ABSTRACT
OBJECTIVE: To establish an HPLC method combined with pulsed amperometric detection for the analysis of ribostamycin sulfate and related substance. METHODS: The HPLC was performed on Thermo AcclaimTMAmG C18 column (4.6 mm×150 mm,3 μm). The mobile phase consisted of acetonitrile and 0.2%(V/V) pentafluoropropionic acid aqueous solution containing 0.15%(V/V) trifluoroacetic acid (1∶99, V/V). The pH of the aqueous solution was adjusted to 1.5 with 50%(m/m) sodium hydroxide solution. The pulsed amperometricdetector was operated with aquadruple-potential wave form at 35 ℃ and the injection volume was 25 μL. RESULTS: Ribostamycin and its related substances were adequately separated under the established HPLC conditions. The LOD and LOQ of ribostamycin were 0.15 μg•mL-1(3.75 ng injected) and 0.375 μg•mL-1(9.38 ng injected), respectively. The linearity of ribostamycin ranged from 0.15 to 40.0 μg•mL-1 with a correlation coefficient of 0.999 3.The repeatability RSDs(n=6)for method validation of the content assay and total impurities test were 0.33% and 1.10%, respectively. CONCLUSION: The established method is characterized by high specificity, sensitivity and good stability. The established method has much lower test cost than the current Ch.P 2015 method and is hopeful to replace it.