Separation of Ofloxacin and Its Six Related Substances Enantiomers by Chiral Ligand-Exchange Chromatography

A chiral ligand-exchange high-performance liquid chromatography method was developed for the enantioseparation of ofloxacin and its six related substances termed impurities A, B, C, D, E, and F. The separation was performed on a conventional C-18 column. Different organic modifiers, copper salts, amino acids, the ratio of Cu2+ to amino acid, pH of aqueous phase, and column temperature were optimized. The optimal mobile phase conditions were methanol-water systems consisting of 5mmol/L copper sulfate and 10mmol/LL-isoleucine (L-Ile). Under such conditions, good enantioseparation of ofloxacin and impurities A, C, E, and F could be observed with resolutions (R-S) of 3.54, 1.97, 3.21, 3.50, and 2.12, respectively. On the relationship between the thermodynamic parameters and structures of analytes, the mechanism of chiral recognition was investigated. It was concluded that ofloxacin and impurities A, C, E, and F were all enthalpically driven enantioseparation and that low column temperature was beneficial to enantioseparation. Furthermore, the structure-separation relationship of these analytes is also discussed. Chirality 27:843-849, 2015. (c) 2015 Wiley Periodicals, Inc.