Stability indicating chromatographic method development and validation for acotiamide HCl trihydrate with comprehensive forced degradation studies and LCMS/MS characterization of related substances

A newly developed stability-indicating reversed-phase high-performance liquid chromatography method was validated for the simultaneous quantification of assay and related substances of Acotiamide Hydrochloride Trihydrate, a gastroprokinetic drug, in pharmaceutical dosage forms. The aim of the study was to establish a simple, rapid, precise, accurate, and cost-effective chromatographic method capable of detecting degradation products through forced degradation studies. Chromatographic separation was achieved using an Inertsil ODS-3 V column (150 mm × 4.6 mm, 5 μm), with a mobile phase comprising methanol and ammonium acetate buffer (pH 6.8) in the ratio of 55:45 (v/v), at a flow rate of 2.0 mL/min. Detection was performed with a photodiode array detector at 280 nm. Validation, as per the International Council for Harmonisation guideline Q2 (R2), demonstrated excellent specificity, linearity (correlation coefficient > 0.999), accuracy (98–102% for the assay), precision (relative standard deviation < 2%), and robustness under variable analytical conditions. The method effectively separated Acotiamide from its degradation products. Forced degradation studies revealed significant degradation under basic and oxidative conditions, while stability was observed under acidic, thermal, and photolytic conditions. A major degradation product observed in basic and oxidative conditions, eluting at 9.5 min, was identified using a TSQ Endura Triple Quadrupole Mass Spectrometer and characterized as 2-[(1,2-dihydroxy-5,6-dimethoxybenzamido)-N-(2-(diisopropylamino) ethyl)-N-hydroxythiazole-4-carboxamide], with prominent mass fragmentation observed at m/z 467. The validated method is suitable for routine quality control and stability assessment of Acotiamide Hydrochloride Trihydrate.