Development of a fast RP-HPLC method for the separation of nifurtimox and its forced degradation products through a design of experiment approach

Abstract

The study depicts the successful development and validation of a simple and fast reverse-phase high-performance liquid chromatography (RP-HPLC) method for the separation of nifurtimox (NFX) and its degradation products. NFX was exposed to different degradation conditions and a RP-HPLC method reported in the literature was used to evaluate different peaks that could interfere with the one from the pure NFX. NaOH generated a degradation product, with a peak that tends to overlap with NFX. Therefore, the NFX exposed to this condition was used during screening and optimization of the RP-HPLC method using a Design of Experiment (DoE) approach. A factorial design was applied to screen different factors, followed by a central composite design for process optimization with those factors that significantly affected the evaluated responses during the screening step. The optimized method was further validated according to International Conference of Harmonization guidelines. Methanol proportion and gradient time to reach it, alongside with mobile phase flow rate, were the factors that had a statistically significant effect on resolution, retention time, and tailing factor in the chromatographic peak. The optimized chromatographic parameters were: 41.6 % of methanol, a gradient time of 2 min, and a flow rate of 0.62 mL/min. Under these chromatographic conditions, the retention time of NFX was 5.3 min, while for the nearest degradation products was 4.5 min and 6.3 min. The resolution of the NFX peak was 5.14 and the tailing was 1.13. The DoE approach allowed the development of an optimized RP-HPLC method capable of successfully separating NFX and its forced degradation products.