Discriminating microplastics from lipid artifacts in the human brain: refined digestion and multi-analytical strategies to overcome matrix effect challenges in neuro-toxicology

The detection of micro- and nanoplastics (MNPs) in the human brain is a critical frontier in neurotoxicology, yet analysis is severely hindered by the brain’s complex, lipid-rich matrix. Recent concerns regarding “false positive” signals from endogenous fats necessitate the development of rigorous, validatable pretreatment protocols. This study optimized pretreatment procedures for post-mortem human dorsal thalamus samples. We compared oxidative (H₂O₂), alkaline-oxidative (NaOH/H₂O₂), and acidic microwave-assisted (HNO₃) digestion. MNP presence and polymer integrity were cross-validated using a multi-analytical suite: Laser Direct Infrared (LDIR) imaging, Optical Photothermal Infrared (O-PTIR) spectroscopy, SEM-EDS, and MALDI-TOF MS. Lipid removal efficiency was quantitatively assessed via GC-MS. Standard oxidative or alkaline digestions were found insufficient, as they promote the formation of insoluble zinc and calcium lipid salts (soaps) that mimic polymer spectra. A critical methodological advancement was achieved by incorporating an ethanol-assisted solvent stage and a 5% acetic acid filter rinse. This dual-action approach significantly facilitated the removal of recalcitrant fatty compounds (e.g., C16:0) and dissociated lipid-metal complexes. While acidic microwave digestion provided rapid matrix destruction and high filtration rates, it caused unacceptable degradation of synthetic polymers (e.g., PS, PET), as confirmed by MALDI-TOF signals in the filtrates. The optimal protocol for maintaining polymer integrity while ensuring a clean matrix was identified as a two-step process: (1) 0.05 M NaOH/30% H₂O₂ (1:2.5, v/v) at 50 °C, followed by (2) ethanol (1:1, v/v) addition and a 5% acetic acid rinse. This study provides a refined analytical framework for neurotoxicological research, ensuring that the reported biodistribution of environmental contaminants in the central nervous system is free from endogenous organic interference. Not applicable.