Improved high-performance liquid chromatographic method for polypeptide antibiotics and its application to study the effects of treatments to reduce microbial levels in bacitracin powder

Abstract

Improvements were made in the high-performance liquid chromatographic (HPLC) method to obtain baseline separation of chromatographic peaks of structurally similar polypeptide components in bacitracin. The improved method uses a 30-cm-long stainless-steel column packed with μBondapak C 18. The theoretical plates of the column are approximately 140,000 per meter for the bacitracin A peak. The resolution function between bacitracins B 1 and B 2 and that between bacitracins A and B 2 have been improved 418 and 225%, respectively. The components of bacitracin, bacitracins A, B, C, D, E, F, and G, were fractionated by the countercurrent distribution technique. These components, together with Compound X, a compound separated on a carboxymethylcellulose column, and bacitracin F, obtained by degrading bacitracn A sample at neutral pH, were used to identify peaks in the HPLC chromatogram. Effects of processing methods used to reduce microbial contamination levels in bacitracin powders were evaluated. Heat treatment caused a significant loss of anti-microbial activity (35% reduction), bacitracins A, B 1, and B 2 were reduced by 37, 22, and 21%, respectively. A significant increase (2.8 times) of bacitracin F, an oxidative degradation compound, was shown. Irradiation by 60Co at 1.8 Mrad caused no loss of potency nor change in any of the bacitracin components. Ethylene oxide treatment, on the other hand, aused considerable (46%) reduction of potency. Substantial reduction of areas under the peaks of bacitracins A, B 1, and B 2 (50, 24 and 37%, respectively) were noted. The chromatograms showed numerous unresolved peaks around bacitracins A, B 1, and B 2; however, no significant increase in the bacitracin F peak, nor appearance of non-UV absorbng peaks were observed. Peptide antibiotics of the polymyxin group, circulin, colistin, and polymyxin, were also analyzed using the μBondapak C 18 column with a linear-gradient elution. A UV monitor was used for polymyxin. A moving-wire flame ionization detector was used to monitor circulin and colistin. A sample of polymyxin, circulin, and colistin may be analyzed in less than 20 min of chromatographic time.