Abstract: Aim ：To establish an HPLC method for the determination of secnidazole and its related substances，and to elucidate the structure of the main related substances. Methods ：HPLC was performed on an Agilent C₁₈(150 mm×4.6 mm，5 μm)column.The mobile phase consisted of acetonitrile and 0.1% formic acid solution(10∶90)．The detection wavelength was set at 320 nm and the column oven was maintained at 30 °C.The mass spectrometry detector was equipped with an ESI source with a temperature of 350 °C，and set at positive ion monitoring model.The spray chamber pressure was 0.34 MPa. The drying gas flow rate was 10 L/min. Results ：Secnidazole was completely separated from the impurities.The calibration curve of secnidazole was linear over the range of 0.10-1 500.0 μg/mL with the correlation coefficient of 0.999 9.The contents of secnidazole in the samples were 100.5%，100.3% and 100.7%，respectively，and those of the related substances were 0.463%，0.488% and 0.465%，respectively.The three related substances were identified by HPLC-UV-MSn as 2-methyl-5-nitroimida-zole and the isomerides of secnidazole，respectively. Conclusion ：The developed method is reliable for the quality control and determination of related substances of secnidazole.