Isolation of isoflavonoids from Amphimas pterocarpoides and structure elucidation using LC-HRMSn and NMR techniques
In an ongoing project directed toward the discovery of novel phytoestrogens, fifteen isoflavonoids have been traced from the methanolic extract of the stem back of Amphimas pterocarpoides (Leguminosae – Papilionoideae) using an ultra high performance liquid chromatography-photodiode array (UHPLC-PDA) system, hyphenated with a hybrid, high resolution Ion trap-Orbital trap (LTQ-Orbitrap Discovery) mass spectrometer equipped with an electrospray ionization (ESI) probe in negative and positive mode. The proposed structures of these isoflavonoids were determined by analysing the chemical information of each peak such as retention time (Rt), λmax, m/z value of the quasi-molecular ion, m/z value of the MS/MS fragment ions, along with the molecular formulas. Furthermore, the high accurate mass spectra together with the isotope peak information allowed the prediction of possible molecular formulas. The UHPLC-ESI-MS guided fractionation of that methanolic extract, followed by various chromatographic techniques including CC, MPLC, prep-TLC and prep-HPLC resulted in the isolation of eight known isoflavonoids, two isoflavonoid glycosides as well as one novel isoflavone (7-methyl dihydrotectorigenin).
The structure elucidation of the purified compounds was performed via 1 and 2D NMR techniques. The NMR data were in accordance with the HRMS and MS/MS data verifying the proposed chemical structures.