Improved liquid chromatographic method with pulsed electrochemical detection for the analysis of kanamycin

This work describes the separation of the main component kanamycin A from its related substances using an improved liquid chromatographic method with pulsed electrochemical detection (LC-PED). Two methods, one using volatile ion pairing agents and the other using non-volatile ones were developed. Using volatile additives, the total run time was rather long with no possibility of developing gradient elution. The non-volatile method was found to be more performant and hence was selected for further quantitative work. This method employed gradient elution in order to reduce the analysis time and to improve the sensitivity of the late eluting peaks. Mobile phase A consisted of sodium sulphate (5.0g/l), sodium octanesulphonate (0.5g/l) and 0.2M phosphate buffer pH 3.0 (50.0ml/l). Mobile phase B was the same as A except for the amount of sodium sulphate which was increased to 15g/l. Using a Platinum EPS column (150mmx4.6mm ID, 3microm) kept at 45 degrees C, 22 components could be separated within 45min indicating that this method is much more selective than other already published ones. Robustness of the method was examined by means of an experimental design. The limit of detection and limit of quantitation were found to be 1.7 and 5ng, respectively. The method was found to be linear in the range LOQ-600ng injected with a coefficient of determination equal to 0.999.