The effect of the stationary phase on the separation of Cinchona alkaloids

Abstract

Abstract Background: The production of enantiopure drugs has become a key focus for pharmaceutical companies. However, the analysis and preparation of pure enantiomers pose significant challenges and are often costly. Chiral separation methods, such as chiral High-Performance Liquid Chromatography (HPLC), have been effective but have their own challenges. In this context, the use of achiral columns as a potential alternative due to their lower cost and effectiveness in separating diastereomers gains significant relevance. Cinchona alkaloids are natural compounds isolated from the bark of Cinchona trees. Quinine, the most important of them, has been used to treat malaria for over 400 years. However, impurities found in quinine formulations have raised questions regarding their potential impact on the potency of quinine and the accuracy of the reported IC50 values available to date. Objective: This thesis combines a systematic literature review and experimental investigation. The literature review examined studies on the use of non-chiral columns for chiral molecule separation and investigated the IC50 values of Cinchona alkaloids in malaria. The experimental objective was to assess the effectiveness of different achiral columns in separating seven chiral Cinchona alkaloids, focusing on achieving optimal separation efficiency and shorter retention times. Method: The literature review involved searching and analyzing for relevant studies on separating chiral molecules using achiral columns, and from 134 screened articles, five articles were included in our study. The other literature review investigated the IC50 values of Cinchona alkaloids in malaria, and from 82 screened articles, 13 articles were included in our study. The experimental part of the study focused on studying the separation of Cinchona alkaloids employing six distinct achiral columns. Furthermore, the investigation included assessing the influence of various mobile phase compositions characterized by differing ratios of water and acetonitrile and exploring the impact of pH on separation efficacy on different columns. Reversed-phase HPLC coupled with Mass Spectrometry (MS) detection in an acidic mobile phase was utilized for the separation and subsequent analysis. Results: The literature review revealed that the IC50 values for quinine varied widely among studies from 5.76 nM to 1911 nM. However, small sample sizes in some studies could affect the reliability of the results. Also, differences in study design, experimental conditions, and assay methods could affect the variability of IC50 values. The other literature review revealed that none of the studies examined successfully separated enantiomers with achiral columns but managed to separate diastereomers and cis and trans isomers. In the experimental part, C18-HL, C18-PFP, and Diphenyl achiral columns showed good baseline separation of Cinchona alkaloids, with the C18-HL column demonstrating the highest selectivity. The mobile phase composition and pH levels (0.1% and 0.5% formic acid) influenced the retention times and separation quality. Notably, a decrease in acetonitrile concentration from 10% to 7% in the mobile phase led to increased retention times across all columns. Furthermore, pH exerted a notable influence on retention times, especially evident in the C18-PFP column, where the retention time was 17.04 minutes shorter in the mobile phase composition of 93:7 water with 0.5% formic acid:acetonitrile compared to the same composition but with 0.1% formic acid. Despite these variations, the elution order of the analytes remained consistent across all tested columns, underscoring the importance of stereochemistry in retention. Conclusion: The shortest retention time (24.76 min), achieving baseline separation for seven peaks, was observed with the C18-HL column using a mobile phase composition of 93:7 water with 0.5% formic acid and acetonitrile. pH exerted varying effects on retention times across all columns, with the most significant impact observed with the C18-PFP column and the least effect observed with the Diphenyl column. This research underscores the potential of achiral columns in separating chiral molecules, offering a promising avenue to streamline the process of analyzing and preparing enantiopure drugs. However, it also highlights the need for further research to fully explore their capabilities, particularly in the area of separating enantiomers, a potential area of growth in this field.