Optimization of a liquid chromatography method based on simultaneous electrospray ionization mass spectrometric and ultraviolet photodiode array detection for analysis of flavonoid glycosides.
Different reversed-phase liquid chromatography (LC) columns of conventional dimensions were coupled to an ultraviolet photodiode array detector (UV-DAD) and a magnetic sector-type spectrometer, equipped with an electrospray ionization (ESI) source, by a laboratory-made flow splitter. A mixture of three flavonoid-O-glycosides was employed to examine the effects of the solvent composition, the flow rate, the stationary phase, the pH and the organic acid added, on the chromatographic separation, the UV-DAD detection, the ESI process and the entire LC system with ESI-MS and UV-DAD detection. In the positive ion mode, methanol containing 1% acetic acid was by far the most sensitive in ESI-MS analysis, whereas an acetonitrile/water mobile phase containing 0.5% formic acid was proved to give the best sensitivity in LC/ESI-MS/UV-DAD analysis. In the negative ion mode, the highest sensitivity was obtained with a mobile phase containing 0.1% formic acid, while addition of bases decreased the sensititvity. The optimal flow rate was higher in negative ESI (20-50 micro L/min) than in positive ESI (5 micro L/min), and the percentage of organic phase had an influence on the sensitivity of ESI-MS detection. With regard to the selection of a suitable C(18) reversed-phase LC column, a column which is well end-capped is to be preferred, because residual silanol groups appear to impair the separation of flavonoid glycosides. The optimized LC/ESI-MS/UV-DAD method was applied to a commercial Crataegus extract, which is used in phytomedicine to treat cardiovascular problems and is known to be rich in flavonoids. It is demonstrated how UV spectra and first-order ESI mass spectra allow a fast characterization of flavonoids, even if reference compounds are not available or at hand.
Cuyckens
Filip
F
University of Antwerp (UIA), Department of Pharmaceutical Sciences, Universiteitsplein 1, 2610 Antwerp, Belgium.
Claeys
Magda
M
D000143:Acids; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D027825:Crataegus / Q000737:chemistry; D004563:Electrochemistry; D005419:Flavonoids / Q000032:analysis* / Q000737:chemistry; D006027:Glycosides / Q000032:analysis* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D010936:Plant Extracts / Q000737:chemistry; D012680:Sensitivity and Specificity; D012997:Solvents; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014466:Ultraviolet Rays
D000143:Acids; D005419:Flavonoids; D006027:Glycosides; D010936:Plant Extracts; D012997:Solvents; C007145:crataegus extract