Identification of isoflavone conjugates in red clover (Trifolium pratense) by liquid chromatography–mass spectrometry after two-dimensional solid-phase extraction Original Research Article

A high-performance liquid chromatography–mass spectrometry (LC–MS) combined with a two-dimensional solid-phase extraction (2D-SPE) enabled the identification of fourteen isoflavone glycoside malonates (daidzin-6″-O-malonate, genistin-6″-O-malonate, orobol-7-O-β-d-glucoside-6″-O-malonate, 3-methylorobol-7-O-β-d-glucoside-6″-O-malonate, calycosin-7-O-β-d-glucoside-6″-O-malonate, pratensein-7-O-β-d-glucoside-6″-O-malonate, pseudobaptigenin-7-O-β-d-glucoside-6″-O-malonate, formononetin-7-O-β-d-glucoside-6″-O-malonate, irilone-4′-O-β-d-glucoside-6″-O-malonate, afrormosin-7-O-β-d-glucoside-6″-O-malonate, biochanin A-7-O-β-d-glucoside-6″-O-malonate, texasin-7-O-β-d-glucoside-6″-O-malonate, 5,7,2′-trihydroxy-6-methoxyisoflavone-7-O-β-d-glucoside-6″-O-malonate, prunetin-4′-O-β-d-glucoside-6″-O-malonate) and six acetyl glycosides (daidzin-6″-O-acetate, formononetin-7-O-β-d-glucoside-6″-O-acetate, pseudobaptigenin-7-O-β-d-glucoside-6″-O-acetate, irilone-4′-O-β-d-glucoside-6″-O-acetate, biochanin A-7-O-β-d-glucoside-6″-O-acetate and prunetin-4′-O-β-d-glucoside-6″-O-acetate) isolated from red clover (Trifolium pratense). Eight isoflavone glycoside malonates and six acetyl glycosides were firstly identified in red clover. The analyses of crude extracts with those using 2D-SPE extracts were compared. UV spectra, mass spectra of protonated molecules and their fragmentation and subsequent conversion to known flavonoid glycosides were the basis for the identification of the substances.