Resolution Of Higenamine Enantiomers Derivatized With Fluorescent Chiral Tagging Reagent By High-performance Liquid Chromatography

Many chiral drugs may differ in their pharmacological activities due to the difference of stereochernistry. Although most natural compounds contain the eliminations, but there is no systematically and deeply biological active and toxicology researches up to now. In an effort to achieve the above aim, separation analysis method of natural eliminations should be developed.Higenamine is an active ingredient of Aconite root, Asarum, embryo of Nelumbo nucifera and Gnetum parvifolium, Chinese herbal medicine. In this paper, A sensitive method for determination of higenamine enantiomers based on a derivatization reaction with a fluorescent chiral tagging reagent, R-(-)/S-(+)-4-(3-isothi ocyanatopyrrolidin-1-yl)-7-(N,N-Dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R-(-)/ S-(+)-DBD-PyNCS] has been developed. The tagging reagent preferably reacts with higenamine enantiomers under mild reaction conditions (at 60℃for 60 min) in the presence of 3%pyridine to produce the corresponding fluorescent diastereomers with an excitation maximum at 450 nm and an emission maximum at 550 nm. The derivatives of higenamine enantiomers were efficiently resolved on a conventional ODS column. The resolution (Rs) values of the corresponding higenamine diastereomers was 1.52. The calibration curves of both R-(+)/S-(-)-higenamine eantiomers were linear over the concentration range of 0.2-400μg/mL. The limits of detection (S/N= 3) per injection were 20 ng/mL. The developed method was applied successfully to the determination of higenamine in embryo of Nelumbo nucifera, a Chinese herbal medicine.