Abstract: A modified QuEChERS coupled with high performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method was developed for the determination of two nitromidazoles and one metabolites in fish. Key parameters that affected impurity removal and recovery of samples were optimized including the sample extraction solvent and amount of purifying agent ZrO₂. The results showed that, the optimal sample pretreatment conditions were achieved as follows:Samples were extracted with acetonitrile contained 0.1% ammonium hydroxide, and then purified with 100 mg of PSA, 40 mg of C₁₈, 600 mg of anhydrous magnesium sulfate and 20 mg zirconium dioxide. The optimal conditions were achieved as follows:The analysis was performed by an HPLC-MS/MS system with C₁₈ (2.1 mm×50 mm) column, the mobile phase consisted of 0.1% acetonitrile and 0. 1% formic acid solution by gradient elution, and multiple reaction monitoring (MRM) mode with positive electrospray ionization was used. The R² of three drugs were higher than 0.998 in the linear range of 0.5~20 ng/mL. At three spiked levels (n=6), the percentage of drugs with recoveries ranging from 88.1% to 107.0%, and the relative standard deviations (RSDs) of 2.43%~8.17%. The limits of detection (LODs) were 0.2~0.5 μg/kg and the limits quantification (LOQs) were 0. 6~1.2 μg/kg.Conclusion:This method was accurate and rapid, and could be widely used for the quantitative detection of nitromidazoles in fish.