Separation and quantitation of protoanemonin in ranunculaceae by normal- and reversed-phase HPLC

A method is described for the extraction and high-performance liquid chromatographic separation and quantitation of the lactone protoanemonin in leaves of Ranunculaceae. For samples with a protoanemonin concentration over 10 microg/g W. W., a reversed-phase technique using a Lichrosorb RP 18 column and a binary solvent system was developed. Alternatively, for samples with a lower lactone concentration, a normal-phase technique with a Lichrosorb Si 60 and quaternary elution system was elaborated. Protoanemonin was detected at 258 nm; its calibration curves were established, and its response factor was calculated using, as standard, the pure compound extracted from HELLEBORUS NIGER. A survey of ten different taxa of Ranunculaceae was performed and it showed the suitability of the method for routine work with high sensitivity limit.