Abstract: OBJECTIVE To detected the related substances in carbazochrome sodium sulfonate and indentified structure to provide reference for process optimization and quality control. METHODS The separation of related substances in forced degradation solution of carbazochrome sodium sulfonate was carried out by using a Shim-pack Scepter C₁₈(4.6 mm×250 mm, 5 µm) with 50 mmol·L^-1 ammonium acetate solution as mobile phase A and acetonitrile as mobile phase B. MS was performed on quadrupole time of flight high-resolution mass spectrometry under positive and negative mode with the mass range of m/z 100-800, MS² was acquired under data dependent acquisition mode. Combined with the exact molecular mass, fragmentation rule and synthesis process, the possible structures of related substances were deduced. RESULTS Eight major impurities were found in carbazochrome sodium sulfonate degradation solutions. Among the identified impurities, 2 were reported and 6 were firstly reported. The force degradation test proved that carbazochrome sodium sulfonate was relatively stable on high temperature and oxidation conditions, while light, strong acids and strong alkali had a greater impact on carbazochrome sodium sulfonate. Related substances 5, 6 and 8 were detected in carbazochrome sodium sulfonate raw materials. Impurity 8 was the main degradation impurity by high temperature. CONCLUSION HPLC method combined with flight high resolution mass spectrometry established in this study has good separation and identification of related substances in carbazochrome sodium sulfonate, which provides a reference for its production process and quality control.