Journal of Chromatography A, 1206 (2008) 11–20 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Analysis of heartsease (Viola tricolor L.) flavonoid glycosides by micro-liquid chromatography coupled to multistage mass spectrometry Viktoria Vukics a,b,1 , Thomas Ringer a,1 , Agnes Kery b , Guenther K. Bonn a , Andras Guttman a,∗ a Horv´ ath Laboratory of Bioseparation Sciences, Institute of Analytical and Radiochemistry, University of Innsbruck, Innrain 52A, A-6020 Innsbruck, Austria b Department of Pharmacognosy, Semmelweis University, Budapest, Hungary article info Article history: Available online 14 May 2008 Keywords: LC–MS n Flavonoids Glycosides Viola tricolor abstract Micro-liquid chromatography (LC) in conjunction with multistage mass spectrometry (MS n ) was intro- duced to study several major heartsease flavonoid glycosides. High-resolution LC separation was achieved by using a monolithic poly(p-methylstyrene-co-1,2-bis(p-vinylphenyl)ethane) column under reversed-phase conditions. The MS/MS and MS 3 analysis of the flavonoid components of interest pro- vided data about their glycosylation type and position, nature of their aglycones, and the structure/linkage information of their glycan moieties. With our LC–MS n approach, four flavonol O-glycosides, nine flavone-C-glycosides, and three flavone C,O-glycosides were characterized in heartsease methanol extract. All of these glycoconjugates were found to be the derivatives of six aglycones: apigenin, chrysoeriol, isorhamnetin, kaempferol, luteolin, and quercetin. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Heartsease, also known as wild pansy (Viola tricolor L., Vio- laceae), has a long history in phytomedicine. It has been utilized to treat various skin disorders, upper-respiratory problems and also used as a diuretic [1]. Most of heartsease’s biological activities are attributed to its antioxidant flavonoid compounds. Flavonoids are important secondary plant metabolites and have been proved useful in the prophylaxis and treatment of car- diovascular problems, complications of diabetes, inflammations, immune disorders, and liver problems, just to list a few indications [2]. The term flavonoids comprise a large group of structurally related compounds with a chromane-type skeleton and a phenyl substituent. In plants, the basic three-ring flavonoid structure is usually modified by means of hydroxylation, methylation or gly- cosylation [3]. Occasionally, aromatic or aliphatic acids, sulphate, prenyl, or isoprenyl groups are attached to the flavonoid agly- cone [4,5]. Glycosylated flavonoids commonly occur as flavonoid O-glycosides, where one or more hydroxyl groups of the aglycone are bound to a sugar unit trough an acid-labile glycosidic O–C bond. While glucose is the most commonly encountered monosac- ∗ Corresponding author. Tel.: +43 512 5075180; fax: +43 512 5072943. E-mail address: andras.guttman@uibk.ac.at (A. Guttman). 1 These authors contributed equally. charide type, galactose, rhamnose, xylose, and arabinose are not uncommon either [6]. Disaccharides were also found in asso- ciation with flavonoids: rutinose (rhamnosyl-(1 → 6)-glucose) and neohesperidose (rhamnosyl-(1 → 2)-glucose) being the most frequent [7]. Trisaccharides and tetrasaccharides have also been reported [7,8] in flavonoids. In addition, sugars can be attached to the flavonoid aglycone via acid-resistant C–C bonds, referred to as flavonoid C-glycosides. Flavonoid C-glycosides are classi- fied into mono-C-glycosyl flavonoids, di-C-glycosyl flavonoids and flavonoid-O,C-glycosides. In this latter category, a hydrolysable sugar is linked either to a phenolic hydroxyl group or a hydroxyl group of the C-glycosyl residue. For structural characterization of natural compounds in crude plant extracts hyphenated chromatographic techniques such as GC–MS, LS–MS or LC–NMR proved to be very efficient. Direct inter- facing of gas chromatography with mass spectrometry is effective for the analysis of free flavonoid aglycones, but is not well suited for the highly polar, thermally labile, and high molecular weight glycosidic conjugates [9]. Liquid chromatography combined with mass spectrometry prevail those applicability limitations. In addi- tion, with LC coupled tandem mass spectrometry information can be obtained on (i) the glycosylation type (O-, C-, or mixed gly- cosides) [7], (ii) the aglycone moiety [10,11], (iii) the types of carbohydrates (hexoses, deoxyhexoses or pentoses) [12], (iv) the sequence of the glycan part [8,13], (v) interglycosidic linkages [8,13,14], and (vi) attachment points of the substituents to the agly- cone [15–20]. 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.05.017