Unique Quality by Design Approach for Developing HPLC and LC-MS Method for Estimation of Process and Degradation...

Pibrentasvir (PIB) was approved for treating hepatitis C patients. A specific, accurate, linear, robust, and stability-indicating method was developed and validated for determining degradation impurities present in the PIB drug substance by studying the quality by design (QbD) principles. All identified degradation impurities were separated with the stationary phase HALO C18, 150 mm × 4.6 mm, 2.7 μm. Mobile phase A contains pH 2.5 phosphate buffer and acetonitrile in the ratio of (70:30, v/v), and mobile phase B contains water and acetonitrile in the ratio of (30:70, v/v), respectively. The chromatographic conditions were optimized, such as flow rate of 0.8 mL/min, UV detection at 252 nm, injection volume of 20 μL, and column temperature of 40 °C. The proposed method was validated per the current ICH Q2 (R1) guidelines. The recovery study and linearity ranges were established from limit of quantification (LOQ) to 300% optimal concentrations. The method validation results were between 98.6% and 106.2% for recovery, and linearity r ² was more than 0.999 for all identified impurities. The method precision results achieved below 5% relative standard deviation (RSD). The forced degradation results demonstrated that the drug was sensitive to chemical stress conditions. During the stress study, degrading impurities were identified by the LC-MS technique and the mechanism pathway. A QbD-based experimental design (DoE) approach was used to establish the robustness of the method.