A Scalable Method for Quantitation of Per-and Polyfluoroalkyl Substances in Human Milk

Breastfeeding is a major route of early-life exposure to per-and polyfluoroalkyl substances (PFAS), yet population-level characterization of this exposure remains limited by the lack of sensitive, high-throughput methods to quantitate PFAS in human milk.To date, PFAS analysis in human milk has required large volumes (1-5 mL) and labor-intensive workflows that are unsuitable for large-scale, population-level biomonitoring.We developed and validated a semi-automated, high-throughput workflow for quantitatively determining 23 legacy and replacement PFAS in 300 µL of human milk, enabling analysis of 72 samples per 20-hr batch.Human milk and quality control (QC) samples are processed in 96-well format using an Opentrons Flex automated liquidhandling system with Enhanced Matrix Removal (EMR) mixed-mode matrix cleanup and analyzed by liquid chromatography with high-resolution mass spectrometry (LC-HRMS).Method detection limits ranged from 0.53 to 5.13 pg/mL, with 20 compounds achieving limits of quantitation ≤5 pg/mL.Average accuracy was 91 ± 10% at 5 pg/mL, the lowest validated concentration, and 91-98% at higher spiking levels.Applied to paired human milk samples collected at 1 and 6 months postpartum from 36 mothers, the method detected 6 PFAS above the LOQ in ≥50% of mothers at one or both time points.Long-chain PFAS concentrations were stable between time points, while short-chain PFAS showed greater between-sample variability.This scalable, low-volume workflow enables high-throughput quantitation of PFAS in human milk and supports large-scale biomonitoring of early-life exposure, with direct implications for exposure assessment, epidemiologic studies, and public health guidance.