JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2002; 37: 1272 – 1279 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.402 Direct stereochemical assignment of hexose and pentose residues in flavonoid O-glycosides by fast atom bombardment and electrospray ionization mass spectrometry † Filip Cuyckens, Abdelaaty A. Shahat, Luc Pieters and Magda Claeys * Department of Pharmaceutical Sciences, University of Antwerp (UIA), Universiteitsplein 1, B-2610 Antwerp, Belgium Received 21 May 2002; Accepted 24 October 2002 Mass spectrometric methods have been developed which allow the direct stereochemical assignment of terminal monosaccharide residues in flavonoid O-glycosides without the need for chemical hydrolysis. Standards containing a glucose, galactose, mannose, xylose, arabinose or apiose residue were examined because these monosaccharides are by far the most commonly encountered in flavonoid glycosides. Following acetylation, the major peracetylated sugar related fragments, generated by fast atom bombardment (FAB) or electrospray ionization (ESI), were selected for collisional activation employing a broad range of collision energies. Both FAB and ESI proved to be useful as ionization techniques. Stereoselective fragmentation was achieved and allowed us clearly to differentiate and characterize isomeric monosaccharide residues. The method developed was successfully applied to an unknown flavonoid containing a terminal pentose and hexose residue which was isolated from Farsetia aegyptia. Copyright  2002 John Wiley & Sons, Ltd. KEYWORDS: flavonoid glycosides; sugar residues; electrospray ionization; fast atom bombardment INTRODUCTION Flavonoid glycosides are predominant forms of naturally occurring flavonoids in plants, representing a large group of secondary plant metabolites. They all contain a C 15 skeleton as an aglycone, are usually divided into O- and C-glycosyl flavonoids and are of interest because they have various biological activities, are useful for chemotaxonomy and are used as tracers in medicinal plant preparations. 1,2 Flavonoids generally occur as glycosides in plants because the effect of glycosylation renders the flavonoid less reactive and more water soluble, permitting storage of the flavonoids in the cell vacuole where they are mostly found. Glucose is by far the monosaccharide most commonly bound to flavonoids, whereas galactose, rhamnose, xylose and arabinose are not uncommon and mannose and apiose are also occasionally encountered. 1 Although 1 H and 13 C NMR techniques are preferred for stereochemical assignment, mass spectrometry (MS) can make a valuable contribution because it can be applied to much smaller quantities of isolated compounds. It provides the molecular mass, in addition to structural information on the flavonoid skeleton, 3–15 attachment points of carbohydrate residues to the aglycone, 6–8 the types of L Correspondence to: Magda Claeys, Department of Pharmaceutical Sciences, University of Antwerp (UIA), Universiteitsplein 1, B-2610 Antwerp, Belgium. E-mail: claeys@uia.ua.ac.be † Paper presented at the 20th Informal Meeting on Mass Spectrometry, Fiera di Primiero, Italy, 12–16 May 2002. carbohydrates present (mono-, di-, tri- or tetrasaccharides and hexoses, deoxyhexoses or pentoses) 9–11 and the type of interglycosidic linkages. 12 – 15 Early investigations of car- bohydrates by means of MS analysis were performed by resorting to acetyl and methyl derivatives. 16,17 Derivatization of the compounds was required in order to obtain sufficient volatility necessary for electron ionization (EI) and chemical ionization (CI). Although more modern soft ionization tech- niques such as fast atom bombardment (FAB) 18 – 20 allow the detection of molecular ion species (i.e. protonated, depro- tonated or sodiated species) of underivatized saccharides, Richter and co-workers 21 – 24 showed that acetylation can still be useful in the structural characterization of oligosaccha- rides and glycoside residues. It was demonstrated that the differentiation between a terminal glucose, galactose and mannose residue of naturally occurring glycosides could be achieved by low-energy collision-induced dissociation (CID) using a triple-quadrupole mass spectrometer equipped with a CI source. In this study, we followed the same approach for developing methodology to differentiate and characterize the terminal monosaccharide residue of flavonoid O-glycosides. Standards containing the major hexoses and pentoses found attached to flavonoids were analyzed utilizing FAB and elec- trospray ionization (ESI). Deoxyhexoses were not included in this study because, to our knowledge, only rhamnose occurs in association with flavonoids and can easily be distinguished from pentoses and hexoses by its different mass. Finally, the monosaccharide residues of an unknown flavonoid, isolated Copyright  2002 John Wiley & Sons, Ltd.