Development and validation of an improved method for the analysis of vancomycin by liquid chromatography: Selectivity of reversed-phase columns towards vancomycin components

The current method prescribed in official monographs for the purity control of vancomycin is inappropriate in that several components are not separated from each other and other components are coeluted with the main component vancomycin B. The method uses an ODS column at pH 3.2. In this study, several changes were introduced in order to improve the separation. The optimization of the separation method at low pH indicated that pH 1.7 was optimum and that the use of dioxane as organic modifier drastically improved the separation. These conditions were used to test a set of more than 40 reversed-phase columns for their selectivity towards vancomycin components. The selection of the most suitable columns was performed by means of principal component analysis. Most of these columns did not allow the separation of didechlorovancomycin from monodechlorovancomycin 1. It was found that neutral to slightly alkaline mobile phases allowed better separation. Further optimization of the separation method and a robustness study were performed by means of experimental design. This optimization indicated that pH 7.7 was optimum and gradient elution was also used to effect complete analysis. The final method uses a Kromasil column and the mobile phase comprises dioxane, water and ammonium formate solution pH 7.7. The separation of monodechlorovancomycin 2 and of some unknown impurities from the main component vancomycin B is described for the first time. The method shows good repeatability, linearity and sensitivity.