A rapid HPLC–DAD method for quantification of amikacin in pharmaceuticals and biological samples using pre-column derivatization with Hantzsch reagent

Abstract

Amikacin (AMK) is an important member of aminoglycoside class, and its determination has therapeutic importance due to its matchless potency against gram –ve pathogens. Due to narrow therapeutic window, its monitoring in clinical samples is inevitable. Direct determination of AMK using HPLC with UV–visible detection is not possible because of its limited absorbance. Herein, Hantzsch reagent (mixture of acetylacetone, formaldehyde and acetate buffer) was used as pre-column derivatization for AMK. UV–visible detection was performed at 340 nm. Separation and identification of derivatized drug (amikacin) were carried out using C-18 column Kromasil 100 (15 cm × 0.46 mm, 5 μm) with isocratic mobile phase elution of pH 5 (acetate buffer 0.01 M):acetonitrile (30:70 v/v) with flow rate of 1 ml/min. The procedure was able to resolve AMK from endogenous compounds and from cephalosporin drug (most prescribed combination) with run time of 10 min. Under optimized conditions; calibration curve was linear in the range 0.10–25.0 µg/mL with LOD and LOQ values of 0.024 and 0.071 µg/mL. Method was also validated for reproducibility, ruggedness and accuracy. The procedure was found sensitive, robust and precise for the comprehensive analysis (qualitative and quantitative) that was applied for determination of AMK in pharmaceuticals, urine and blood samples.