Tsuji K, Robertson J H
J Chromatogr. 1975 Oct 29;112:663-72. doi: 10.1016/s0021-9673(00)99995-3.
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-stell column packed with muBondapak C18. The theoretical plates of the column are approximately 140,000 per meter for the bacitracin A peak. The resolution function between bacitracins B1 and B2 and that between bacitracins A and B2 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 bacitracin 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 antimicrobial activity (35% reduction), bacitracins A, B1, and B2 were reduced by 37, 22, and 21%, respectively. A significant increase (2.8 times) of bacitracin F, an oxidative degradation compound, was show. 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, caused considerable (46%) reduction of potency. Substantial reduction of areas under the peak of bacitracins A, B1, and B2 (50, 24 and 37%, respectively) were noted. The chromatograms showed numerous unresolved peaks around bacitracins A, B1 and B2,; however, no significant increase in the bacitracin F peak, nor appearance of non-UV absorbing peaks were observed. Peptide antibiotics of the polymyxin group, circulin, colistin, and polymyxin, were also analyzed using the muBondapak C18 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.
对高效液相色谱（HPLC）方法进行了改进，以实现杆菌肽中结构相似的多肽成分色谱峰的基线分离。改进后的方法使用一根30厘米长、填充有μBondapak C18的不锈钢柱。对于杆菌肽A峰，该柱的理论塔板数约为每米140,000。杆菌肽B1和B2之间以及杆菌肽A和B2之间的分离度函数分别提高了418%和225%。采用逆流分配技术对杆菌肽的成分，即杆菌肽A、B、C、D、E、F和G进行了分离。这些成分与在羧甲基纤维素柱上分离得到的化合物X以及通过在中性pH下降解杆菌肽A样品获得的杆菌肽F一起，用于识别HPLC色谱图中的峰。评估了用于降低杆菌肽粉末中微生物污染水平的加工方法的效果。热处理导致抗菌活性显著丧失（降低35%），杆菌肽A、B1和B2分别降低了37%、22%和21%。氧化降解产物杆菌肽F显著增加（增加2.8倍）。用60Co以1.8兆拉德进行辐照不会导致效力丧失，也不会使任何杆菌肽成分发生变化。另一方面，环氧乙烷处理导致效力大幅降低（46%）。注意到杆菌肽A、B1和B2峰下的面积大幅减少（分别为50%、24%和37%）。色谱图显示在杆菌肽A、B1和B2周围有许多未分离的峰；然而，未观察到杆菌肽F峰显著增加，也未出现非紫外吸收峰。还使用μBondapak C18柱和线性梯度洗脱对多粘菌素类的肽抗生素、环杆菌素、粘菌素和多粘菌素进行了分析。对多粘菌素使用紫外监测器。使用移动丝火焰离子化检测器监测环杆菌素和粘菌素。多粘菌素、环杆菌素和粘菌素的样品可在不到20分钟的色谱时间内进行分析。
