Low Level Quantification Of Potential Genotoxic Impurity In Daclatasvir Hydrochloride By RP-HPLC Method

Abstract

The goal of our research work was to develop new specific analytical method for the determination of 4 4'-bis(2-bromoacetyl) biphenyl at genotoxic level in Daclatasvir dihydrochloride drug substances. The method was developed by using Reverse phase High performance liquid chromatography technique (RP-HPLC). The comprehensive method development was done to accomplish right combination of chromatographic conditions and validation was completed as per ICH guidelines. The method utilizes waters X-select CSH C-18 (250 mm x 4.6 mm ID, 5.0 μm) HPLC column maintained at 45⁰C temperature and detected by ultra violet detector at 210nm. The separation of  4'-bis(2-bromoacetyl) biphenyl was attained by gradient elution mode where mobile phase A was purified water pH-3 adjusted with Ortho-phosphoric acid and mobile phase B was acetonitrile. Flow rate was used 1.5 mL/min and injection volume was 20 µL. The method was entirely screened with all the key validation parameters likewise system suitability, specificity, linearity, sensitivity (LOD/LOQ), precision, accuracy, robustness, and solution stability. All the related substances of sample were specific with impurity and no blank interference was found. The achieved limit of detection (LOD) were 3 μg/ml and limit of quantitation (LOQ) 10 μg/ml with respect to sample. The calibration curve exhibited good linearity from LOQ to 150% level and the correlation coefficient was found 0.999. The accuracy in terms of % recovery of the added known amount found in the range of 95-102 %. Based on experimental result developed analytical method can be used for quantitation of 4,4'-bis(2-bromoacetyl) biphenyl genotoxic impurity in Daclatasvir dihydrochloride drug substances at low level.