Determination of the component ratio of commercial gentamicins by high-performance liquid chromatography using pre-column derivatization.

Commercial samples of gentamicin from different origins were analyzed by paired-ion high-performance liquid chromatography (HPLC) on a C18 bonded phase. The procedure uses pre-column derivatization with a omicron-phthalaldehyde-mercaptoacetic acid reagent and UV detection (350 nm). The ratios of the four gentamicin components (C1, C1a, C2a and C2) were determined and compared with the compositions obtained by an independent method based on 13C NMR spectrometry. Quantitation by HPLC, based on peak heights and peak areas, was performed with the aid of an external standard, which was an artificial mixture of the four components. The latter were prepared by separation of the gentamicins C1, C2 + C2a and C1a by chromatography on silica gel, followed by chromatography of the C2 + C2a fraction on a cellulose phosphate column.