Abstract：Objective The purification and identification of minor components in colistin E. Methods Each single component was purified by using reverse-phase chromatography twice, and derivatization using the reagent FDAA was performed to analyze the amino acid composition and the stereochemistry of each component. Structural identification was based on the secondary mass spectrometry and amino acid composition. Results The amino acid composition and molecular weight of colistin E2 were consistent with those published reports. Both impurities 3 and 9 contained L-Leu residues and hydroxylated fatty acid chains. The impurity 6 contained an L-Val residue. Conclusion Impurity 3, impurity 6, and impurity 9 were 3-OH-E2, Val-E2, and 3-OH-E1, respectively.