Development and Validation of HPLC Fluorescence and UPLC/DAD Stability-Indicating Methods for Determination of Hepatitis C Antiviral Agent Daclatasvir.

Abstract

Background: Few stability-indicating chromatographic methods were published for determination of daclatasvir. All used UV detection. Objective: This work aimed to develop rapid, specific, and novel stability-indicating methods using HPLC with fluorescence detection and ultra performance liquid chromatography (UPLC) with UV detection for the determination of daclatasvir in bulk powder and in its dosage form. Methods: The drug was subjected to hydrolysis (acidic and alkaline) as per International Conference on Harmonization (ICH) guidelines. The fragmentation pattern of the drug was studied using LC-MS. Separation was carried out first by HPLC using Thermo BDS Hypersil Phenyl (300 mm × 4 mm, 5 μm) column and a mobile phase consisting of ammonium phosphate buffer (0.02 M) pH 3-methanol (40+60, v/v) at flow rate 1 mL/min. Quantitation was achieved by fluorescence detection at 305 and 457 nm for excitation and emission, respectively. The second method used UPLC equipped with diode-array detector. Acquity BEH C18 (50 mm × 2.1 mm, 1.7 μm) column was used with the same mobile phase at flow rate 0.4 mL/min and detection wavelength at 305 nm. ICH guidelines were used for validation. Results: The mean percentage recovery ± SD values for tablet assay were found to be 101.12 ± 0.655 and 99.78 ± 0.632 by HPLC and UPLC methods, respectively. The assay results showed a good agreement with the reported method. Conclusions: The developed HPLC and UPLC methods provide simple, accurate, and reproducible analysis of daclatasvir without interference from degradates. Highlights: This is the first research using fluorescence detection in a stability-indicating chromatographic method for determination of daclatasvir.