doi:10.1016/j.chroma.2005.01.015 
Copyright © 2005 Elsevier B.V. All rights reserved.
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
H.B. Xiaoa, b, M. Kruckera, K. Putzbacha and K. Alberta, 
, 
aUniversity of Tuebingen, Institute of Organic Chemistry, Auf der Morgenstelle 18, D-72076 Tuebingen, Germany
bDalian Institute of Chemical Physics, Chinese Academy of Sciences, 161 Zhongshan Road, 116011 Dalian, PR China
Available online 29 January 2005.
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Abstract
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.
Keywords: Capillary LC–NMR; Natural products; Radix astragali; Structure elucidation; HPLC–MS
Fig. 1. Structures of four known major isoflavones in R. astragali identified in this study.
Fig. 2. HPLC–UV traces for the isoflavone fractions of R. astragali obtained by matrix solid-phase dispersion method. (A) HPLC separation of one isoflavone fraction on a 250 mm × 4.6 mm i.d. Bischoff ProntoSIL Eurobond C18 5 μm column at 1.0 mL/min, (B) capillary HPLC separation of the sum of 10 fractions on a lab-made 150 mm × 250 μm i.d. fused-silica capillary column packed with Bischoff EUROBond C18 (120 Å, 5 μm) particles at 5.0 μL/min. See text for the detailed chromatographic conditions and Table 1 for peak identifications.
Fig. 3. Multi-stage mass spectrometry of peaks 1–4 in Fig. 2 obtained by HPLC–MSn. (A) ESI–MS, MS–MS (MS2) and triple MS (MS3) of peak 1. (B) ESI–MS and MS–MS (MS2) of peak 2. (C) ESI–MS, MS–MS (MS2) and triple MS (MS3) of peak 3. (D) ESI–MS and MS–MS (MS2) of peak 4. The peaks were ionized using positive electrospray ionization (ESI) and the MS–MS and triple MS were obtained by collision-induced dissociation (CID) in ion trap. See text for the detailed mass spectrometric conditions and deductions about fragmentation procedures.
Fig. 4. Retro-Diels-Alder (RDA) fragmentation procedure (m/z 285 → 137) of peak 3 (calycosin (3)).
Fig. 5. Stopped-flow 1H NMR spectrum of peak 1 (calycosin-7-O-β-d-glucopyranoside (1)) in Fig. 2 obtained with capillary HPLC–NMR equipped with a 1.5 μL solenoidal NMR probe. The spectrum was recorded with a 30 k transients corresponding to an overnight acquisition time, a time domain of 64 k and a sweep width of 12,195 Hz and referenced to the solvent signal of residual CH3CN at δ 1.93 ppm. See Table 1 for peak assignment. *Ghost signal from system.
Fig. 6. The aromatic regions of the 1H NMR spectra of peaks 1–4 in Fig. 2 obtained by direct stopped-flow capillary HPLC–NMR. (A) 30 k transients for peak 1. (B) 12 k transients for peak 2. (C) 1 k transients for peak 3. (D) 1.4 k transients for peak 4. All spectra were recorded with a time domain of 64 k and sweep width of 12,195 Hz and referenced to the solvent signal of residual CH3CN at δ 1.93 ppm. See Table 1 for peak assignment. *Ghost signal from system.
Table 1.
1H NMR data (chemical shifts and coupling constants) and comparison with literature values for peaks 1–4 shown in Fig. 2
a Identified structure.
b Chemical shift differences calculated by subtracting the chemical shifts measured in this study from the values reported in reference
[32].
c Chemical shift differences calculated by subtracting the chemical shifts measured in this study from the values reported in reference
[33].
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