1 H-NMR Fingerprinting of Vaccinium vitis-idaea Flavonol Glycosides Kaisu R. Riihinen, a,b * Velitchka V. Mihaleva, c,d Tanja Gödecke, e Pasi Soininen, f Reino Laatikainen, f Jacques M. Vervoort, c,d David C. Lankin e and Guido F. Pauli a,e ABSTRACT: Introduction – The fruits of Vaccinium vitis-idaea L. are a valuable source of biologically active flavonoid derivatives. For studies focused on the purification of its quercetin glycosides (QGs) and related glycosides from plants and for the purpose of biological studies, the availability of numeric datasets from computer-assisted 1 H iterative full spin analysis (HiFSA), that is, 1 H-NMR finger- printing, can replace and assist the repetitive and tedious two-dimensional NMR identification protocol required for both known and new compounds, respectively. Objective – To fully interpret the complex 1 H-NMR fingerprints of eight QGs obtained from the berries of V. vitis-idaea and provide complete and unambiguous signal assignments. Methods – Vaccinium vitis-idaea QGs were purified in a single run by long-bed gel permeation chromatography and identified by comparison with commercially available compounds using LC–MS combining ion-trap and time-of-flight detection and one- or two-dimensional NMR. The HiFSA analysis yielded full sets of 1 H chemical shifts and proton–proton coupling constants, allowing for field-independent spectral simulation. Results – Signal assignments were achieved for the reference standards and the QGs that dominated in purified fractions. How- ever, even mixtures of two to three QGs could be fitted using the HiFSA approach. In the case of the overlapped sugar resonances, the initial fitting of the 1 H spectra of reference compounds, together with values extracted from the two-dimensional NMR data and literature data, assisted in the process. Conclusion – The HiFSA method revealed for the first time the presence of Q-3-O-b-glucopyranoside and Q-3-O-b- glucuronopyranoside in the berries of V. vitis-idaea, and unambiguously confirmed the structures of Q-3-O-[4 00 -(3-hydroxy-3- methylglutaroyl)]-a-rhamnopyranoside, Q-3-O-a-rhamnopyranoside, Q-3-O-b-galactopyranoside, Q-3-O-a-arabinofuranoside, Q- 3-O-b-xylopyranoside and Q-3-O-a-arabinopyranoside. Copyright © 2013 John Wiley & Sons, Ltd. Supporting information can be found in the online version of this article. Keywords: 1 H-NMR fingerprint; 1 H iterative full spin analysis; HiFSA; food and dietary supplement analysis; berries; flavonol glycosides Introduction The fruits of Vaccinium vitis-idaea L. (Family Ericaceae), which are commonly known as lingonberries and cowberries, are recognised as an important source of natural flavonols in the Finnish diet (Ovaskainen et al., 2008). Data to support the characterisation and quantification of flavonols and other flavonoids in berries and berry products are of considerable interest due to the reported health benefits when the berries are part of a plant-rich diet (Crozier et al., 2009). The characterisation of flavonol glycosides also enhances our understanding of their biosynthetic pathways and their functional roles in the producing plants (Treutter, 2005). The wild growing berries of V. vitis-idaea are generally consumed fresh, dried, or as juices, jams and jellies in their natural growing habitats of northern, central and eastern Europe and Russia. Moreover, V. vitis-idaea is marketed world- wide as a dietary supplement (Mane et al., 2011). The berry flavonols are characterised by a double bond between C-2 and C-3 as well as the presence of a hydroxyl group at C-3 (Fig. 1). They are primarily found as 3-O-glycosides in fruits and other plant-derived foods (Macheix et al., 1990; Williams, 2006). The most frequently encountered glycosidic moieties are glucose, galactose, glucuronic acid, xylose, rhamnose and arabinose, of which the first four occur normally in the D-configuration, whereas the last two are typically found to * Correspondence to: K. Riihinen, Institute of Public Health and Clinical Nutri- tion, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland. Email: Kaisu.Riihinen@uef.fi a Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, Illinois, 60612-7231, USA b Institute of Public Health and Clinical Nutrition, University of Eastern Finland, P.O. Box 1627, 70211, Kuopio, Finland c Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703, HA Wageningen, The Netherlands d Netherlands Metabolomics Centre, Einsteinweg 55 2333, CC, Leiden, The Netherlands e Department of Medicinal Chemistry and Pharmacognosy, College of Phar- macy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, Illinois, 60612-7231, USA f School of Pharmacy, University of Eastern Finland, P.O. Box 1627, 70211, Kuopio, Finland Phytochem. Anal. 2013 Copyright © 2013 John Wiley & Sons, Ltd. Research Article Received: 26 January 2013, Revised: 17 March 2013, Accepted: 30 March 2013 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/pca.2444 1