Flavonoids are secondary plant metabolites of great structural variety and high medicinal significance. The search for new chemical entities and the quality control of flavonoid containing natural products require easy-to-use but reliable and robust analytical methodologies. For structural elucidation of flavonoids and their glycosides, nuclear magnetic resonance (NMR) and mass spectroscopy (MS) are the generally used techniques. In phytochemical analyses, however, high amounts of flavonoids are difficult to isolate for NMR, thus low sample volume requiring MS based methods are emerging. This review summarizes and compares currently available methods for structural elucidation of flavonoids by LC-MS and LC-MS(n), and focuses on the identification options of unknown flavonoid glycosides in complex samples (e.g., plant extracts) with the emphasis on the differentiation of isomeric compounds.

Sephadex LH-20 column chromatography was used to separate flavonoid components in a heartsease methanol extract. One of the main components was identified by NMR as violanthin (6-C-glucosyl-8-C-rhamnosylapigenin). As a first approximation, the other main flavonoid component was considered to be rutin (3-O-rhamnoglucosylquercetin), based on comprehensive comparison of retention times and UV spectra of reference molecules, as well as molecular mass and fragmentation patterns obtained by mass spectrometry. The minor flavonoids were separated by polyamide column and analyzed by LC-MS. The antioxidant capacity of different flavonoid fractions was determined using both Trolox equivalent antioxidant capacity (TEAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) in vitro antioxidant assays. The highest electron-donor capacity was found for the major flavonoid component (rutin), whereas one minor component-rich flavonoid fraction exhibited the highest hydrogen-donor activity.

Heartsease (Viola tricolor L.) is a well-known medicinal plant. Its biological activities are supposed to be related to its antioxidant capacity. Garden pansies (Viola x wittrockiana Gams.) have been crossbred from heartsease and are applied as ornamental plants only. In this study, the mother and the daughter species are compared from a phytochemical point of view. Their flavonoid and anthocyanidin contents are determined by spectroscopic methods recommended by the European Pharmacopoeia 5.0. The compositions of the samples (heartsease and garden pansy varietas of several petal color) are analyzed by high-performance liquid chromatography with UV detection and their antioxidant capacity is determined by trolox equivalent antioxidant capacity assay. Our results suggest that garden pansy, especially its flower, is a promising source of natural antioxidants. In addition, a significant correlation is found between the flavonoid content and antioxidant activity.