Comparative evaluation of enzymatic, acidic, and alkaline digestion protocols for organic matrix removal from lyophilized and frozen mussel samples (Mytilus galloprovincialis).

Efficient removal of organic matter from biological matrices is a critical step in the analysis of microplastics in marine organisms. In this study, enzymatic (Kreon®25,000), acidic (HNO₃ + H₂O₂), and alkaline (KOH + H₂O₂) digestion protocols were comparatively evaluated for the treatment of lyophilized and frozen Mytilus galloprovincialis samples. Digestion efficiency was assessed gravimetrically, while the effects of the protocols on polymer integrity were examined using ATR-FTIR, HQI analysis, and microscopic observations on representative polymers (HDPE, PA, PET, PVC). All three methods demonstrated high digestion efficiencies (>96%). Acidic digestion provided rapid and stable removal of organic matter within 20 min, whereas enzymatic digestion required longer incubation times (2-24 h) but exerted the least impact on polymer integrity. Frozen samples consistently showed slightly higher digestion efficiencies compared to lyophilized ones, likely due to preserved tissue hydration facilitating reagent penetration. Microscopic and spectroscopic analyses revealed that HDPE and PET maintained their structural and chemical integrity under all treatments, whereas PA and PVC exhibited surface alterations after acidic digestion. Enzymatic and alkaline protocols did not produce visible or spectral changes in any polymer type. Based on these findings, the enzymatic protocol was selected for recovery experiments. Mass-corrected recovery values ranged from 92.87% to 95.36% for PA, PET, and PVC, and 75.69% for HDPE, indicating that the method allows effective isolation of most polymers while preserving their integrity. The results demonstrate that although all digestion methods are efficient in removing organic matter, enzymatic digestion provides the most reliable approach for microplastic analysis in Mytilus galloprovincialis, ensuring both high digestion efficiency and preservation of polymer characteristics.