Fast separation of bacitracin on monolithic silica columns

Abstract

The development of isocratic and gradient stability indicating HPLC methods for bacitracin (Bc) and bacitracin zinc (BcZn), which are complex mixture of several related polypeptides, is described. The methods are based on a new type of reversed phase (RP-18e) monolithic silicagel stationary phase. Chromatographic experimental conditions used on conventional column with microparticles were adopted and further modified to achieve efficient separation of Bc. The influence of methanol and acetonitrile in combination with phosphate buffer was thoroughly studied to separate microbiologically active components A, B1, B2, B3 and their oxidative degradation products F, H1, H2 and H3. Chromatographic peaks of all the mentioned components were identified using compounds isolated previously by preparative HPLC. Applying isocratic or gradient approach, highly efficient separation was achieved together with drastically reduced analysis times (ca. 6 min) compared to all published HPLC methods up to date. With thus developed HPLC methods, it is possible to evaluate not only the main degradation product F, but for the first time also several other oxidative degradation products of Bc (H1, H2 and H3). Such methods are also suitable for routine quality control and stability testing. Validation of both isocratic and gradient methods confirmed the selectivity and efficiency comparable to that on microparticulate columns, yet contrary to conventional columns with highly reduced analysis time.