Development of a chiral HPLC method for the separation and quantification of hydroxychloroquine enantiomers

Abstract

Hydroxychloroquine (2-[[4-[(7-Chloroquinolin-4-yl) amino]pentyl](ethyl) amino]-ethanol, HCQ), an effective anti-malarial drug, has been applied in the clinics for potential treatment of severe coronavirus disease 2019 (COVID-19). Although the clinical benefits of HCQ require extensive clinical data to confirm, the existence of a chiral center in the molecule to possess two optical isomers suggests that there might be an enantiomeric difference on the treatment of COVID-19. Due to poor resolution and the inability of quantification by previously reported methods for the analysis of HCQ enantiomers, it is necessary to develop an analytical method to achieve baseline separation for quantitative and accurate determination of the enantiomeric purity in order to compare the efficacy and toxicity profiles of different enantiomer. In this study, we developed and validated an accurate and reproducible normal phase chiral HPLC method for the analysis of two enantiomers of HCQ, and the method was further evaluated with biological samples. With this newly developed method, the relative standard deviation of all analytes was lower than 5%, and the limit of quantification was 0.27 μg/ml, 0.34 μg/ml and 0.20 μg/ml for racemate, R- and S-enantiomer, respectively. The present method provides an essential analytical tool for preclinical and clinical evaluation of HCQ enantiomers for potential treatment of COVID-19.