Evaluation of dynamics of derivatization and development of RP-HPLC method for the determination of amikacin sulphate

The absence of chromophore and/or conjugated system, prerequisite for UV and florescent light detection, or absorbance at very low wavelength necessitates the development of simple and reliable methods for the determination of amikacin sulphate. Therefore, the present study describes for the first time dynamics of the drug derivatization using ninhydrin reagent and development and validation of a simple RP-HPLC method, using diode array detector (DAD). The variables such as heating time, heating type, drug-reagent ratio, reagent composition and storage temperature of the derivative were optimized. The analyte and aqueous ninhydrin solution upon heating for 2.00-5.00 min produced the colored drug-derivative which was stable for one month at refrigeration. The derivatized drug (20.00μL) was eluted through a column - Eclipse DB-C18 (5.00 µm, 4.60×150.00 mm), maintained at 25°C- using isocratic mobile phase comprising water and acetonitrile (70:30, v/v) at a flow rate of 1.00 mL/min, and detected at 400 nm. The method was found to be reliable (98.08-100.72% recovery), repeatable (98.02-100.72% intraday accuracy) and reproducible (98.47-101.27% inter day accuracy) with relative standard deviation less than 5%. The results of the present study indicate that the method is easy to perform, specific and sensitive, and suitable to be used for the determination of amikacin sulphate in bulk and pharmaceutical preparations using less expensive/laborious derivatization.