Development and Validation of Reverse Phase High-Performance Liquid Chromatographic Method Enabling Simultaneous Estimation of Polmacoxib and its Process-Related Impurities: Exploring Degradation Pathways and Elucidating Degradant Structures via LC–MS/MS and NMR Spectroscopy

A simple, sensitive, and selective reverse phase high-performance liquid chromatography (HPLC) method was developed and validated for the estimation of Polmacoxib (POL) and its process-related impurities. Chromatographic separation was achieved on a Kromasil C18 column (250 mm x 4.6 mm, 5 μm) using gradient elution with mobile phase containing 10 mM ammonium acetate buffer and acetonitrile in the initial ratio of 90:10 (% v/v). Chromatographic parameters were optimized: flow rate of 1.2 mL/min, UV detection at 232 nm, and injection volume of 10 μL. The developed method was validated as per International Council for Harmonization Q2 (R1) guidelines. For the stability study, the drug was subjected to various stress conditions. The results showed that was sensitive to the alkaline and degraded. Under alkaline condition, impurity-I and II were detected at retention time of 2.61 and 21.09 min, respectively. To identify the structures of the detected impurities, were initially isolated using a preparative HPLC and then collected fractions were injected into liquid chromatography with tandem mass spectrometry for structural identification. Further structure elucidation was performed using 1D-NMR (1H and 13C) and 2D-NMR (1H-1H correlation spectroscopy and 1H-13C heteronuclear single quantum coherence), and explore the degradation pathway. The proposed method is applicable for assay estimation of POL in dosage form and is suitable for quality control analysis.