Development of a validated UPLC-MS³ method for trace-level azoxystrobin determination in zebrafish liver tissues

Azoxystrobin is a widely used strobilurin fungicide. Its environmental persistence and potential toxicity to aquatic organisms demand accurate trace-level quantification in biological tissues. However, its sensitive and specific determination in small tissue samples using conventional methods remains challenging. In this study, a novel high-sensitivity analytical method based on ultra-performance liquid chromatography coupled with triple-stage mass spectrometry (UPLC-MS³) was established for the trace determination of azoxystrobin in zebrafish liver tissues. Following protein precipitation extraction, chromatographic separation was performed on a C₁₈ column using a gradient of 0.1% aqueous formic acid and acetonitrile. Detection relied on an optimized MS³ transition (m/z 404.0 → 371.9 → 344.2). The method exhibited excellent linearity (r > 0.9984) from 0.1 to 20 ng mL^-1, with accuracy between -3.33% and 3.67% and precision (CV) between 5.57 and 10.19%. Consistent recoveries (94.93-106.64%) and minimal matrix effects (99.45-104.79%) were achieved across all tissue matrices. Compared to conventional MRM, MS³ scanning significantly enhanced specificity by reducing endogenous interference. The validated approach was successfully applied to tissue distribution studies in zebrafish, confirming its reliability for environmental toxicology research and providing a robust platform for investigating fungicide biodistribution in aquatic organisms.