title: Preparative purification of gentamicin components using high-speed counter-current chromatography coupled with electrospray mass spectrometry.
abstract: We developed a useful and preparative method based on high-speed counter-current chromatography with mass spectrometry (HSCCC/MS) to purify gentamicin C1a, C2/2a and C1 from standard powder. The analytes were purified on the HSCCC model CCC-1000 (multi-layer coil planet centrifuge) with a volatile two-phase solvent system composed of n-butanol/10% aqueous ammonia solution (50:50, v/v) and detected on an LCMS-2020EV quadrupole mass spectrometer fitted with an electrospray ionization (ESI) source system in positive ionization following scan mode (m/z 100-500). The HSCCC/ESI-MS peaks indicated that gentamicin C1a (m/z 450: [M+H](+)), C2/2a (m/z 464: [M+H](+)) and C1 (m/z 478: [M+H](+)) have the peak resolution values of 1.3 and 1.7 from 30 mg of loaded gentamicin powder. The HSCCC yielded 3.9 mg of gentamicin C1a, 12.6 mg of gentamicin C2/2a and 12.0 mg of gentamicin C1. These purified substances were analyzed by LC/MS with scan positive-mode. Based on the LC/MS chromatograms and spectra of the fractions, analytes were estimated to be over 95% pure. These gentamicin isomers of C1a, C2/2a and C1 were evaluated for their antibacterial activities. The overall results indicate that this approach of HSCCC/MS is a powerful technique for the purification of gentamicin components.
authors.affiliation: Department of Physical and Analytical Chemistry, School of Pharmacy, Kinjo Gakuin University, Nagoya, Aichi, Japan. kinoue@kinjo-u.ac.jp
authors.forename: Koichi
authors.initials: K
authors.lastname: Inoue
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Hattori
authors.forename: Masakazu
authors.initials: M
authors.lastname: Horie
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Hino
authors.forename: Hisao
authors.initials: H
authors.lastname: Oka
mesh_terms: D003377:Countercurrent Distribution / Q000379:methods*; D005839:Gentamicins / Q000737:chemistry / Q000302:isolation & purification*; D007536:Isomerism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D005839:Gentamicins