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

BEDOGNI, GISELLE; AZCÁRATE, FEDERICO; SEREMETA, KATIA; OKULIK, NORA; SALOMON, CLAUDIO; BOSCHETTI, CARLOS

JOURNAL OF MOLECULAR LIQUIDS

ELSEVIER SCIENCE BV

Año: 2024

0167-7322

The study depicts the successful development and validation of a simple and fast reverse-phasehigh-performance liquid chromatography (RP-HPLC) method for the separation of nifurtimox(NFX) and its degradation products. NFX was exposed to different degradation conditions and aRP-HPLC method reported in the literature was used to evaluate different peaks that could interferewith the one from the pure NFX. NaOH generated a degradation product, with a peak that tends tooverlap with NFX. Therefore, the NFX exposed to this condition was used during screening andoptimization of the RP-HPLC method using a Design of Experiment (DoE) approach. A factorialdesign 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 thescreening step. The optimized method was further validated according to International Conferenceof Harmonization guidelines. Methanol proportion and gradient time to reach it, alongside withmobile 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 chromatographicparameters 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 thenearest degradation products was 4.5 min and 6.3 min. The resolution of the NFX peak was 5.14and the tailing was 1.13. The DoE approach allowed the development of an optimized RP-HPLCmethod capable of successfully separating NFX and its forced degradation products.