Hyphenation of centrifugal partition chromatography with electrospray ionization mass spectrometry using an active flow-splitter device for characterization of flavonol glycosides.

Online coupling of centrifugal partition chromatography to electrospray ionization mass spectrometry (CPC/ESI-MS) was investigated for the separation and characterization of flavonol glycosides. Structural identification and purification monitoring of analytes on milligram scale were demonstrated to be possible by using an active flow-splitter device which transfers automatically and successively, at discrete frequencies, small aliquots of the chromatographic effluent to an independent auxiliary stream directed to an ESI quadrupole mass spectrometer. The CPC protocol used a biphasic solvent system composed of ethyl acetate/ethanol/water (4.5:1:4.5, v/v/v) in isocratic mode. During the separation process, continuous acquisition of mass spectral data of the isolated flavonols from the effluent was performed in the negative ion mode with an auxiliary stream composed of 50 mM ammonium acetate/ethanol (2:8, v/v) delivered by a secondary pump. To demonstrate the potential of this hyphenated technique, flavonol glycosides from an apple peel extract were identified, purified and quantitatively analyzed. Calibration curves and limits of detection are also detailed. Copyright (c) 2009 John Wiley & Sons, Ltd.