Development and validation of a stability indicating LC-PDA-MS/MS method for separation, identification and characterization of process related and stress degradation products of rivaroxaban

Abstract

An isocratic reversed phase high-performance liquid chromatography coupled with a photo diode array and electrospray ionization tandem mass spectrometry (LC-PDA-ESI-MS/MS) method was developed for the identification and characterization of stress degradation products (DPs) and an unknown process related impurity (PR) of rivaroxaban (RVR) in bulk drugs. RVR, a selective and direct factor Xa inhibitor, was subjected to hydrolysis (acidic, alkaline and neutral), photolysis, thermolysis, and oxidation as per ICH guidelines and found susceptible to acid and base hydrolytic stress conditions. In total, three DPs (DP-1, DP-2, and DP-3) were formed. All the DPs, PR and RVR were well separated on a Kinetex C₁₈ (150 × 4.6 mm, 5 μm) column using a mobile phase consisting of 20 mM ammonium acetate and acetonitrile (65 : 35 v/v) at a flow rate of 1.0 ml min⁻¹. To elucidate the structures of PR and DPs, MS/MS experiments were carried out to study the molecular ion fragmentation of RVR, PR, and DPs. The product ions of PR and DPs were compared with those of the RVR molecular ion to assign most possible structures. Potential impurities were isolated by semi-preparative HPLC and subjected to NMR spectroscopy. The LC-PDA method was validated with respect to specificity, linearity, accuracy, precision and robustness as per ICH guidelines.