Abstract: For quantitative analysis of related substances in TSD-1 active pharmaceutical ingredient, structures of prepared impurities were confirmed by NMR and UHPLC-MS, and a high performance liquid chromatographic method was established to determine the related substances in TSD-1. The analytical column was an Agilent ZORBAX Eclipe XDB-C8 (250 mm×4.6 mm, 5 μm). The mobile phase A was 50 mmol·L^-1 ammonium acetate solution (adjusted pH to 5.8 with acetic acid) and the mobile phase B was acetonitrile. The whole run was carried out by gradient elution at a flow rate of 1.0 mL·min^-1. The detection wavelength was set at 240 nm and the column temperature was 30 ℃. The resolutions among peaks of TSD-1, impurity A, impurity B, TSD-D, and TSD-F were good. The calibration curves (n = 7) of TSD-1, impurity A, impurity B, TSD-D and TSD-F were linear in their respective weight ranges of 0.242-48.4 μg·mL^-1 (r = 1.000 0), 0.244-9.75 μg·mL^-1 (r = 0.999 9), 0.244-4.80 μg·mL^-1 (r = 0.999 9), 0.254-1.02 μg·mL^-1 (r = 0.999 9), and 0.247-0.987 μg·mL^-1 (r = 0.999 9). The lower limits of quantitation were 0.244, 0.244, 0.254, and 0.247 μg·mL^-1 for impurity A, impurity B, TSD-D, and TSD-F, respectively, and the average recovery of each impurity ranged from 99.08% to 103.00% with high accuracy. TSD-D and TSD-F were not detected in the three batches of TSD-1 active pharmaceutical ingredients, and impurity A and impurity B were not detected beyond the limit. The established HPLC method is simple, accurate, and suitable for determination of related substances of TSD-1, which can provide a valuable reference for the subsequent development of TSD-1.