Eco-friendly stability-indicating HPLC method development, validation, and LC-MS-based characterization of...

Imeglimin (IMG) is a recently developed cyclic biguanide compound used for the management of hyperglycaemia. To comprehend the stability behavior of the polar drug IMG under various degradation environments, an HPLC method was developed, and the major degradation products (DPs) were characterized using mass-spectrometric studies. The mobile phase comprising a 20 mM potassium dihydrogen phosphate (KH₂PO₄) buffer (pH 3.1) along with ethanol in a simple isocratic mode (98:02, v/v) was employed for optimum resolution of the drug and its DPs using a reverse-phase Luna C18(2) (250 × 4.6 mm, 5 µm) column. Structural characterization of IMG and its DPs was performed using triple-quadrupole LC-MS, employing electrospray ionization (ESI) mode. IMG degradation occurred under acidic, alkaline, peroxide-mediated oxidation, and, to a smaller extent, nitrosation stress conditions, forming twelve DPs. Peak-purity analysis using HPLC-PDA depicted that the IMG peak remained pure under all stressed and stability conditions. Mass-spectrometric analysis enabled the identification of seven previously unreported DPs, thereby expanding the known degradation profile of IMG. The degradation pathways were elucidated based on the plausible structures of the DPs. Consequently, the probable root cause of DP4 formation in the drug product was identified through an experiment involving the enzymatic neutralization of hydrogen peroxide (H₂O₂). Following ICH Q2(R2) guidelines, the developed method demonstrated suitability for quality control testing of the IMG drug substance and the finished tablet product.