Preparative purification of gentamicin components using high‐speed counter‐current chromatography coupled with electrospray mass spectrometry

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.