engleski

2D LC HRMS analysis of bivalirudin and its degradation products

Bivalirudin is a direct thrombin inhibitor which is used as anticoagulant for patients with acute coronary syndromes or patients undergoing percutaneous coronary intervention [1]. It is a synthetic oligopeptide, consists of 20 amino acids (D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly- Asp- Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu), and specifically binds to the thrombin at two sites, catalytic site and anion binding site. Bivalirudin is administrated intravenously, and have a rapid anticoagulant effect. An UPLC method for determination of bivalirudin in the presence of its degradation products has been developed using quality-by-design approach. Novel method development approach was applied - two software packages, Fusion AE and DryLab, were used in conjunction to obtain method that can provide best selectivity in a very short analysis time. This approach has shown to be fast, systematic and effective. Method development was carried out in two phases: development phase - performed using Fusin AE software and the optimization phase - conducted using DryLab software. Main goal of the development phase was to define chromatographic parameters that will separate all degradation impurities and the main goal of optimization phase was to preserve separation power but at about five times shorter run time. Developed UPLC method was validated as well, and was proven to be an effective method for separating bivalirudin from its degradation products. Forced degradation studies were performed using acid, base, H2O2, heat and light exposure as recommended by the International Conference on Harmonization (ICH). High resolution MS and MS/MS analysis were performed to propose the structure of degradation products generated under force degradation conditions. 2D-UPLC instrument was used to remove non-volatile buffer used as a mobile phase. Molecular structure was proposed for 14 degradation impurities.

Bivalirudin, LC-HR MS

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