Enhanced Sensitivity and Selectivity for the Quantification and Organ-Specific Distribution of Imidacloprid in Zebrafish Tissues via UHPLC–MS

Neonicotinoid insecticides, such as imidacloprid, pose significant risks to aquatic ecosystems due to their widespread use and potential for accumulation. In this study, a sensitive and robust analytical strategy based on ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS 3 ) was developed and validated for the determination of imidacloprid in multiple complex zebrafish biological matrices, including the muscle, liver, intestine, heart, brain, and gills. By harnessing the unique selectivity of the MS 3 acquisition mode, the method effectively eliminated endogenous matrix interferences and achieved a 243-fold enhancement in signal intensity compared to the conventional multiple reaction monitoring (MRM) mode. The sample pretreatment utilized a streamlined protein precipitation approach with methanol, ensuring high extraction efficiency and reproducibility across all tissues. The method was fully validated according to FDA guidelines, demonstrating excellent linearity, accuracy, and precision. Furthermore, the practical application of this assay in a tissue distribution study revealed a non-uniform accumulation pattern of imidacloprid, with the highest concentrations observed in the muscle and intestine, and the lowest in the heart. These findings provide a powerful tool and essential empirical data for further biodistribution investigations and environmental risk assessments of trace-level contaminants in micro-gram level biological samples.