Desenvolvimento e otimização de método cromatográfico para separação de três ativos e suas respectivas impurezas com aplicação de planejamento fatorial

Abstract

An essential part of the development of a new drug is the development of methods to analyze the products during the stage of development and throughout their useful life to ensure quality and safety for patients. The analytical method must be indicative of stability, that is, capable of detecting, over time, changes in the physical, chemical or microbiological properties of a substance. Other important characteristics describe that they are specific methods able of accurately measuring the dosage of the active pharmaceutical ingredient (mg/tablet), degradation products (% of impurity) and other components of interest, without interference. In order to identify and quantify different components of a product's formulation, chromatographic techniques are widely used. By means of this technique it is possible to properly separate the substances present in the formulation and quantify several analytes in the same analytical run. Several factors interfere in the separation of components in the chromatographic method, highlighting the pH of the mobile phase, the type of material in the stationary phase, the type of solvent and its concentration in the mobile phase, among others. The goal was to use chemometric tools to explore the principles of separation of three drugs and their respective impurities and to develop an indicative method of stability by HPLC-DAD. The initial tests were performed on a sample built with the presence of the three actives and their impurities, which were added or generated through sample stress. Chromatographic separation was evaluated in screening and optimization tests. Screening was performed with a wide range of mobile phase pH and different chromatographic columns. Different organic solvents and rates of increase in organic solvent concentration (gradient time) along the analytical run were also evaluated. Data were processed using Fusion and Octave software. The analytical method indicative of stability for the separation of the three actives was efficiently developed and validated, presenting itself as selective, linear, accurate and precise. The use of chemometric tools proved to be efficient to detect the limitations of the method and to define the variables to obtain the best performance (greater separation and shorter analysis time).