Separation and isolation of the isomers of bacitracin by high-performance liquid chromatography and their relationship to microbial activity.

Bacitracin, a polypeptide antibiotic produced from strains of Bacillus licheniformis, is one of the most commonly used antibiotics in the world. Actually, the various products generally referred to as 'bacitracin' are mixtures of similar polypeptides which may differ by only one amino acid. The approved method of analysis for bacitracin is microbial. To correlate the microbiological method with an HPLC method, bacitracin was chromatographed using a YMC basic column with UV detection. Adequate separation of the isomers were obtained to scale up this procedure to preparative HPLC using a 250 x 21 mm YMC basic column. The various fractions were separated, isolated and examined for microbial activity. The chromatograms can accurately predict in minutes the microbiologically-determined potency which usually takes 16-24 h to develop. The chromatographic procedure also provides information on the amounts of isomers and degradation products present in the sample, whereas the microbiological assay only provides activities or potencies of the antibiotic. The reported HPLC method also possesses some advantages over some other published HPLC methods in terms of accuracy and time of analysis.