Optimization and Validation in Liquid Chromatography Using Design of Experiments

Elbasvir (ELB) and grazoprevir (GZP) were two new approved drugs and their co-formulation presents one of the more recently approved combinations for treatment of hepatitis C virus. A stability indicating reversed phase-high performance liquid chromatography (RP-HPLC) method was developed for simultaneous determination of ELB and GZP and their degradation products under hydrolysis and oxidative stress conditions as per International Conference on Harmonization [ICH Q1A (R2) guidelines]. Adequate chromatographic separation with well-defined peaks was achieved using a Waters Spherisorb phenyl Column (150 mm × 4.6 mm I.D, 5 µm particle size) with a temperature maintained at 40 °C ± 2 °C. The mobile phase consists of acetonitrile: 5 mM ammonium formate buffer (+ 0.1% v/v of trimethylamin, pH was adjusted to 3.2 by formic acid) (60:40 v/v) at a flow rate of 0.8 mL min−1. A validation study was performed according to the accuracy profile methodology. Specificity, suitability, robustness, precision and trueness of the developed method were confirmed. The proposed method was also successfully compatible to identification of molecular structure of degradation products by liquid chromatography-mass spectrometry (LC–MS) coupling. A quadrupole-time of flight mass analyzer equipped with an electrospray ionization source was used to characterizing degradation products based on the MS spectra and accurate mass measurements.

Sami Jebali

Papers

RP-HPLC Stability Indicating Method Development and Validation for Simultaneous Determination of Grazoprevir and Elbasvir