SEPARATION AND IDENTIFICATION OF PHYTOALEXINS FROM LEAVES OF GROUNDNUT (ARACHIS-HYPOGAEA) AND DEVELOPMENT OF A METHOD FOR THEIR DETERMINATION BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Leaves of groundnut, Arachis hypogaea, infected with the early leaf spot fungus, Cercospora arachidicola, were extracted in aqueous ethanol and the phytoalexins partitioned into ethyl acetate. Flash chromatography of the ethyl acetate extract on silica gel yielded fractions with one to five compounds from which the phytoalexins could be isolated by semipreparative reversed-phase high-performance liquid chromatography (HPLC). The major phytoalexins were demethylmedicarpin, formononetin, 7,4'-dimethoxy-2'-hydroxyisoflavanone and medicarpin. Minor components were 7,2'-dihydroxy-4'-methoxyisoflavanone and daidzein. Compounds were identified by cochromatography and comparison of their ultraviolet and mass spectra with authentic samples using an HPLC system equipped with a diode-array detector, HPLC mass spectrometry and gas chromatography - mass spectrometry of their trimethylsilyl derivatives. A solid-phase extraction method was developed for processing large numbers of samples. Acetonitrile eluates from C18 cartridges were separated by reversed-phase HPLC and the phytoalexins quantified by reference to external standards of the authentic compounds.