Trace analysis of tobramycin in human plasma by derivatization and high-performance liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method is established for the trace determination of tobramycin in human plasma by derivatization. The method is based on the chemical derivatization of aminoglycoside antibiotic, tobramycin in human plasma, with 1-naphthyl isothiocyanate (NITC) in pyridine at 70 degrees C. After derivatization reaction, a methylamine/acetonitrile solution was added to the reaction mixture to eliminate the excess derivatizing agent and shorten the analysis time. The resulting derivative was separated using a Purospher STAR RP-18e column and a water-acetonitrile (50:50, v/v) mobile phase (detection at 230 nm). Optimization conditions for the derivatization of tobramycin were investigated by HPLC. The linear range for the quantitation of tobramycin in spiked plasma was over 0.93-9.34 mg/l; the detection limit (signal-to-noise ratio=3; injection volume, 10 microl) was about 0.23 mg/l. The relative standard deviation was less than 2.1% for intra-day assay (n=6) and 5.2% for inter-day assay (n=6) and relative recoveries were found greater than 99%.