Method Development and Validation for the Estimation of Liraglutide and Its Related Substances by using RP-HPLC

Abstract

A robust and precise gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous estimation of Liraglutide and its related impurities Des-acetyl Liraglutide (Impurity A) and Glycosylated Liraglutide (Impurity B). The optimized chromatographic conditions employed an Inertsil C18 column (250×4.6 mm, 3μm) with a mobile phase of NAH₂PO₄ buffer (pH 4) and methanol under gradient elution. System suitability parameters, including resolution (>2), tailing factor (<2), and theoretical plate count (>2000), confirmed the method’s efficiency and reliability. Linearity was demonstrated across concentration ranges of 40–200 µg/mL for Liraglutide and 4–20µg/mL for impurities, with correlation coefficients (R²>0.999) indicating excellent linearity. The method exhibited high precision (%RSD <2%) and accuracy (recovery 98–102%) across multiple concentration levels. Sensitivity was confirmed with acceptable limits of detection and quantification. Robustness was validated through deliberate variations in flow rate and mobile phase composition, showing minimal impact on performance. Forced degradation studies under various stress conditions verified the method’s stability-indicating capability, effectively separating and quantifying degradation products. This validated RP-HPLC method is suitable for routine quality control and stability testing of Liraglutide formulations, ensuring reliable detection of impurities and degradation products.