ABSTRACT
OBJECTIVE: To develop a suitable HPLC method combined with component preparation to analyze the related substances and effective contents in bacitracin.METHODS: The separation was performed on Agilent HPLC system with a Diamonsil Plus C18 column (4.6 mm×250 mm, 5 μm) using 50 mmol•L-1 ammonium formate (pH adjusted to 4.0 with formic acid) as mobile phase A and acetonitrile as mobile phase B at a flow rate of 1 mL•min-1 under gradient elution. The detection wavelength was set at 254 nm. The HPLC method was carried out by adjusting the pH of ammonium formate solution, the ratio of mobile phase and the solvent. Furthermore, the specificity, linearity, precision, repeatability, stability and durability were studied. The known components like bacitracin A, B1/B2/B3, C1/C2/C3 and F were collected by preparative HPLC according to the HPLC method for bacitracin in USP 40. They were positioned in ammonium formate-acetonitrile HPLC system.RESULTS: The established HPLC method was verified to be suitable for analyzing the related substances and effective contents in bacitracin. Baseline separation between the known components and adjacent impurities was achieved, especially the B1/B2. The elution positions of the known component were clearly defined. The contents of the known components in different batches of bacitracin were compared. More detailed sample information was provided in this research.CONCLUSION: The HPLC method can simultaneously analyze the related substances and effective contents in bacitracin, and is suitable for the quality control of the product.