Abstract: Objective High performance liquid chromatography was used to study the related substances of adenosine cobalt amine for injection, and mass spectrometry analysis and toxicity prediction were performed on the related impurities. Methods Determination by high-performance liquid chromatography, the chromatographic conditions were using octadecylsilane bonded silica gel as the filling agent in the chromatographic column, with a detection wavelength of 260 nm, a column temperature of 35℃, and an injection volume of 10 μL. 0.01 mol·L^-1 ammonium acetate solution (adjusted to pH 4.0 with glacial acetic acid) was used as mobile phase A, and methanol was used as mobile phase B for gradient elution. The established method was used to evaluate the related substances in 105 batches of adenosine cobalt amine for injection. Unknown impurities with a content of ≥0.1% were scanned by high-resolution mass spectrometry and genotoxicity prediction was performed on the unknown impurities. Results The impurity peaks of the established method were effectively separated from the main peak, with a detection limit of 1.497×10^-4 mg·mL^-1. The methodological validation of the detection method was conducted, and the results met the requirements of the pharmacopoeia. Conclusion The established method has strong specificity, high accuracy, good repeatability, and can effectively obtain comprehensive impurity information. It is suitable for impurity detection and control of adenosine cobalt amine for injection.

Key words: adenosine cobalt amine for injection, related substances, high-performance liquid chromatography, mass spectrometry, impurity, methodology