Abstract: AimTo study the four diastereomers of leucogen and structure of the related substances. MethodsLC-DAD, LC-MS/MS and sLC-¹H NMR were used. LC was carried out with a Phenomnex Luna C₁₈ (250 mm×4.60 mm ID, 5 μm) column and a mobile phase of water-acetonitrile-glacial acetic acid (58∶42∶0.3). ResultsThe structures of leucogen and its related substances were identified. Leucogen and the related substances were found to have four diastereomers in solution state separately. The stability and transformation of the four diastereomers were analyzed and 3r4s5r was found to be more stable than the others according to quantum calculations. ConclusionLeucogen have four diastereomers in solution state and it can transform from one diastereomer to the others, and the 3r4s5r is more stable than the others.