LC Analysis of Kanamycin in Human Plasma, by Fluorescence Detection of the 9-Fluorenylmethyl Chloroformate Derivative

Abstract

High-performance liquid chromatography with fluorescence detection has been used for simple and efficient analysis of the aminoglycoside kanamycin in human plasma. Kanamycin sulfate was derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl) in borate buffer (0.2 M, pH 8.5) for 30 min at 25 °C. The derivative was chromatographed on a 150 mm × 4.6 mm, 5-μm particle, C₈ column with 70% acetonitrile in water as mobile phase at a flow rate of 1.0 mL min⁻¹. Under these conditions the retention time of kanamycin-FMOC was 6.2 min. The fluorescence excitation and emission wavelengths were 268 and 318 nm, respectively. Recoveries ranging from 92.3 to 100.8% were obtained. The correlation coefficient was >0.999 over the range 0.1–4.0 μg mL⁻¹. The within-day precision of the method, as coefficient of variation, was 2.9–5.5% (n = 5). The kanamycin-FMOC derivative was stable in the reaction solution for 24 h at room temperature. Validation of the method is reported.