Asymmetric flow field-flow fractionation (AF4) coupled to multiple on-line detectors for the biomonitoring of polystyrene nanoparticles in human urine and serum.

Asymmetric flow field-flow fractionation (AF4) technique coupled to multiple on-line detectors represents a powerful approach for the separation and characterization of nanoplastics in complex biological matrices. This study developed the AF4-based multi-detection method for the biomonitoring of polystyrene (PS) nanoparticles in human urine and serum. Optimized sample preparation protocols were developed to reduce matrix interferences while preserving nanoparticle integrity and minimizing contamination, aggregation and/or particle loss. The AF4 was coupled with complementary detectors, including multi-angle light scattering (MALS), dynamic light scattering (DLS), and ultraviolet (UV) detection, enabling size-resolved detection and semi-quantification of the PS nanoparticle mixture at 20, 60, 100, 200, and 400 nm. Method performances were assessed in terms of sensitivity, recovery and repeatability on mass and size of the PS mixture in human urine and serum. In addition AF4 was not only effective for size-based separation into monodisperse fractions, but also allowed the fraction collection for further complementary and confirming analyses, including size distribution and concentration by nanoparticle tracking analysis (NTA) and single particle inductively coupled plasma mass spectrometry (SP-ICP-MS). The results demonstrated the suitability and robustness of AF4 coupled to multiple on-line detectors for the human biomonitoring application, providing reliable exposure data to support human health risk assessment and regulatory decision-making in the context of nanoplastic contamination.