Abstract: The aim of the current study was to establish an HPLC method for the determination of related substances of milrinone injection, and identify the structures of the main impurities by spectral techniques. The structural identification of impurity B was done for the first time. The separation was performed on a Waters Symmetry C₁₈ column(250 mm×4. 6 mm, 5 μm), and the mobile phase was phosphate buffer-methanol(75 ∶25). The detection wavelength was 254 nm, and the flow rate was 1. 0 mL/min. Milrinone and its related substances were separated, and the calibration curves were linear over the concentration ranges of 0. 05-3. 2 μg/mL(r=0. 999 5)for milrinone, 0. 05-3. 2 μg/mL(r=0. 999 8)for milrinone impurity A, and 0. 05-3. 2 μg/mL(r=0. 999 4)for milrinone impurity B, respectively. The LOD values of milrinone, milrinone impurity A and milrinone impurity B were all 0. 017 μg/mL. This method is simple, sensitive and specific. It is suitable for the quality control of milrinone injection.