Filip Cuyckens

The Application of Liquid Chromatography-Electrospray Ionization Mass Spectrometry and Collision-Induced Dissociation in the Structural Characterization of Acylated Flavonol O-Glycosides from the Seeds of Carrichtera Annua:

The flavonoid fraction from the seeds of Carrichtera annua was studied using high-performance liquid chromatography simultaneously coupled to a photodiode array detector (LC/UV-DAD) and a mass spectrometer equipped with an electrospray source (LC/ESI-MS). Collision-induced dissociation (CID) mass spectral data obtained off-line by nanospray (nano-ESI) analysis provided a wealth of complementary structural information, which was consistent with structures established by NMR or led to the proposal of base structures of the flavonol O-glycosides present in the Carrichtera annua seed extract. The flavonoid fraction was found to contain 12 structurally related flavonol O-glycosides. Eleven flavonoids, of which several were new compounds, were acylated with one or more benzoyl, feruloyl or sinapoyl groups. These acyl groups gave rise to characteristic product ions in the [M + H]+ and [M + Na]+ CID spectra as well as to radicalar acid-related product ions at high-energy collisional activation. In addition to the characterization of the acyl substituents, the mass spectral data allowed the identification of the aglycone, the determination of the base structure and the differentiation of several positional isomers.

Characterization of metal complexes with metallothioneins in the liver of the carp Cyprinus carpio by reversed-phase HPLC with ICP-MS and electrospray ionization (ESI)-MS

Inductively coupled plasma time-of-flight mass spectrometry (ICP-TOFMS) and electrospray ionization mass spectrometry (ESI-MS) were used in parallel in combination with two-dimensional chromatography (size-exclusion followed by reversed-phase HPLC) for the characterization of metal complexes with metallothionein (MT) isoforms in hepatic cytosols of Cd exposed carp. The cytosolic MT fraction was partially purified by size-exclusion (SE) HPLC, preconcentrated and de-salted by centrifugal microfiltration. The separation of the metal complexes with MT isoforms was performed by reversed-phase (RP) HPLC using a gradient up to 30% methanol in acetate buffer (pH 7.4). Two major and several minor chromatographic peaks were distinguished by RP-HPLC-ICP-TOFMS within a chromatographic run of 20 min. Applying ESI-MS as a detector for RP HPLC allowed the identification of the two major signals detected by ICP-MS on the basis of the molecular mass determined on-line for the metallocomplex(es) and for the ligand. Mass spectra taken at the peak apices indicated the co-elution of different metal (Cd, Cu, Zn and Pb) complexes with MT isoforms within each peak. A simple “in-source” protein oxidation procedure was used to obtain the ligand of the complexes (oxidized form) on arrival at the detector; direct information was obtained on the molecular mass of two MT isoforms in carp liver, allowing their identification. The efficiency and robustness of this method were demonstrated using a rabbit liver MT1 standard as test sample.

Mass spectrometric methods for characterization and differentiation of isomeric **O**-diglycosyl flavonoids

Tandem mass spectrometric methods have been evaluated for the characterisation of the type and the differentiation of the interglycosidic linkage of isomeric flavonoid O-diglycosides. Based on the occurrence of internal monosaccharide residue loss and the relative abundances of Y-type ions formed by fragmentation at glycosidic bonds, four pairs of isomeric flavonoid O-diglycosides can be unambiguously differentiated. The different techniques used, i.e. linked scanning at constant B/E without collisional activation and low-energy collision-induced dissociation using methane or helium as collision gas, have been shown to be useful for distinguishing the two most common (1, 2- and 1, 6-) interglycosidic linkages, e.g. flavonoid O-neohesperidosides and O-rutinosides.