Bioanalysis of Ametryn by UHPLC‐MS ³ Coupled With Multiple Fragmentation to Decrease Interference and Enhance Sensitivity

Rationale Ametryn poses significant risks to aquatic ecosystems, but conventional MRM methods suffer from matrix interference and limited sensitivity. To overcome these limitations, we established a more selective and sensitive UHPLC‐MS 3 approach that effectively suppresses background noise and enhances signal‐to‐noise ratio, enabling reliable quantification of ametryn in zebrafish tissues. Methods A novel UHPLC‐MS 3 method with electrospray ionization was developed. Quantification employed the MS 3 transition m/z 228.0 → 186.1 → 158.0. Five zebrafish tissues (liver, muscle, brain, intestine, gill) were analyzed following sample preparation. Fast chromatographic separation was achieved in 2.8 min. Results Compared with MRM, the MS 3 method substantially reduced matrix interference, enhanced signal intensity from 6.4 × 10 4 to 1.6 × 10 7 cps, and increased the signal‐to‐noise ratio from 6.3 to 45. The assay exhibited excellent linearity (10–1000 pg/mL), and validation results for accuracy, precision, recovery, matrix effects, and stability all met FDA bioanalytical requirements. Conclusions This study presents the first application of UHPLC‐MS 3 for quantitative analysis of ametryn in multiple zebrafish tissues, providing a reliable high‐throughput platform for residue monitoring and mechanistic toxicology research.