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A novel high performance liquid chromatographic (HPLC) method with a pre-column derivatization reaction has been developed and validated. The method was used for the determination of the aminoglycoside antibiotic amikacin sulphate (AMK) in the presence of its synthetic precursor kanamycin sulphate in pure form and in different pharmaceutical preparations. The pre-column derivatization was based on Hantzsch condensation reaction and the obtained coloured products were separated using an isocratic reversed-phase high performance liquid chromatographic method. The separation was achieved on a Spherisorb C18 ODS2 (250 × 4.6 mm, 5 μm) column using a mobile phase composed of acetonitrile–0.1 M sodium acetate buffer (pH 5.0; 25:75, v/v). The column temperature was adjusted at 35°C and the flow rate at 2 mL min −1 . The detection was carried out at 330 nm by using photo-diode array detector. Different conditions for the optimization of the derivatization reaction as well as for the HPLC measurement were studied. Moreover, AMK was subjected to forced degradation by oxidation, hydrolysis, photolysis and dry heat. Degradation products did not interfere with the assay, which can thus be considered selective and specific. The proposed method was validated for linearity, precision, accuracy, specificity and robustness. Also, it was used to check the purity of AMK in the presence of KAN (related impurity) at the pharmacopoeial limit (0.5%).

Liquid Chromatographic Determination of Amikacin Sulphate after Pre-Column Derivatization