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Enzyme-linked immunosorbent assays in combination with semi-preparative high-performance liquid chromatography (HPLC) and analytical HPLC with mass spectroscopy in the selective ion monitoring mode were used for the determination of selected isoflavones, daidzein, genistein, biochanin A and their homologues, in 20 representatives of the Rutaceae family. Species belonging to five genera were studied, namely Citrus, Fortunella, Poncirus, Ruta and Severinia. The enzyme immunoassays used were based on polyclonal antibodies raised against isoflavonoid conjugates with bovine serum albumin (BSA), namely biochanin A-7-BSA, daidzein-7-BSA, daidzein-4'-BSA, genistein-7-BSA and genistein-4'-BSA. Aglycones as well as glycosides were detected, and methoxyisoflavones appeared to be more abundant than hydoxyisoflavones. The content of individual isoflavonoids ranged from 0 to 2.6 mg/kg (dry weight); the sum of all measured substances reached up to 5.9 mg., ((c) 2007 John Wiley & Sons, Ltd.)

Several types of compounds with immunoreactivity similar to isoflavonoids were detected in water: ethanol extracts of leaves of Fortunella obovata Hort. ex Tanaka, Murraya paniculata Jack. and four Citrus species, namely C. aurantium L, C. grandis Osbeck, C. limonia Osbeck., and C. sinensis Osbeck (Rutaceae). The chromatographic mobilities of the immunoreactive substances were compared with those of authentic standards, revealing a spectrum of isoflavonoid metabolites in all plants studied. Aglycones as well as glycosides were recognized, namely daidzin, genistin, daidzein, genistein, formononetin, biochanin A, prunetin, and several incompletely characterized isoflavonoids. A subsequent HPLC-MS study verified the identities of the main immunoreactive isoflavonoids and established the identities of several others, viz. glycitein, glycitin, ononin and sissotrin, including the malonylated and acetylated isoflavonoid glucosides. The estimated content of the individual immunoreactive entities ranged from a few microg to about 2 mg/kg (dry weight). It is concluded that the isoflavonoid metabolic pathway is present throughout the Rutaceae family.