LC-HRMS and NMR studies for characterization of forced degradation impurities of ponatinib, a tyrosine kinase inhibitor, insights into in-silico degradation and toxicity profiles

Abstract

The degradation profile of ponatinib was established during the present study by exposing it to various stress conditions. In-silico degradation pattern of ponatinib was outlined by using Zeneth software. Five degradation impurities were formed during the stress testing of ponatinib. High performance liquid chromatographic method was developed to separate these degradation impurities which includes ammonium acetate of pH 4.75 (A) and methanol (B) as mobile phase in gradient elution mode and Waters Reliant C18 (4.6 × 250 mm, 5 µm) column as stationary phase. Optimised flow rate, injection volume and detection wavelength of the HPLC method were 1.0 mL/min, 10 µL and 254 nm, respectively. Chemical structures of degradation impurities were proposed by high resolution mass spectrometry further, major degradation products were isolated, enriched and investigated thoroughly with the aid of nuclear magnetic resonance spectroscopy studies. The degradation impurities were identified as 4-aminophthalaldehyde (DP 1), 4-((4-methylpiperazin-1-yl)methyl)− 3-(trifluoromethyl) benzenamine (DP 2), 3-(2-(imidazo[1,2-b]pyridazin-3-yl)acetyl)− 4-methylbenzoic acid (DP 3), 3-(2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl)− 4-methylbenzoic acid (DP 4) and N-oxide impurity (DP 5) which are new and were not reported in the literature till date. Additionally, toxicity and mutagenicity profiles of ponatinib and its degradation impurities were predicted in-silico by using DEREK and SARAH software. This whole study gives meaningful insights about chemical stability of ponatinib which is useful in its drug development lifecycle.