Determination of the glycosylation site of flavonoid monoglucosides by metal complexation and tandem mass spectrometry

Differentiation between two isomers of hydroxypyridine N-oxide according to the metal cation adducts generated by electrospray ionization (ESI) was investigated for different metal cations, namely Mg (II), Al (III), Ca (II), Sc (III), Fe (III), Co (II), Ni (II), Cu (II), Zn (II), Ga (III), besides the diatomic cation VO(IV). Protonated molecules of the isomeric hydroxypyridine N-oxides as well as the singly/doubly charged adducts formed from neutral or deprotonated ligands and a doubly/ triply charged cation were produced in the gas phase using ESI, recording mass spectra with different metal ions for each isomer. While complex formation was successful for 2-hydroxypyridine N-oxide with trivalent ions, in the case of 3-hydroxypyridine N-oxide, only peaks related to the protonated molecule were present. On the other hand, divalent cations formed specific species for each isomer, giving characteristic spectra in every case. Hence, differentiation was possible irrespective of the metal cation utilized. In addition, quantum chemical calculations at the B3LYP/6-31+G(d,p) level of theory were performed in order to gain insight into the different complexation of calcium(II) with the isomers of hydroxypyridine N-oxide. The relative stability in the gas phase of the neutral complexes of calcium made up of two ligands, as well as the singly charged and doubly charged complexes, was investigated. The results of these calculations improved the understanding of the differences observed in the mass spectra obtained for each isomer.

Section: Ionized Complexes Stabilitysupporting

confidence: 74%

Section: Ionized Complexes Stabilitymentioning

confidence: 99%

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Differentiation of Isomeric HydroxypyridineN-Oxides Using Metal Complexation and Electrospray Ionization Mass Spectrometry

Butler

Máñez

Cabrera

2011

“…We have developed several methods for identifying the conjugation positions and saccharide identities of flavonoid glycosides using tandem mass spectrometry [23][24][25][26][27][28][29]. These methods exploit the natural chelating ability of flavonoids to form metal complexes in solution, which can then be analyzed in a mass spectrometer for structural identification.…”

mentioning

confidence: 99%

Regioselectivity of human UDP-glucuronosyl-transferase 1A1 in the synthesis of flavonoid glucuronides determined by metal complexation and tandem mass spectrometry

Davis

Brodbelt

2008

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A three-part tandem mass spectrometric strategy that entails MS n analysis and a post-column LC-MS cobalt complexation method is developed to identify flavonoid monoglucuronide metabolites synthesized using the 1A1 isozyme of human UDP-glucuronosyltransferase (UGT). Ten flavonoid aglycons were used as substrates, spanning the subclasses of flavones, flavonols, and flavanones. The products were characterized by LC-MS and LC-MS n , with post-column cobalt complexation employed to pinpoint the specific sites of conjugation. The dissociation of complexes of the form [Co(II) (flavonoid glucuronide Ϫ H) (4,7-diphenyl-1,10-phenanthroline) 2 ] ϩ allowed identification of the products and differentiation of isomers. The correlation between glycosylation site and elution order is used to provide additional structural confirmation. Flavonoids lacking a 3= hydroxyl group were glucuronidated only at position 7, while those containing this functionality also formed 3=-O-glucuronides and sometimes 4=-O-glucuronides, thus supporting the conclusion that the presence or absence of the 3=-OH group is the major determinant of the regioselectivity of glucuronidation. Moreover, the specific distribution of multiple glucuronide products ( The currently accepted paradigm involves the consumption of flavonoid glycosides in plant-based food products, deglucosylation in the small intestine by ␤-glucosidase or lactose phloridzin hydrolase, and rapid metabolism by Phase I and (especially) Phase II enzymes [1][2][3]. Glucuronidation and sulfation are important metabolic routes for most flavonoids, while methylation or hydroxylation may also occur depending on the structure of the molecule in question [2,3]. There has also been a report of glutathionerelated metabolites in human urine [4]. As a result of these rapid conjugation reactions, neither the original flavonoid glycosides (except anthocyanins) nor the aglycon forms (except catechins) are found in plasma [5][6][7][8]. Early reports of unmodified flavonoid glycosides circulating in the bloodstream [9 -11] were likely mistaken identifications of flavonoid glucuronides, which have similar chromatographic and ultraviolet (UV) spectroscopic characteristics [7,12]. Flavonoids that fail to be absorbed in the small intestine may be broken down by microflora in the large intestine [1][2][3]. This process may release the free aglycons, which can then be absorbed and metabolized, but mostly results in the release of small phenolic acids, which are expelled in the urine [1][2][3]. Quantitative in vivo studies generally show that only a small percentage of consumed flavonoid glycosides is recovered in urine as conjugated Phase II metabolites [13][14][15]. Walle et al. used 14 Clabeled quercetin to show that up to 81% of the administered dose ultimately is exhaled in the form of carbon dioxide [16]. There remains considerable interest in the conjugated metabolites as they may retain some of the bioactivity of the original molecules [5,17].In spite of breakthroughs in the field of flavonoid metabol...

