Flavonoids are very common and widespread secondary plant metabolites. They have a wide range of biological and physiological activities and serve as chemotaxonomic marker compounds. Therefore, they have been extensively investigated both in the past and during recent years. The interest in them is still increasing. In the search for new compounds, and also in quality control, there is a need to have reliable methodology for the analysis of flavonoids. Mass spectrometry can make an invaluable contribution because of its high sensitivity, possibilities of coupling with liquid chromatography and the availability of powerful tandem mass spectrometric techniques. A review of currently available mass spectrometric methodology used in the structure elucidation of flavonoids is presented. Sample preparation, liquid chromatographic/mass spectrometric analysis and tandem mass spectrometric procedures for the characterization of flavonoid aglycones, O-glycosides, C-glycosides and acylated glycosides are considered.

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In the present study, we have characterized in detail the MS(2) and MS(3) fragmentation behaviors, using electrospray ionization (ESI) in the negative ion mode, of previously identified sulfated isoprene secondary organic aerosol compounds, including 2-methyltetrols, 2-methylglyceric acid, 2-methyltetrol mononitrate derivatives, glyoxal and methylglyoxal. A major fragmentation pathway for the deprotonated molecules of the sulfate esters of 2-methyltetrols and 2-methylglyceric acid and of the sulfate derivatives of glyoxal and methylglyoxal is the formation of the bisulfate [HSO(4)](-) anion, while the deprotonated sulfate esters of 2-methyltetrol mononitrate derivatives preferentially fragment through loss of nitric acid. Rational interpretation of MS(2), MS(3) and accurate mass data led to the structural characterization of unknown polar compounds in K-puszta fine aerosol as organosulfate derivatives of photooxidation products of unsaturated fatty acids, i.e. 2-hydroxy-1,4-butanedialdehyde, 4,5- and 2,3-dihydroxypentanoic acids, and 2-hydroxyglutaric acid, and of alpha-pinene, i.e. 3-hydroxyglutaric acid. The deprotonated molecules of the sulfated hydroxyacids, 2-methylglyceric acid, 4,5- and 2,3-dihydroxypentanoic acid, and 2- and 3-hydroxyglutaric acids, showed in addition to the [HSO(4)](-) ion (m/z 97) neutral losses of water, CO(2) and/or SO(3), features that are characteristic of humic-like substances. The polar organosulfates characterized in the present work are of climatic relevance because they may contribute to the hydrophilic properties of fine ambient aerosol. In addition, these compounds probably serve as ambient tracer compounds for the occurrence of secondary organic aerosol formation under acidic conditions.

Isomeric flavonoid O-diglycosides were analyzed by positive and negative nano-electrospray ionization (ESI) ion trap mass spectrometry (ITMS) in order to evaluate whether the two most common interglycosidic linkage types, i.e. 1 --> 2 and 1 --> 6, found for glycosides containing a rhamnosylglucose glycan part can be differentiated. In the positive ion mode the degree of internal glucose residue loss was found to be strongly dependent on the aglycone type and was very pronounced for aglycones of the flavanone type. The relative abundance of the Y-type ions formed by fragmentation at glycosidic bonds only allows one to infer the interglycosidic linkage types in the case of flavone O-diglycosides. In contrast, the negative ion mode makes a clear differentiation between a rutinoside (1 --> 6) and a neohesperidoside (1 --> 2) glycan residue possible for all aglycone types. The neohesperidose-containing compounds could be characterized by additional product ions. When the compounds were dissolved in pure methanol a molecular radical ion was found to be the base peak in nano-ESI.

The influence of the glycosylation site on the fragmentation behavior of 18 flavonoid glycoside standards was studied using positive and negative electrospray ionization mass spectrometry in combination with collision-induced dissociation and tandem mass spectrometry. The glycosylation position is shown to affect the relative abundance of the radical aglycone ions that can be observed in the [M-H]- collision-induced dissociation spectra. In particular, the radical aglycone ions are very abundant for deprotonated flavonol 3-O-glycosides. Collisional activation of the radical aglycone ions produced from positional isomers revealed minor differences: m,nB0- product ions are pronounced for 7-O-glycosides, whereas m,nA0- product ions are relatively more abundant for 4'-O-glycosides. In addition, the ratio between the radical aglycone and the regular aglycone ions in the [M+Na]+ high-energy collision-induced dissociation spectra gives an indication about the glycosylation site. This ion ratio allows the differentiation between flavonoid 3-O- and 7-O-glycosides or can be useful in the comparison of unknown compounds with standards. Unambiguous differentiation between O-glycosylation at the common positions of flavonoid O-glycosides, i.e. the 3-, 4'- and 7-positions, is achieved by collisional activation of sodiated molecules at high collision energy. The presence of a B-ring product ion containing the sugar residue indicates 4'-O-glycosylation, whereas the loss of the B-ring part from the aglycone product ion is characteristic of 3-O-glycosylation and the loss of the B-ring part from both the [M+Na]+ precursor ion and the aglycone product ion points to 7-O-glycosylation.