Abstract: To solve the blocking problem the current official methods，a new HPLC method for the determination of methacycline and its related substances in the bulk material of methacycline hydrochloride and its preparations was established.The method was carried out using a column packed with octadecysilane bonded silica gel and a mixture of the buffer solution containing 100 volumes of 0.25 mol/L ammonium acetate solution，10 volumes of 0.1 mol/L ethyldiaminetetraacetic acid disodium salt solution and 1 volume of triethyamine adjusted pH to 8.3 with glacial acetic acid -acetonitrile (87∶13) as the mobile phase，and with a column temperature of 35 ℃，a detection wavelength of 280 nm，a flow rate of 1 mL/min.A good resolution with baseline separation was obtained.The linear range was 0.02-0.14 mg/mL.The limit of detection was 0.01 μg/mL，and the limit of quantity was 0.03 μg/mL.The average recovery data of the tablet and capsule methods were 99.3%(n=9，RSD=0.5%) and 99.7%(n=9，RSD=0.5%)，respectively.Methacycline was stable in 0.01 mol/L hydrochloric acid solution for more than 24 h.The developed method is better than the current methods of China Pharmacopeia 2005(ChP 2005) and United States Pharmacopeia 32nd ed and suitable for the determination of methacycline and its related substances in the bulk material of methacycline hydrochlorid and its preparations.