Determination of R-(+)-higenamine enantiomer in Nelumbo nucifera by high-performance liquid chromatography with a fluorescent chiral tagging reagent

A sensitive method for determination of higenamine enantiomers based on a derivatization reaction with a fluorescent chiral tagging reagent, R-(−)/S-(+)-4-(3-isothiocyanatopyrrolidin-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 °C 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 ODS column by an isocratic elution with water–acetonitrile–methanol mobile phase containing 0.2% formic acid. The resolution (Rs) values of the corresponding higenamine diastereomers was 1.52. The calibration curves of both R-(+)/S-(−)-higenamine enantiomers were linear over the concentration range of 0.2–400 μg/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.