Abstract: By silica gel column chromatography, solvent extraction and preparative high performance liquid chromatography (HPLC), four new related substance were isolated and purified from the mass production and preparation process of alogliptin benzoate. Then it was analyzed and confirmed by various spectrum identification methods such as nuclear magnetic resonance (NMR) spectroscopy, high-resolution mass spectrometry (HR-MS) and Fourier-transform infrared spectroscopy (FTIR) according to its physical and chemical properties. The chemical structures of the four related substances produced in each step of the synthesis process of alogliptin benzoate were determined, and they were named as impurities L, M, T, and V. These four related substances were new impurities which were found for the first time. The isolation and identification of these impurities are of great importance to the quality control of alogliptin benzoate, and the optimization of manufacturing process.