ABSTRACT
We described the main pathways of bacitracin (Bc) decomposition, chromatographically set the position of its major degradation products and evaluated microbiological activity of isolated components of Bc and its degradation products. All processes of Bc decomposition under stress and accelerated test conditions were monitored with HPLC, performed mainly on a new type reversed-phase (RP-18e) monolithic silica column (Chromolith) enabling fast separation times and some of them also on conventional HPLC columns. Diode array detection, preparative HPLC and FAB mass spectrometry were used for identification of individual Bc components. We found that the major decomposition mechanism in water solutions of Bc is oxidation, and in alkaline solutions, deamidation. In oxidation process the components B1, B2 and B3 and A are oxidized into their corresponding oxidative products H1, H2, H3 and F respectively by the same mechanism. A detailed study of oxidative degradation products revealed that HPLC separation with an acid mobile phase caused splitting of peaks of components H2, H3 and F into two peaks but the peak of component H1 did not split due to its special structural properties. For the component A we confirmed gradual formation of desamido product through an intermediate. We found oxidative degradation products of Bc to be relatively stable, and desamido degradation products to be rather unstable. The estimation of kinetics of Bc decomposition was presented with a semi-quantitative model. Microbiological activity of individual isolated active components of Bc was established and the negligible antimicrobial activity of the degradation products was confirmed.
Subject(s)
Anti-Bacterial Agents/chemistry , Bacitracin/chemistry , Peptides/chemistry , Amides/metabolism , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Bacitracin/metabolism , Bacitracin/pharmacology , Chromatography, High Pressure Liquid/methods , Drug Stability , Fermentation , Hydrochloric Acid/chemistry , Hydrochloric Acid/metabolism , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Mass Spectrometry/methods , Microbial Sensitivity Tests/methods , Micrococcus luteus/drug effects , Micrococcus luteus/growth & development , Micrococcus luteus/metabolism , Molecular Structure , Oxidation-Reduction , Peptides/metabolism , Peptides/pharmacology , TemperatureABSTRACT
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.
