Two validated spectrofluorimeteric and high performance liquid chromatography (HPLC) methods with fluorescence detection for the analysis of a new anti-hepatitis C drug, daclatasvir hydrochloride, in raw material or tablet form and in biological fluids.

Abstract

Two sensitive and accurate methods have been developed for the estimation of daclatasvir (DAC) in its raw material, dosage form and in biological fluids. Method I is based on the measurement of DAC native fluorescence in methanol at 385nm after excitation at 315nm. The relationship between fluorescence intensity and concentration was found to be rectilinear over the linearity range (3.0-30.0ng/ml). There were good per cent recoveries both in the dosage form (99.87±0.84) and in spiked human plasma (99.96±1.54%). Method II utilized reversed-phase high performance liquid chromatography to estimate the antiviral agent daclatasvir hydrochloride against hepatitis C within 4.0min on a C18 column (Eurosphere. 100-5 C18, 150×4.6mm, Germany) using a mobile phase consisting of acetonitrile and 0.1M sodium dihydrogen phosphate (30:70, v/v) at pH3.0 and with a fluorescence detector adjusted to 315nm and 385nm for excitation and emission respectively. The calibration curve was linear over the range (20.0-200.0ng/ml) with SD 1.38%, error 0.56%, recovery 99.99±1.30% in tablets, recovery 100.28±1.73% in spiked urine and recovery 99.63±2.72% in spiked plasma.The new developed methods were successfully applied to the assay of the daclatasvir in tablet form and extended to its determination in real plasma, spiked human plasma and urine. The analytical performance of the proposed method was validated according to International Conference on Harmonization (ICH) guidelines. The proposed methods were compared with the results of a comparison method and it was found that there was no significant difference between the methods, as revealed by Student's t-test and variance ratio F-test.