Simplified high-performance liquid chromatographic method for the determination of gentamicin sulfate in a microsample of plasma: comparison with fluorescence polarization immunoassay

Abstract

The authors describe a simplified high-performance liquid chromatographic (HPLC) method for the determination of gentamicin sulfate (GEN) in microsamples of plasma using 9-fluorenylmethyl chloroformate (FMOC) as a derivatizing agent and neomycin sulfate as the internal standard (IS). The drug and IS were separated on a 4 microm (particle size), 8 x 100 mm Nova-Pak C18 radial compression cartridge using a mixture of 84.5% acetonitrile and 15.5% water at a flow rate of 2.5 mL/min. The compounds were detected fluorometrically in the effluent at excitation and emission wavelengths of 260 nm and 315 nm, respectively. Sample preparation was performed on 50 microL of plasma using a simple liquid-liquid extraction followed by a room-temperature derivatization procedure. No interference from any endogenous substance or concurrently used drug was observed, and the retention times of the IS and three major components of GEN were 12.4, 19.5, 23.6, and 27.6 min, respectively. The concentration of the GEN in plasma for the range of 0.2-20.0 microg/mL was linearly (r > .997) related to the peak height ratio of the sum of the three major GEN peaks to that of the IS, with CV value at 0.3, 7.5, and 15 microg/mL being <3.61%. A comparison of the results from this assay versus fluorescence polarization immunoassay (TDx) showed a close agreement between the two methods with r = 0.994. This assay is currently being used to monitor GEN and investigate its pharmacokinetics in pediatric patients.