Ultrasound-assisted solid-phase extraction coupled with photodiode-array and fluorescence detection for chemotaxonomy of isoflavone phytoestrogens in Trifolium L. (Clover) species.

Detailed chemotaxonomic studies were undertaken to establish the qualitative profile and real amounts of the pharmacologically active isoflavone aglycones genistein, daidzein, formononetin, and biochanin A in aerial parts of thirteen Trifolium L. (clover) species, native to Poland. A newly elaborated micropreparative technique--SPE--on BakerBond octadecyl, cyclohexyl, and phenyl cartridges was used in combination with ultrasound-assisted extraction for isolation of isoflavone aglycones from hydrolyzed samples. The effectiveness of all three SPE sorbents in the purification of plant extracts was compared and very high recoveries (>96%) were documented for four isoflavones. Classical photodiode-array and very sensitive fluorescence detection, coupled with reversed-phase high-performance liquid chromatography (RP-HPLC), were employed to obtain the most reliable qualitative and quantitative results. Chemotaxonomic differences combined with flower color variability were demonstrated within thirteen clover species. Concentration levels of particular isoflavones in ten Trifolium species possessing flowers with white, pink, or purple-red corolla ranged from approximately 3 to approximately 3300 microg/g dry weight, while in three yellow flowering clovers (T. aureum, T. dubium, and T. campestre) isoflavone compounds have not been detected at all. RSD values, determined for intra- and inter-day precision of the quantitative results, were not higher than 6.2% and 7.1%, respectively.