Ultra-sensitive quantification of fipronil in zebrafish tissues via UPLC-MS³

A sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry cubed (UPLC-MS³) assay was established for detection of fipronil in zebrafish tissue samples. Following protein precipitation pretreatment, the concentrations of fipronil in various zebrafish tissue samples were quantified by the developed UPLC-MS³ assay. Fipronil was monitored by MS³ transition (m/z 435.0 → 398.9 → 330.0) with a ± 1.0 Da isolation window for secondary product ions. Chromatographic separation was achieved with 0.1 % formic acid-water (v/v) as the aqueous phase and acetonitrile as the organic phase. Gradient elution with C₁₈ column was employed for separation of fipronil and probenecid (internal standard, IS). Method validation demonstrated excellent linearity over the range of 0.1-10 ng/mL (r² > 0.995), with the accuracies ranging from -10.33 % to 3.17 % and precisions between 3.25 % and 11.34 %. Consistent extraction recoveries (89.92-113.49 %) and acceptable matrix effects (92.54-110.27 %) were observed across all tested matrices. The implementation of MS³ scanning significantly enhanced specificity by eliminating endogenous interference compared to conventional MRM detection. The successful application of this method in zebrafish tissue distribution studies confirmed its reliability for environmental toxicology research. This MS³-based approach provides a robust analytical platform for investigating biodistribution of fipronil in aquatic models.