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

摘要

Background: Amikacin belongs to aminoglycosides family, commonlyadministered in the treatment of systemic infections due to gram negativebacteria. Its narrow therapeutic index results in adverse effects like nephrotoxicityand ototoxicity. Objective: Optimize an ultra-high performanceliquid chromatography (UHPLC) based analytical method for the determinationof amikacin sulfate in human serum using derivatizaon with FMOCCland glycine. Methods: Pre-column derivatization reaction of amikacinperformed 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 solutionafter completion of the derivatization reaction for stabilization of fluorescentcomplex product. Fluorimetric detection of amikacin was performed atexcitation and emission wavelength of 265 nm and 315 nm respectively,using C18 UHPLC column. The reported method was validated by performinglinearity, precision, recovery and ruggedness. Results: The optimummobile phase composition was found to be Acetonitrile:water in the ratioof 70:30 (v/v) at flow rate of 0.4 ml/min. A linear response of amikacin inserum samples ranging from 0.5-10 μg/ml was obtained, with correlationco-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 lessthan 10%. Conclusion: The developed UHPLC method will be useful forpre-clinical and pharmacokinetic study of amikacin in human serum.