LC-MS/MS characterization of forced degradation products of ambrisentan: development and validation of a stability-indicating RP-HPLC method

Abstract

The current study reports the characterization of degradation products of ambrisentan by liquid chromatography-tandem mass spectrometry, and development and validation of a stability-indicating reversed phase high performance liquid chromatographic method for determination of ambrisentan in the presence of its process related impurities in bulk drugs. The drug was subjected to various stress conditions such as hydrolysis, oxidation, photo- and thermal degradations to investigate the stability-indicating ability of the method. Significant degradation was observed during acidic and oxidative stresses. Ambrisentan was well resolved from its process related impurities and degradation products formed under stress conditions. The chromatographic separation was accomplished on an Agilent XDB C₁₈ column (150 × 4.6 mm; 5 μm) with the mobile phase consisting of 10 mM NH₄OAc (pH = 5.2) and CH₃CN in a gradient elution mode at a flow rate of 1.0 ml min⁻¹. The eluents were monitored by a photodiode array detector at 215 nm and quantitation limits were obtained in the range of 0.07–0.25 μg ml⁻¹ for ambrisentan and all its process related impurities. The developed liquid chromatographic method was validated with respect to accuracy, precision, linearity, robustness and limits of detection and quantitation. The degradation products were characterized by comparing their collision induced dissociation mass spectral data with that of ambrisentan and the most possible degradation and fragmentation pathways were proposed.