Ultra‐Selective Ultra‐High‐Performance Liquid Chromatography‐Triple‐Stage Mass Spectrometry Assay for Quantification of Pyraclostrobin in Zebrafish Liver Tissues Via Multistage Fragmentation to Enhance Sensitivity

Pyraclostrobin, a widely applied methoxyacrylate fungicide, poses considerable risks to aquatic ecosystems due to its environmental persistence and bioaccumulation potential. Understanding its tissue-specific residue profile in aquatic organisms such as zebrafish is essential for elucidating ecotoxicological mechanisms and evaluating environmental safety. In this study, a novel, rapid, and sensitive ultra‑high‑performance liquid chromatography-triple‑stage mass spectrometry (UHPLC‑MS³) method was developed and fully validated for the quantification of pyraclostrobin in zebrafish liver tissue. The assay utilized an MS³ transition (m/z 388.0 → 194.0 → 164.0), which significantly enhanced selectivity and sensitivity. The method achieved fast separation (3 min per sample) and exhibited excellent linearity from 1 to 300 ng/mL (R² ≥ 0.998). Precision (intra‑ and inter‑day coefficient of variation <10.2%), recovery (94.4%-95.4%), and matrix effects (99.2%-108.9%) all met rigorous bioanalytical criteria. This work represents the first application of UHPLC‑MS³ for strobilurin analysis in aquatic tissues, providing a reliable high‑throughput platform for residue monitoring and mechanistic toxicology research.