Fluorimetric determination of gentamicin in dosage forms and biological fluids through derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl)

A highly sensitive fluorimetric method has been developed for the determination of gentamicin. The method is based on its coupling with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in 50% (v/v) methanolic phosphate buffer (pH 7.2) to give an intensely fluorescent product having excitation and emission wavelengths at 465 nm and 530 nm, respectively. The experimental parameters were carefully studied and incorporated into the procedure. The fluorescence-concentration plot is rectilinear over the range 0.56-2.8 mug/mL (r = 0.999) with minimum detectability (S/N = 2) of 0.11 mug/mL (1.6 x 10(-7) mol/L). The method was applied successfully to the determination of the drug in pharmaceutical dosage forms and the percentage recoveries were satisfactorily accurate and precise. The method was further applied to spiked human plasma samples, the percentage recovery was 97.9 +/- 3.4. The interference encountered from endogenous amino acids could be eliminated through selective complexation with freshly prepared copper (II) hydroxide. A proposal of the reaction pathway is presented. The method can measure the intact drug, and can be used in presence of possible interference.