Enantioselective Properties of Neglected Drug Nifurtimox on Polysaccharide and Macrocyclic Glycopeptide Chiral Stationary Phases by Green HPLC Separation Methods.

Nifurtimox (NFX) is a chiral drug used for the treatment of Chagas Disease. Little attention has been paid to the enantioselective properties of chiral drugs used for neglected tropical diseases, highlighting the need for further studies in this area. In this work, the enantioselective properties of NFX were carefully investigated by HPLC using different chiral stationary phases (CSPs) and chromatographic modes. Two enantioseparation HPLC methods were successfully developed and validated. Polar ionic, polar organic, and RP modes were tested, and the polar organic mode proved to be suitable for the enantioseparation. Chromatographic resolution was achieved using the polysaccharide-based Chiralpak AD CSP and the macrocyclic glycopeptide-based Chirobiotic V CSP. Both methods employed ethanol as a mobile phase, contributing to greener analytical practices. The influence of the CSPs temperature on the retention of the enantiomers was investigated, and the analysis temperatures were set at 25°C. Thermodynamic parameters were also studied, and the enantioseparation process was found to be enthalpy-driven. Furthermore, molecular docking studies were conducted to identify the interactions of NFX with the CSPs constituents and to predict the elution order of the enantiomers. The elution order of the enantiomers was determined by comparing the experimental electronic circular dichroism (ECD) spectra with the theoretical ECD spectra, which aligned with the predictions made through molecular docking. The environmental impact of the developed methodologies was evaluated using the green metrics MoGAPI and AGREE, confirming their eco-friendly nature. The methods can be employed in future enantioselectivity studies and drug analysis.