Development of a sequential digestion workflow for dual-platform microplastic analysis in pasteurized donor human milk using µ-FTIR and Py-GC/MS.

Reliable determination of microplastics in complex biological matrices remains analytically challenging because of high lipid and protein content, low particle abundance, and the risk of polymer degradation during sample preparation. This study developed and evaluated a sequential digestion and extraction workflow for isolating and characterizing microplastics in pasteurized donor human milk (PDHM). The workflow combined hexane-based lipid removal, proteinase K digestion, and controlled hydrogen peroxide treatment. Particle-count recovery was assessed using polypropylene (PP), polyethylene terephthalate (PET), and nylon 6,6 fragments measuring 0.8-1.3 mm. Recoveries ranged from 96.3% to 100%, indicating minimal loss of these fragments during digestion and filtration. The recovered fragments were analyzed by µ-FTIR. Py-GC/MS-based mass recovery was not evaluated because the dimensions of the recovered reference fragments were incompatible with the capacity of the pyrolysis sample cup, precluding their quantitative transfer and analysis. Blank-corrected microplastic abundance in PDHM ranged from 2.3 to 15.3 particles per 30 mL, equivalent to 76.7-509.9 particles L⁻1. µ-FTIR identified polyamide (PA), polyethylene (PE), PET, polyurethane (PU), polytetrafluoroethylene (PTFE), polystyrene (PS), poly(methyl methacrylate) (PMMA), and rayon, predominantly in the <100 µm size class. Py-GC/MS analysis indicated polycarbonate at 1.5 µg per sample in two of the three replicates, whereas the remaining replicate was below the laboratory reporting limit. This finding was interpreted as preliminary because independent peak-level confirmation was not possible using the analytical information available. The developed workflow provides a basis for microplastic isolation and particle-level characterization in PDHM but requires further size- and polymer-specific validation.