The elucidation of flavonoid isomers is accomplished by electrospray ionization tandem mass spectrometry (ESI-MS/MS) via formation and collisional activated dissociation (CAD) of metal/flavonoid complexes containing an auxiliary ligand. Addition of a metal salt and a suitable neutral auxiliary ligand to flavonoids in solution results in the formation of [M(II) (flavonoid-H) ligand]+ complexes by ESI which, upon collisional activated dissociation, often result in more distinctive fragmentation patterns than observed for conventional protonated or deprotonated flavonoids. Previously, 2,2'-bipyridine was used as an auxiliary ligand, and now we compare and explore the use of alternative pyridyl ligands, including 4,7-diphenyl-1,10-phenanthroline. Using this technique, three groups of flavonoid glycoside isomers are differentiated, including glycosides of apigenin, quercetin, and luteolin.

Two electrochemical catalytic systems for the determination of insulin were developed. The homogeneous system was based on the oxidation of insulin by chloro complexes of iridium(IV). Kinetic studies revealed that the aquation of iridium complexes activated them toward the oxidation of insulin in acidic solutions; e.g., the rate constant was equal to 25, 900, and 8400 L mol -1 s -1 for the oxidation of insulin by the IrCl 6 2-, Ir(H 2 O)Cl 5 -, and Ir(H 2 O) 2 Cl 4 complexes, respectively. The inertness of the iridium complexes argued for the outer-sphere mechanism of the homogeneous oxidation reaction. Electroplating of aquated iridium complexes on the glassy carbon electrode resulted in the formation of the iridium oxide (IrOx) surface film, which was used in the heterogeneous detection system for insulin. The catalytic activity of the IrOx film toward insulin oxidation was ascribed to a combination of electron-transfer mediation and oxygen transfer which was related to the acid/base chemistry of the film. The IrOx film electrode was used as an amperometric detector for flow injection analysis of insulin in pH 7.40 phosphate buffer. Linear least-squares calibration curves over the range 0.05-0.50 µM (five points) had slopes of 35.2 ( 0.4 nA µM -1 and correlation coefficients of 0.999. The detection limit for insulin was 20 nM using the criterion of a signal of 3 times the peak-to-peak noise. The advantageous properties of the detector based on the IrOx film are its inherent stability at physiological pH, high catalytic activity toward insulin oxidation, and simplicity of preparation.

A simplified method for determining the sequence and branching of oligosaccharides using infrared multiphoton dissociation (IRMPD) in a quadrupole ion trap (QIT) is described. An IR-active boronic acid (IRABA) reagent is used to derivatize the oligosaccharides before IRMPD analysis. The IRABA ligand is designed to both enhance the efficiency of the derivatization reaction and to facilitate the photon absorption process. The resulting IRMPD spectra display oligosaccharide fragments that are formed from primarily one type of diagnostic cleavage, thus making sequencing straightforward. The presence of sequential fragment ions, a phenomenon of IRMPD, permit the comprehensive sequencing of the oligosaccharides studied in a single stage of activation. We demonstrate this approach for two series of oligosaccharides, the lacto-N-fucopentaoses (LNFPs) and the lacto-N-difucohexaoses (LNDFHs).

A strategy for increasing the efficiency of infrared multiphoton dissociation (IRMPD) in a quadrupole ion trap (QIT) is described. IR-active ligands (IRALs) are incorporated into noncovalent complexes of the type [M2+(analyte) IRAL]+, where M is a transition metal such as copper or cobalt and IRAL is an auxiliary ligand with an IR-active phosphonate functional group. The complexes are formed via self-assembly in solution directly prior to ESI-MS analysis. We demonstrate this new IRMPD approach for the structural characterization of flavonoids. The fragment ions obtained by IRMPD are similar to those obtained by CAD and allow facile isomer differentiation of flavonoids. Fourier transform infrared absorption attenuated total reflectance (FTIR-ATR) and energy-variable CAD experiments indicate that the high IRMPD efficiencies stem from the very large IR absorptivities of the IR-active ligands.

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...