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

Abstract

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, 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, with accuracy between -3.33% and 3.67% and precision (CV) within 5.57-10.19%. 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. Keywords: Azoxystrobin; Zebrafish; UPLC-MS3; Zebrafish tissues.