An Optimised Method for Routine Separation and Quantification of Major Alkaloids in Cortex Cinchona by HPLC Coupled with UV and Fluorescence Detection.

Abstract

Authentication of herbal products to ensure efficacy and safety require efficient separation and quantification of constituents. Standard assays for Cinchona bark used for the treatment of malaria and production of quinine, either use only spectrophotometry to detect two pairs of diastereoisomers of quinine and cinchonine type alkaloids (European Pharmacopoeia, Ph.Eur.) or liquid chromatography primarily optimised for detection of the four major alkaloids. However, numerous minor alkaloids occur in Cinchona and related species and efficient separation including gradient elution is necessary in order to obtain the full pattern of constituents in bark samples. To develop an optimised HPLC method for separation and quantitative analysis of the four major alkaloids in Cinchona bark using UV detection. Dimethyl sulphoxide (DMSO) extracts of 50mg of pulverised barks were prepared using ultrasonication. The chromatographic separation was performed on an XB-C18 column packed with 2.6μm particles. Gradient elution using an ammonium formate buffer and methanol as organic modifier over 26min was based on non-chiral separation of the diastereoisomers and the high solvent selectivity of methanol. Post column UV detection was performed at 250nm and 330nm. Fluorescence detection was performed using 330nm for excitation and 420nm for emission. The optimised HPLC method facilitates efficient separation and quantification of the four major alkaloids in 26min with a limit of quantification of 5μg/g from 50mg bark sample. The optimised HPLC method offers a simple and efficient quantification of the four major alkaloids. Copyright © 2017 John Wiley & Sons, Ltd.