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 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.
Inoue
Koichi
K
Department of Physical and Analytical Chemistry, School of Pharmacy, Kinjo Gakuin University, 2-1723 Omori, Moriyama-ku, Nagoya, Aichi 463-8521, Japan. kinoue@kinjo-u.ac.jp
Hattori
Yasuko
Y
Hino
Tomoaki
T
Oka
Hisao
H
D000900:Anti-Bacterial Agents / Q000032:analysis*; D003377:Countercurrent Distribution / Q000295:instrumentation; D004355:Drug Stability; D004867:Equipment Design; D009862:Online Systems / Q000295:instrumentation; D010455:Peptides / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation; D010456:Peptides, Cyclic
D000900:Anti-Bacterial Agents; D010455:Peptides; C044447:enramycin; D010456:Peptides, Cyclic