HPLC结合组分制备分析杆菌肽中的有关物质及有效组分含量

Analysis of the Related Substances and Content of Effective Bacitracin by HPLC Combined with Component Preparation

目的建立基于HPLC结合组分制备对杆菌肽中有关物质及有效组分含量测定的分析方法。方法采用Agilent HPLC,选择Diamonsil Plus C18色谱柱(4. 6 mm×250 mm,5μm),以50 mmol·L^(-1)甲酸铵溶液(p H经甲酸调至4. 0)为流动相A,乙腈为流动相B,流速为1 m L·min-1,线性梯度洗脱,检测波长为254 nm。经调整甲酸铵溶液的p H、流动相比例、溶剂等确定了分析方法,开展了专属性、线性、精密度、重复性、稳定性、耐用性等方法学研究。根据半制备液相色谱技术收集了杆菌肽A、B1/B2/B3、C1/C2/C3、F(采用《美国药典》40版中杆菌肽液相分析方法),并在甲酸铵-乙腈液相体系中进行定位及确认。结果建立了适用于杆菌肽有关物质的液相分析方法,方法学试验结果良好。已知组分与相邻杂质之间实现良好分离,特别是B1/B2的分离,明确了各组分的洗脱位置。对比了不同杆菌肽样品中各组分的含量,能够提供更加详细的样品信息。结论该液相分析方法可同时测定杆菌肽中的有关物质及有效组分含量,适用于该产品的质量控制。

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 (4.6mm ×250mm,5 μm)using 50mmol ·L^-1 ammonium formate (pH adjusted to 4.0 with formie acid)as mobile phase A and aeetonitrile as mobile phase B at a flow rate of 1mL ·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 bacitraein 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 effeetive contents in baeitracin.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 bacitraein 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.