Analysis of Amikacin in Human Serum By UHPLC With Fluorescence Detector Using Chloro-Formate Reagent With Glycine

Background: Amikacin belongs to aminoglycosides family, commonly administered in the treatment of systemic infections due to gram negative bacteria. Its narrow therapeutic index results in adverse effects like nephrotoxicity and ototoxicity. Objective: Optimize an ultra-high performance liquid chromatography (UHPLC) based analytical method for the determination of amikacin sulfate in human serum using derivatizaon with FMOCCl and glycine. Methods: Pre-column derivatization reaction of amikacin performed using fluorescence reagent 9-fluorenylmethyl chloroformate (FMOC-Cl) at ambient temperature in the presence of borate buffer (0.2 M). Stabilizing reagent glycine (0.1 M) added into the reaction mixture solution after completion of the derivatization reaction for stabilization of fluorescent complex product. Fluorimetric detection of amikacin was performed at excitation and emission wavelength of 265 nm and 315 nm respectively, using C18 UHPLC column. The reported method was validated by performing linearity, precision, recovery and ruggedness. Results: The optimum mobile phase composition was found to be Acetonitrile:water in the ratio of 70:30 (v/v) at flow rate of 0.4 ml/min. A linear response of amikacin in serum samples ranging from 0.5-10 μg/ml was obtained, with correlation co-efficient of 1.00. The limit of detection (LOD) was found to be 50 ng/ml. Both inter- and intra-day analysis co-efficient values were found to be less than 10%. Conclusion: The developed UHPLC method will be useful for pre-clinical and pharmacokinetic study of amikacin in human serum. Key words: Amikacin Sulphate, Ultra High Performance Liquid Chromatography (UHPLC), 9-Fluorenylmethyl chloroformate (FMOC-Cl) reagent, Borate buffer, Glycine, Fluorescence detector.