An approach to on-line electrospray mass spectrometric detection of polypeptide antibiotics of enramycin for high-speed counter-current chromatographic separation.
An approach to on-line electrospray mass spectrometric detection of polypeptide antibiotics of enramycin for high-speed counter-current chromatographic separation.
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DOI：
10.1016/j.jpba.2009.11.010
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发表时间：
2010-04
影响因子：
3.4
通讯作者：
K. Inoue;Yasuko Hattori;T. Hino;H. Oka
中科院分区：
文献类型：
--
作者：
K. Inoue;Yasuko Hattori;T. Hino;H. Oka
在多肽的药物和生物医学分析领域，一种快速的在线检测和鉴定方法已经成为发现新的生物活性产物的必要条件。本研究建立了一种高速逆流色谱-电喷雾质谱（HSCCC/ESI-MS）在线检测和纯化多肽抗生素enramycin-A和-B的方法。采用正丁醇/己烷/0.05%三氟乙酸水溶液（43/7/50,V/V/V）组成的两相挥发性溶剂，在HSCCC型号cc -1000（多层螺旋行星离心机）上进行纯化，在配备ESI源系统的LCMS-2010EV四极质谱仪上进行正离子扫描模式（m/z 100-2000）检测。HSCCC/ESI-MS峰分析结果表明，15mg载药的enramycin- a（主要成分m/z 786 [m +3H]3+，次要成分m/z 1179 [m +2H]2+）和enramycin- b（主要成分m/z 791 [m +3H]3+，次要成分m/z 1185 [m +2H]2+）的峰分辨率为2.9。各峰段的HSCCC采集量分别为4.3mg （enramycin-A）和5.9mg （enramycin-B）。采用扫描阳性模式的LC/ESI-MS对纯化物质进行分析。根据LC/ESI-MS色谱图和谱图，估计enramycin-A和-B的纯度在95%以上。结果表明，HSCCC/ESI-MS是一种纯化和鉴定生物活性肽的有效方法。
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 15mg of loaded enramycin powder. The HSCCC collected amounts of the peak fractions were additionally 4.3mg (enramycin-A), and 5.9mg (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.