Direct determination of kanamycin in raw materials, veterinary formulation and culture media using a novel liquid chromatography–evaporative light scattering method Original Research Article

A novel method for the direct determination of kanamycin A and its minor component kanamycin B was developed and validated based on reversed phase liquid chromatography with evaporative light scattering detector (ELSD). ELSD response to kanamycins was found to be enhanced by: (a) decrease of peak width and asymmetry (obtained by controlling the mobile phase acidity or ratio of organic solvent to water), (b) use of ion-pairing acidic reagents of increased molecular mass, and (c) increase of mobile phase volatility. Utilizing an Spherisorb ODS-2 C18 column, the selected optimized mobile phase was water–acetonitrile (60:40, v/v), containing 11.6 mM heptafluorobutyric acid (isocratic elution with flow rate of 1.0 ml min−1). Kanamycin A was eluted at 3.9 min and kanamycin B at 5.0 min with a resolution of 2.7. Logarithmic calibration curves were obtained from 0.6 to 28 μg ml−1 (r > 0.9998) for kanamycin A and 4–36 μg ml−1 (r > 0.9994) for kanamycin B, with a LOD equal to 0.20 and 1.4 μg ml−1, respectively. In kanamycin acid sulfate pharmaceutical raw materials, the simultaneous determination of sulfate (tR = 2.1 min, LOD = 2.3 μg ml−1, %R.S.D. = 1.7, r > 0.9998) and kanamycins was feasible. No significant difference (t-test) was found between the results of the developed LC–ELSD method and those of reference methods, while recovery from kanamycin B spiked samples ranged from 95 to 105%. The developed method was also applied with very good accuracy for the determination of kanamycin A in veterinary formulation (%recovery 95–103, %R.S.D. < 1.4, n = 3) and for the determination of kanamycins A and B in bacteria culture media (%recovery 102 and 99, respectively).