Optimization of HPLC method for stability testing of bacitracin.

A stability indicating HPLC assay for bacitracin has been developed and validated. The assay is based on a gradient elution, reversed phase column and UV diode array detection. On the basis of our previous analytical work several additional systematic HPLC tests for optimization of analytical method were performed. In order to achieve the highest selectivity of HPLC method, tests were conducted with extremely complex samples -- zinc bacitracin feed grade as food additive for animals. The influence of pH of mobile phase and type of columns on chromatographic separation of active (A, B(1) and B(2)) and inactive (F) polypeptide components of bacitracin were investigated in detail. It was found also that the peak B(1) comprises three and the peak F two subunits -- probably isomers. The obtained analytical procedure proved to be very selective and effective for the simultaneous determination of active polypeptide A, B(1) and B(2), impurities, known and unknown degradation products and ballast material.