A sequential H₂O₂–KOH digestion protocol for microplastic extraction from cured fish matrices: development, optimization and validation

Reliable isolation of microplastics (MPs) from cured fish products remains challenging because their complex matrices, characterized by high lipid, protein, salt, and mineral contents, hinder organic matter digestion and compromise polymer recovery. In this study, a sequential hydrogen peroxide (H₂O₂)–potassium hydroxide (KOH) digestion protocol was developed and optimized for efficient MPs extraction from diverse cured fish matrices. A preliminary evaluation of different H₂O₂:KOH ratios identified 30:70 (v/v) as the optimum reagent combination, while Response Surface Methodology (RSM) coupled with a Central Composite Design optimized digestion temperature and reagent volume. The developed quadratic models showed high predictive performance (R² = 0.9515 for digestion efficiency and R² = 0.9785 for filtration time) with non-significant lack-of-fit. The optimum conditions (60 °C and 36.39 mL g⁻¹ reagent volume) achieved 99.33% digestion efficiency and a filtration time of 11.33 s. The optimized protocol was subsequently validated using four cured fish matrices with digestion efficiencies exceeding 96%. Micro-Raman spectroscopy confirmed efficient removal of lipid-associated spectral signatures following digestion, while gravimetric measurements, microscopic observations, dimensional analysis, and Raman spectral characterization demonstrated that the optimized sequential H₂O₂–KOH treatment preserved the mass, morphology, size, and chemical integrity of polypropylene, polyethylene, polyvinyl chloride, polyethylene terephthalate, polystyrene, and polyamide particles. The proposed matrix-adapted digestion protocol provides a rapid, reliable, and reproducible approach for MPs isolation from cured fish products and establishes a standardized optimization framework that can facilitate method development for other complex food matrices.