Development and Application of a UPLC–MS ³ Platform for the Ultratrace Analysis of Clothianidin in Zebrafish

ABSTRACT Rationale Neonicotinoid insecticides like clothianidin pose significant ecological risks due to their environmental persistence and toxicity to nontarget organisms. Accurate monitoring of ultratrace residues in complex biological matrices is essential for risk assessment. This study aims to develop a highly selective analytical platform to overcome matrix interferences and sensitivity limitations inherent in traditional methods. Methods A novel method was developed using ultrahigh performance liquid chromatography coupled with triple‐stage mass spectrometry (UPLC–MS 3 ) on a QTRAP 6500+ system. Clothianidin was extracted from zebrafish tissues via protein precipitation with acetonitrile. The MS 3 transition ( m/z 250.1 → 168.9 → 110.0) was optimized to enhance the signal‐to‐noise ratio compared with conventional multiple reaction monitoring (MRM). Results The UPLC–MS 3 method provided a 13‐fold improvement in signal‐to‐noise ratio (713.5) over MRM (54.5). The limit of quantification was 0.05 ng/mL across all tissues. Validation showed excellent linearity ( r > 0.995), precision (CV < 8.4%), and recovery (90.64%–111.98%). Exposure studies revealed tissue‐specific bioaccumulation, with the highest clothianidin concentrations found in muscle (6.06 ng/mg) and heart (5.42 ng/mg). Conclusions The developed UPLC–MS 3 platform offers unparalleled sensitivity and specificity for trace‐level monitoring of clothianidin in complex biological systems. These findings provide a robust tool for investigating the environmental fate and toxicological mechanisms of neonicotinoids in vertebrates.