Capillary liquid chromatography–microcoil 1H nuclear magnetic resonance spectroscopy and liquid chromatography–ion trap mass spectrometry for on-line structure elucidation of isoflavones in Radix astragali

Miniaturization and hyphenation of chromatographic separation techniques to nuclear magnetic resonance spectroscopy is being increasingly demanded in the field of biomedical, drug metabolite and natural product analysis. Herein, capillary liquid chromatography was coupled on-line to microcoil 1H nuclear magnetic resonance spectroscopy (capLC–NMR) equipped with a 1.5 μL solenoidal probe for structure elucidation of isoflavones in Radix astragali. The extract was screened by HPLC–UV–MS as the preliminary step and four major peaks were identified tentatively by ion trap mass spectrometry molecular weights and characteristic fragments. Then, stopped-flow capLC–UV–NMR was performed using 33 μg extract injected on-column. The four peaks were parked manually in the micro probe one by one and corresponding 1H NMR spectra were recorded with good resolutions under the applied capLC–NMR conditions (120 and 220 ng injected on-column for peaks 2 and 4, respectively). All aromatic regions of 1H NMR spectra correlated well to the characteristic signals of isoflavone aglycone protons. And the signal corresponding to the anomeric proton of the glucopyranoside of isoflavone glycoside was also obtained for peak 1. Therefore, these four peaks are determined as calycosin-7-O-β-d-glucopyranoside (1), ononin (2), calycosin (3) and formononetin (4) unambiguously. The capLC–NMR results indicate that this hyphenated technique could be used for the determination of a great variety of natural products from small sample amounts, e.g., only 5 g R. astragali in this study.