摘要
目的开展低含量成分的富集和纯化研究,从Glarea lozoyensis菌体的发酵产物(即纽莫康定B0粗品)中制备出杂质B1和B5,并对其进行结构鉴定。方法建立了基于制备高效液相色谱(prep-HPLC)技术的两步纯化法,第一步使用亲水(HILIC)模式固定相对目标杂质进行富集,第二步采用反相(RPLC)模式固定相对杂质进行制备。得到的两个杂质采用质谱(MS)、核磁(NMR)进行鉴定,确定化合物结构。结果两个杂质为B1和B5,相对分子质量均为1049 Da,色谱纯度分别为97.83%和98.13%。经核磁鉴定,B1为B0的高酪氨酸类似物,B5为B0的鸟氨酸类似物。结论本研究发展的基于prep-HPLC的两步纯化方法为低含量成分制备提供参考,第一步分离实现目标成分富集,第二步利用正交性的色谱柱提高分离选择性,成功制备出杂质B1与B5,有助于B0杂质谱的建立和进一步的质量控制研究。
Objective This research was carried out on the enrichment and purification of components with low content.The purification of impurities B1 and B5 from Glarea lozoyensis fermentation broth(i.e.,the crude product of pneumocandin B0)was prepared,following their structures identification.Method A two-step purification method based on preparative high performance liquid chromatography(prep-HPLC)was established.The HILIC mode stationary phase was used in the first step in order to enrich the targeted impurities,and the RPLC mode stationary phase was used in the second step in order to purify the impurities.The two impurities were identified by mass spectrometry(MS)and nuclear magnetic resonance(NMR).Results The two impurities were B1 and B5,with a relative molecular mass of 1049 Da and chromatographic purity of 97.83% and 98.13%,respectively.By NMR identification,B1 is a homo tyrosine analogue of B0 and B5 is an ornithine analogue of B0.Conclusion The two-step purification method based on prep-HPLC developed in this study provides a reference for the isolation of components of low content.The first-step separation was to enrich the target components.The second-step separation that used an orthogonal column to improve the separation selectivity.As a result,impurities B1 and B5 were successfully isolated.It is helpful in establishing the impurity profile of B0 and furthering its quality control.
作者
张洪志
傅青
金郁
Zhang Hongzhi;Fu Qing;Jin Yu(School of Pharmacy,East China University of Science and Technology,Shanghai 200237)
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2024年第1期57-64,共8页
Chinese Journal of Antibiotics
关键词
纽莫康定
杂质
制备高效液相色谱
纯化
结构鉴定
Pneumocandin
Impurity
Preparative high performance liquid chromatography
Purification
Structure identification
