Received: 22 November 2010 Revised: 14 December 2010 Accepted: 14 December 2010 Published online in Wiley Online Library: 2011 Approach to the study of C-glycosyl flavones acylated with aliphatic and aromatic acids from Spergularia rubra by high-performance liquid chromatography-photodiode array detection/electrospray ionization multi-stage mass spectrometry Federico Ferreres 1 * , Angel Gil-Izquierdo 1 , Juliana Vinholes 2 , Clara Grosso 2 , Patrı ´cia Valenta ˜o 2 and Paula B. Andrade 2 ** 1 Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), P.O. Box 164, 30100 Campus University Espinardo, Murcia, Spain 2 REQUIMTE/Laboratory of Pharmacognosy, Faculty of Pharmacy, Porto University, R. Anı ´bal Cunha, 164, 4050-047 Porto, Portugal The use of mass spectrometry (MS) coupled to liquid chromatography (LC) as working tool for the study of the C-glycosyl flavones acylated with aliphatic and aromatic acids has allowed the tentative characterization of these compounds in Spergularia rubra and the establishment of the position of the acylation on the sugar moiety of the C-glycosylation by use of MS data. The combination of retention time (Rt), ultraviolet (UV) and MS n data of the compounds revealed their C-glycosyl flavone nature, being luteolin, apigenin and chrysoeriol derivatives. Ten non-acylated flavones were identified, from which six are described for the first time (one 7-O-glycosyl-6, 8-diC-glycosyl flavone, four 6,8-diC-glycosyl flavones and one 2’’-O-glycosyl-6-C-glycosyl flavone). Twenty-six acylated derivatives were also found for the first time. These compounds are grouped in three classes, namely, C-glycosyl flavones acylated with aliphatic acids, with aromatic acids or with a mixed acylation. The first group is characterized by the presence of one 6,8-diC-(acetyl)glycosyl flavone, four 6,8-diC-(malonyl)glycosyl flavones and two 7-O-glycosyl-6,8-diC-(malonyl)glycosyl flavones, while in the second one twelve 6,8-diC-(acyl)glycosyl flavones and two 7-O-glycosyl-6,8-diC-(acyl)glycosyl flavones are described. The last class contained five 6,8-diC-(malonyl,acyl) glycosyl flavones. No previous work has described the presence of C-glycosyl flavones acylated with aliphatic acids in this genus. Copyright ß 2011 John Wiley & Sons, Ltd. The analysis of C-glycosyl flavonoids is difficult due to the resistance to hydrolysis of the C–C bond. Their structural characterization has highly advanced by the use of mass spectrometry (MS) coupled to liquid chromatography (LC) allowing an important approach to the study of these compounds, without their previous isolation and clean-up. Mass spectrometry offers the molecular weight as primary information, which is of great value for characterizing C-glycosides. Indeed, the [M þ H] þ or [M  H]  ions are the most intense fragments resulting from the soft ionization interfaces when the usual voltages and energies are applied. The use of multi-stage mass spectrometry (MS n ) in the identification of C-glycosyl flavones has been recently reviewed [1] and this technique allows elucidation of the type of sugar moiety of C-glycosylation, its position (C-6/C-8) in mono-C-glycosyl isomers, and the differentiation of the position (C-6/C-8) of the sugar moieties in diC-glycosyl asymmetric flavones. [2,3] In addition, it can be also used for the identification of acylated O-glycosyl-C-glycosyl flavones. [4] In opposition to O-glycosides, whose rupture is easy and characterized by the loss of the sugar moiety, the fragmentations in C-glycosides occur preferentially on the glycidic moiety. The kind and number of fragments formed indicate the position of the linkage between the sugar and the aglycone. [3] Spergularia (Caryophyllaceae) is a cosmopolitan genus distributed from temperate to subtropical climatic zones of all continents, excepting Antarctica. [5,6] Several species have been studied from the chemical and pharmacological points of view revealing them to be active as hypoglycemic, diuretic, antihypertensive and cholesterol-lowering agents. [7–12] Nevertheless, little is known about the phenolic composition of S. rubra. In the 1970s the occurrence of some C-glycosyl flavones was reported in this species. Six C-glycosyl flavones compounds were identified, comprising apigenin, luteolin and chrysoeriol derivatives, namely 6,8-diC-b-D-glucopy- ranosyl apigenin (vicenin-2), 6,8-diC-b-glucopyranosyl luteolin (lucenine-2), 6,8-diC-b-glucopyranosyl chrysoeriol (stellarin-2), 7,2’’-diO-b-D-glucopyranosyl-6-C-a-L-arabinopyranosyl api- Rapid Commun. Mass Spectrom. 2011, 25, 700–712 (wileyonlinelibrary.com) DOI: 10.1002/rcm.4910 Research Article * Correspondence to: F. Ferreres, Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), P.O. Box 164, 30100 Campus University Espinardo, Murcia, Spain. E-mail: federico@cebas.csic.es ** Correspondence to: P. B. Andrade, REQUIMTE/Laborato ´ rio de Farmacognosia, Departamento de Quı ´mica, Faculdade de Farma ´cia, Universidade do Porto, R. Anı ´bal Cunha 164, 4050-047 Porto, Portugal. E-mail: pandrade@ff.up.pt Rapid Commun. Mass Spectrom. 2011, 25, 700–712 Copyright ß 2011 John Wiley & Sons, Ltd. 700