mL. TPRs were 0.86 for standards and 0.76 for fortified plasma. Forty-five PFAS were annotated at confidence levels 2a-4 in 615 plasma samples from children aged 4, 8, and 14 years in two Basque Country regions (Urola and Goierri). Twenty PFAS families were detected, with sulfonate esters, benzyl halides, amides, esters, and ketones being most frequent. PFAS profiles varied by age, sex, and region. At age 4, girls had more short-chain PFAS and boys had more long-chain ones. By ages 8 and 14, both sexes showed increased short-chain PFAS, particularly in Urola.