Nanoplastics (NPs) are cause for increasing concern due to their ability to cross biological barriers, enhanced reactivity, and potentially greater toxicity than microplastics. However, their quantification in biological samples such as animal tissues remains challenging, primarily due to complex matrix interference and contamination risks. Reliable quantification of NPs in tissues is critical for understanding their bioavailability, bioaccumulation, and associated toxicological risks to animals, as well as their potential ecological risks. Here, we developed a simple yet robust method for quantifying polystyrene (PS) NPs in animal tissues using silica gel column chromatography coupled with pyrolysis–gas chromatography/mass spectrometry. Tissue samples were homogenized in dichloromethane (DCM) to extract and dissolve PS NPs in DCM and then subjected to alkaline digestion. The extract was subsequently purified using pre-heat-treated silica gel columns. The method showed high recovery rates (102.0 % and 91.2 % at 0.3 and 1.7 µg g⁻¹, respectively), with limits of detection and quantification of 3.0 ng and 7.8 ng, respectively. Validation with liver samples from Japanese quail orally exposed to 50 nm PS NPs revealed significantly elevated levels of PS NPs on Days 1 and 3 post-exposure (33.8 ± 1.5 and 34.1 ± 5.2 ng g⁻¹) compared to controls (13.2 ± 0.3 and 14.5 ± 2.0 ng g⁻¹) (p < 0.005). This method demonstrates effective removal of biological matrix components, enabling accurate and reproducible quantification of PS NPs in avian liver tissues. While the application to other biological samples will be explored in future studies, the method’s simplicity and minimal contamination make it a valuable tool for advancing risk assessments of NPs in biological systems.
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