Stability-Indicating Liquid Chromatography Method for Rifaximin and LC-MS Characterization of its Potential Degradants

Abstract

Rifaximin (RFX) is a structural analog of rifampin that has been shown to be a gastrointestinal-selective antibiotic with broad-spectrum antibacterial properties, a pronounced safety profile, and limited drug interactions. Crohn’s disease, diarrhea, hepatic encephalopathy, irritable bowel syndrome, and traveler’s diarrhea caused by non-invasive diarrheagenic Escherichia coli, can all be treated with rifaximin. FDA has designated RFX as an orphan drug for the treatment of hepatic encephalopathy. There is a substantial need for analytical quality monitoring of Rifaximin to ensure the safety and efficacy of treatment. To generate degradants, forced degradation study of the drug was carried out in acid, alkali, oxidative, thermal, and photolytic stress conditions. The optimum separation of the drug, and its degradants was achieved on an Inertsil ODS-3V C18 column (250 X 4.6 mm X 5 μm) under gradient elution conditions using, 10 mM potassium dihydrogen orthophosphate (pH 5.0 ± 0.05) as mobile phase A, and acetonitrile as mobile phase B, at a flow rate of 1.0 mL/min. The run time was 41 minutes and the detection wavelength was 258 nm. The method was validated in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human use Guidelines. The drug was stable in neutral hydrolysis, photo and dry heat degradation conditions. The percentage degradation observed during acid, alkali hydrolysis and oxidative stress conditions were 70.46, 15.11 and 24.18, respectively. The degradants were investigated by LC-MS which indicated the presence of four (m/z 784.2, 744.5, 784.3, 753.8), three (m/z 744.5, 784.3, 753.8) and five (m/z 772.4, 838.4, 744.5, 753.8, and 801.9) degradants in acid, alkali and oxidative stress conditions, respectively. The structures of degradants were elucidated using Liquid chromatography–mass spectrometry (LC-MS) and Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses.