Non-targeted identification of PPARγ-active contaminants in human breast milk by affinity selection-mass spectrometry

Human breast milk surveillance is essential for assessing infants and early-childhood exposure to endocrine-disrupting chemicals. However, current biomonitoring is challenged by the growing number and chemical complexity of emerging contaminants, leaving substantial gaps in data on their occurrence and contamination profiles in breast milk. To address this, we developed a peroxisome proliferator-activated receptor γ (PPARγ) affinity selection-mass spectrometry (PPARγ-ASMS) assay for the non-targeted identification of bioactive ligands in this matrix. We successfully identified 18 potential PPARγ-binding contaminants in ten human breast milk samples. Among these, seven were confirmed with high confidence (Schymanski level 1). Their semi-quantitative concentrations ranged from 0.67 to 303.37 ng/mL and highlighted the potential for co-exposure to multiple PPARγ-active contaminants for both mothers and infants. Notably, octyl hydrogen phthalate (OHP) was validated as a direct PPARγ ligand via docking (-7.3 kcal/mol) and surface plasmon resonance (SPR) binding assays (K_D = 0.149 µM). A dual-luciferase reporter gene (DLR) assay further demonstrated OHP acted as PPARγ agonist, exhibiting dose-dependent activity with an EC₅₀ of 7.5 µM. Overall, this study established a reliable, effect-directed affinity screening approach for emerging contaminants in human breast milk biomonitoring and delineated the risk profile associated with chemical mixture exposure.