Pure samples of gentamicin C1, C1a, C2 and seven minor components were isolated from a commercial mixture of gentamicin sulphate using column chromatography on TLC grade silica gel. A preparative HPLC method based on this separation is also described. Sufficient quantities of the minor components were isolated for spectroscopic examination. With one exception they were less biologically active against Bacillus pumilus than the 3 major components. Quantitation of gentamicin in commercial samples by spectrofluorimetric and titrimetric methods based on the primary amino groups of gentamicin were investigated but neither proved to be suitable for routine analysis. A high-pressure liquid chromatographic method for routine control of the composition of gentamicin in commercial samples and formulations is described. The method utilises pre-column derivatisation followed by reversed phase chromatography with fluorescence detection and uses an internal standard for quantitation. Analysis of nineteen samples of gentamicin from various sources indicates that the ratio of major components and the content of minor constituents varies with the geographical origin of the sample. The results were compared with those of a microbiological assay and the B.P. nuclear magnetic resonance (NMR) spectroscopic limit test of the same samples. The microbiological assay may be influenced by biologically active impurities whilst the NMR assay was insensitive to the presence of minor components. The method described offers a discrimating and flexible means of monitoring the composition of gentamicin. The same HPLC method was applied to the determination of gentamicin in plasma though it was not possible to use the internal standard. The method is rapid and specific and involves no extraction of gentamicin from the plasma. It would be suitable for both routine clinical monitoring and pharmacokinetic studies.
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