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A forced degradation study on novel glaucoma drug netarsudil was performed as per ICH and WHO guidelines. All the degradation products were separated from the drug on a C-18 column utilizing ammonium acetate buffer (10 mM, pH 4.90) as a mobile phase-A and acetonitrile: methanol (70:30) v/v as mobile phase-B, by employing gradient elution mode. The injection volume was 10 µL with a 0.8 mL/min flow rate. The detection wavelength was 245 nm and the study was executed at a constant column temperature of 40 °C. Initially, the mass fragmentation pathway of the drug was postulated followed by characterization of degradation products from LC-MS-TOF/MS data. This was followed by LC-MS/TOF studies on the degradation products. Significant degradation was witnessed in alkaline and acidic conditions whereas no signification decline in the drug was observed in other decomposition environments. A total of four degradation products were formed and characterized with the aid of high-resolution mass spectrometry. The mechanistic pathway of formation of all DPs from netarsudil is established. In addition, comparative toxicity properties of the drug and degradation products were established using the online web server Pro Tox II.

Identification and characterization of novel hydrolytic degradation products of netarsudil by LC-Q-TOF-MS/MS: In silico toxicity prediction