“…Although flavonoids deprotonate efficiently, the resulting fragmentation patterns often do not give diagnostic ions adequate for differentiation of isomers. Metal complexation is an alternative ionization strategy that has been explored in our group for improved analysis of flavonoids [17][18][19][20][21][22][23], and we have explored metal complexation both with and without the use of an auxiliary …”

mentioning

confidence: 99%

Tunable transition metal-ligand complexation for enhanced elucidation of flavonoid diglycosides by electrospray ionization mass spectrometry

Pikulski

Aguilar

Brodbelt

2007

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ifferentiation of isomers remains a challenging analytical problem and confirming structures of isomers is difficult even when they can be adequately separated from mixtures. Tandem mass spectrometry has been used extensively for structural elucidation of isomers [1][2][3][4][5][6][7][8][9][10][11][12] and there continues to be interest in applying existing tandem mass spectrometric methods, such as MS n [9,13,14], and developing new ones, such as STEP [15,16], a statistically based method that relies on comparison of abundances of fragment ions obtained for different collision activation conditions, to differentiate isomers. Our group has focused on the development of metal complexation strategies to enhance the differentiation of isomers [17][18][19][20][21][22][23] because metal complexation often alters the dissociation pathways of ions, thus giving additional diagnostic fragment ions. We have reported several metal complexation strategies that have been useful for distinguishing flavonoid isomers and, in this study, we expand this approach by developing a "tunable" collisionally activated dissociation (CAD) method based on the use of rationally selected auxiliary ligands to form metal complexes. The concept of this tunable CAD strategy is applied for the differentiation of flavonoid isomers.Flavonoids are phytochemicals found in almost all plants, including fruits and vegetables. Several studies have indicated their antioxidant, chemopreventive, antiviral, antibacterial, and radical-scavenging properties [24 -28]; moreover, they have been implicated in cardiovascular protection [29 -36]. Although all flavonoids are structurally similar, many exist as glycosides and there are subtle differences in their structure that lead to important changes in their biological activity. These differences include hydroxylation position of the aglycon, glycosylation site, sequence of glycosylation, and interglycosidic linkages of the glycan portion. It is because of these subtle differences that there is a need for sensitive analytical techniques to elucidate these similar compounds.Mass spectrometry has become an ideal tool for the analysis of flavonoids, and two recent reviews provide an excellent background of the techniques used and the analyses performed [37,38]. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) has been used extensively to differentiate several sets of flavonoid isomers over the past decade. For example, for a series of flavonoid diglycosides, CAD has been used to pinpoint the intersaccharide linkages (rutinosides: 1 ¡ 6 disaccharides versus neohesperidosides: 1 ¡ 2 disaccharides) in flavonoid-O-glycosides based on relative abundances of the fragment ions obtained from protonated flavonoids [7]. However, in general it has been noted that flavonoids do not protonate particularly well because they are acidic, phenolic compounds. Although flavonoids deprotonate efficiently, the resulting fragmentation patterns often do not give diagnostic ions adequate for differentiation of isomers. Metal com...