RP-HPLC separation and characterization of unknown impurities of a novel HIV-protease inhibitor Darunavir by ESI-MS and 2D NMR spectroscopy

A selective RP-HPLC method for separation and determination of darunavir from its process related substances has been developed and validated. The separation was accomplished on a Hiber, LiChrospher® 60, RP-select B, C8 (250 mm × 4.6 mm, 5 μm) column connected to a photo diode array detector (PDA) using 10 mM phosphate buffer with 0.1% of triethylamine (pH: 4.0 adjusted with orthophosphoric acid)/acetonitrile as a mobile phase under gradient elution. Two unknown impurities of darunavir were isolated and characterized by 1H, 13C, 2D-NMR and mass spectrometry. The method was validated in terms of accuracy, precision, linearity, robustness, LOD and LOQ.

► Separation and determination of darunavir from its ten process related substances by RPLC. ► Isolation and characterization of two unknown impurities of Darunavir by 1H, 13C, 2D-NMR and mass spectrometry. ► Until now, there is no validated LC method reported for determination of process related substances of DRV in literature.