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

ABSTRACT 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‑MS 3 ) method was developed and fully validated for the quantification of pyraclostrobin in zebrafish liver tissue. The assay utilized an MS 3 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 ( R 2 ≥ 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‑MS 3 for strobilurin analysis in aquatic tissues, providing a reliable high‑throughput platform for residue monitoring and mechanistic toxicology research.