Stability-Indicating Gradient RP-LC Method for the Determination of Process and Degradation Impurities in Bosentan Monohydrate: An Endothelin Receptor Antagonist

Abstract

Described is a simple, rapid, selective, and stability-indicating RP-LC method for the determination of process and degradation-related impurities of bosentan monohydrate. Chromatographic separation was achieved on Zorbax SB-Phenyl column thermostated at 35 ° C under gradient elution by a binary mixture of solvent A (60% phosphate buffer, pH 2.5, and 40% methanol) and solvent B (acetonitrile) at a flow rate of 1.0 mL/min. Forced degradation was carried out under acidic, alkaline, oxidative, photolytic, and thermal conditions. Significant degradation is observed in acid and alkali stress conditions. Process- and degradation-related impurities were characterized by mass spectrometry, 1 H NMR, and FT-IR spectral data. Validation of the developed method was carried out as per ICH requirements. Regression analysis shows an “ r ” value (correlation coefficient) of greater than 0.999 for bosentan and five potential impurities. This method was capable to detect the five impurities at 0.01% of the test concentration of 1.0 mg mL -1 .