Liquid chromatography coupled to nuclear magnetic resonance spectroscopy for the identification of isoflavone glucoside malonates in T. pratense L. leaves.
Previous studies revealed that the main isoflavones in extracts of leaves of T. pratense L. are biochanin A and formononetin, their 7-O-glucosides, and two glucoside malonate isomers of each of them. Since LC-MS(/MS) did not provide sufficient information to distinguish the glucoside malonate isomers, in the present paper LC-NMR as well as off-line two-dimensional NMR were used to obtain further structural information. Matrix solid-phase dispersion (MSPD) was applied to obtain sufficiently high analyte concentrations to perform LC-NMR. Stop-flow reversed-phase LC-NMR was performed using a gradient of deuterated water and deuterated acetonitrile. Offline COSY and NOESY experiments were carried out to determine the positions of the glucose moiety on the flavonoid aglycone, and of the malonate moiety on the glucose. Based on the fragmentation patterns in MS/MS and the NMR spectra, the two formononetin glucoside malonate isomers were identified as 7-O-beta-D-glucoside 6"-O-malonate and 7-O-beta-D-glucoside 4"-O-malonate; i.e. they only differ in the substitution position of the malonate group on the glucoside ring. The biochanin A glucoside malonate isomers, however, have quite different structures. The main and later eluting isomer is biochanin A 7-O-beta-D-glucoside 6"-O-malonate, and the minor and earlier eluting isomer is 5-hydroxy-7-methoxyisoflavone 4'-O-beta-D-glucoside 4"-O-malonate: the positions of the methoxy group and the glucoside 6"-O-malonate group on the flavonoid skeleton are interchanged.
de Rijke
Eva
E
Vrije Universiteit, Department of Analytical Chemistry and Applied Spectroscopy, de Boelelaan 1083, 1081 HV Amsterdam, the Netherlands.
de Kanter
Frans
F
Ariese
Freek
F
Brinkman
Udo A Th
UA
Gooijer
Cees
C
D002853:Chromatography, Liquid / Q000379:methods; D005960:Glucosides / Q000737:chemistry*; D007529:Isoflavones / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008314:Malonates / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D015394:Molecular Structure; D018515:Plant Leaves / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013997:Time Factors; D029921:Trifolium / Q000737:chemistry*
D005960:Glucosides; D007529:Isoflavones; D008314:Malonates