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

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-beta -D-glucoside-6-O-malonate, 3-methylorobol-7-O-beta -D-glucoside-6-O-malonate, calycosin-7-O-beta -D-glucoside-6-O-malonate, pratensein-7-O-beta -D-glucoside-6-O-malonate, pseudobaptigenin-7-O-beta -D-ghucoside-6-O-malonate, formononetin-7-O-beta -D-glucoside-6-O-malonate, irilone-4'-O-beta -D-glucoside-6-O-malonate, afrormosin-7-O-beta -D-glucoside-6-O-malonate, biochanin A-7-O-beta -D-glucoside-6-O-malonate, texasin-7-O-beta -D-glucoside-6-O-malonate, 5,7,2'-trihydroxy-6-methoxyisoflavone-7-O-beta -D-glucoside-6-O-malonate, prunetin-4'-O-beta -D-glucoside6-O-malonate) and six acetyl glycosides (daidzin-6-O-acetate, formononetin-7-O-beta -D-glucoside-6-O-acetate, pseudobaptigenin-7-O-beta -D-glucoside-6-O-acetate, irilone-4'-O-beta -D-glucoside-6-O-acetate, biochanin A-7-O-beta -Dglucoside-6-O-acetate and prunetin-4'-O-beta -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. (C) 2001 Elsevier Science B.v. All rights reserved.