Abstract: A new, simple and sensitive method based on pre-column derivatization by reversed-phase high performance liquid chromatography (HPLC) is described for the separation and quantification of netilmicin in plasma, using 9-fluorenylmethyl chloroformate (FMOC-Cl) as the derivatization reagent. Its pharacokinetics is also presented. The derivatization modes and chromatographic conditions were optimized. The separation was performed on an Agilent ZORBAX Eclipse XDB-C8 column (150 mm×4.6 mm, 5 μm) with a mixture of water-acetonitile (15:85, v/v) as mobile phase and the flow rate was 1.0 mL/min. The excitation wavelength was 265 nm and the emission wavelength was 315 nm. The linear range was 0.045~8.88 mg/L and the correlation coefficient (r) was 0.9993. The limit of detection (LOD) (S/N=3) was about 0.01 mg/L, and the limit of quantification was 0.03 mg/L (3LOD) for netilmicin. The relative standard deviation was less than 3% for intra-day assay (n=5) and 3.5% for inter-day assay (n=5) and the relative recovery was in the range of 96.62%~100.84%(n=3). The plasma volume of 30 μL was sufficient for the determination of netilmicin. The method provides a reliable bioanalytical methodology to carry out netilmicin pharmacokinetics in rat plasma.

Key words: 9-fluorenylmethyl chloroformate (FMOC-Cl), high performance liquid chromatography (HPLC), netilmicin, pharmacokinetics, rat plasma , pre-column derivatization