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

The authors describe a simplified high-performance liquid chromatographic ( HPLC) method for the determination of gentamicin sulfate (GEN) in microsamp les of plasma using 9-fluorenylmethyl chloroformate (FMOC) as a derivatizin g agent and neomycin sulfate as the internal standard (IS). The drug and IS were separated on a 4 mu m (particle size), 8 x 100 mm Nova-Pak Cls radial compression cartridge using a mixture of 84.5% acetonitrile and 15.5% wate r at a flow rate of 2.5 mL/min. The compounds were detected fluorometricall y in the effluent at excitation and emission wavelengths of 260 nm and 315 nm, respectively. Sample preparation was performed on 50 mu L of plasma usi ng a simple liquid-liquid extraction followed by a room-temperature derivat ization procedure. No interference from any endogenous substance or concurr ently used drug was observed, and the retention times of the IS and three m ajor components of GEN were 12.4, 19.5, 23.6, and 27.6 min, respectively. T he concentration of the GEN in plasma for the range of 0.2-20.0 mu g/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 mu g/ mL being <3.61%. A comparison of the results from this assay versus fluores cence polarization immunoassay (TDx) showed a close agreement between the t wo methods with r = 0.994. This assay is currently being used to monitor GE N and investigate its pharmacokinetics in pediatric patients.