An approach to on-line electrospray mass spectrometric detection of polypeptide antibiotics of enramycin for high-speed counter-current chromatographic separation

In the field of pharmaceutical and biomedical analysis of peptides, a rapid on-line detection and identification for a methodology have been required for the discovery of new biological active products. In this study, a high-speed counter-current chromatography with electrospray mass spectrometry (HSCCC/ESI-MS) was developed for the on-line detection and purification of polypeptide antibiotics of enramycin-A and -B. The analytes were purified on HSCCC model CCC-1000 (multi-layer coil planet centrifuge) with a volatile solvent of two-phase system composed of n-butanol/hexane/0.05% aqueous trifluoroacetic acid solution (43/7/50, V/V/V), and detected on an LCMS-2010EV quadrupole mass spectrometer fitted with an ESI source system in positive ionization following scan mode (m/z 100–2000). The HSCCC/ESI-MS peaks indicated that enramycin-A (major m/z 786 [M+3H]³⁺ and minor m/z 1179 [M+2H]²⁺) and enramycin-B (major m/z 791 [M+3H]³⁺ and minor m/z 1185 [M+2H]²⁺) have the peak resolution value of 2.9 from 15 mg of loaded enramycin powder. The HSCCC collected amounts of the peak fractions were additionally 4.3 mg (enramycin-A), and 5.9 mg (enramycin-B), respectively. These purified substances were analyzed by LC/ESI-MS with scan positive mode. Based on the LC/ESI-MS chromatograms and spectra of the fractions, enramycin-A and -B were estimated to be over 95% purity. The overall results indicate that this approach of HSCCC/ESI-MS is a powerful technique for the purification and identification of bioactive peptides.     

恩拉霉素高产菌株选育的研究#br#

目的 进一步提高恩拉霉素发酵生产水平。方法 对杀真菌链霉菌RN16-7进行ARTP(常压室温等离子体)等离子与庆大霉素复合处理。结果 经大量筛选，得到了恩拉霉素高产突变株RN16-242，其遗传性状稳定，发酵效价达7423u/mL，比出发菌株RN16-7(4703u/mL)提高了57.8%。菌株保存，采用甘油管低温保藏法、沙土管保藏法和冷冻真空干燥保藏法进行了比较，结果是甘油管低温保藏法最好。     

HPLC法测定预混剂中恩拉霉素的含量

目的采用高效液相色谱法测定饲料中恩拉霉素的含量。方法测定色谱条件为：色谱柱为C18（5um,4．6mm×250mm）,流动相为0．05mol／L磷酸二氢钠-乙腈（70：30）,检测波长为267nm。结果恩拉霉素A及恩拉霉素B在12．5～200ug/mL范围内线性关系良好,相关系数R。分别为0．9960和0．9945;恩拉霉素A的回收率为96．4％-101．6％,恩拉霉素B的回收率为96．8％～101．2％。恩拉霉素A及恩拉霉素B的RSD分别为0．65％和1．12％。结论该方法线性范围宽、分析时间短、样品前处理简便、定量结果准确、重复性好,为其质量控制提供了依据。     

恩拉霉素质量标准的建立

目的 建立恩拉霉素质量标准。方法 采用HPLC-PDA法建立鉴别及恩拉霉素组分检查方法，采用效价法建立恩拉霉素含量测定方法，并根据药品质量标准分析方法验证指导原则的要求，进行相应方法验证。结果 鉴别恩拉霉素组分检查方法专属性良好，在100~350u/mL范围内浓度与组分A与B峰面积之和具有良好的线性关系(r=0.9999)；效价含量测定方法专属性良好，在10~48u/mL范围内浓度与抑菌圈直径具有良好的线性关系(r=0.9995)，方法准确度回收率96.3%，RSD为2.9%。 结论 该质量标准考察项目全面，方法简单准确，可作为恩拉霉素质量控制的方法。