Reversed-phase liquid chromatography with electrospray mass detection and H-1 and C-13 NMR characterization of new process-related impurities, including forced degradants of Efavirenz: Related substances correlated to the synthetic pathway

摘要

In this study, a stability-indicating reversed-phase liquid chromatographic electrospray mass spectrometric method was developed and validated for the determination of process-related impurities and forced degradants of Efavirenz in bulk drugs. Efavirenz was subjected to acid, alkaline hydrolysis, H2O2 oxidation, photolysis, and thermal stress. Significant degradation was observed during alkaline hydrolysis, and the degradants were isolated on a mass-based purification system and characterized by high-resolution mass spectrometry, positive electrospray ionization tandem mass spectrometry, and H-1 and C-13 NMR spectroscopy. Accurate mass measurement and NMR spectroscopy revealed the possible structure of process-related impurities and degradant under stress conditions. The acceptable separation was accomplished on Waters bondapak C-18 column (250 mm x 4.6 mm; 5 mu m), using 5 mM ammonium acetate and acetonitrile as a mobile phase in a gradient elution mode at a flow rate of 1.0 mL/min. The eluents were monitored by diode array detector at 247 nm and quantitation limits were obtained in the range of 0.1-2.5 mu g/mL for Efavirenz, degradants, and process-related impurities. The liquid chromatography method was validated with respect to accuracy, precision, linearity, robustness, and limits of detection and quantification as per International Conference on Harmonization guidelines.