Rafał Frański

Monitoring changes in anthocyanin and steroid alkaloid glycoside content in lines of transgenic potato plants using liquid chromatography/mass spectrometry

Transgenic potato plants overexpressing and repressing enzymes of flavonoids biosynthesis were created and analyzed. The selected plants clearly showed the expected changes in anthocyanins synthesis level. Overexpression of a DNA encoding dihydroflavonol 4-reductase (DFR) in sense orientation resulted in an increase in tuber anthocyanins, a 4-fold increase in petunidin and pelargonidin derivatives. A significant decrease in anthocyanin level was observed when the plant was transformed with a corresponding antisense construct. The transformation of potato plants was also accompanied by significant changes in steroid alkaloid glycosides (SAG) level in transgenic potato tuber. The changes in SAGs content was not dependent on flavonoid composition in transgenic potato. However, in an extreme situation where the highest (DFR11) or the lowest (DFRa3) anthocyanin level was detected the positive correlation with steroid alkaloid content was clearly visible. It is suggested that the changes in SAGs content resulted from chromatin stressed upon transformation. A liquid chromatography/mass spectrometry (LC/MS) system with electrospray ionization was applied for profiling qualitative and quantitative changes of steroid alkaloid glycosides in tubers of twelve lines of transgenic potato plants. Except alpha-chaconine and alpha-solanine, in the extracts from dried tuber skin alpha-solamargine and alpha-solasonine, triglycosides of solasonine, were identified in minor amounts, triglycosides of solanidine dehydrodimers were also recognized.

A central fission pathway in alkylphenol ethoxylate biodegradation

A representative alkylphenol Triton X-100 (having 9.5 oxyethylene subunits) was treated over 40 days under the conditions of the continuous flow activated sludge simulation test in a plant with aeration and denitrification chambers. Treated sewage was separated by sequential extraction with ethyl acetate and chloroform. The extracts were analysed by high-performance liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-ESI-MS). Single ion chromatograms of the chloroform extracts showed the presence of neutral, mono- and di-carboxylated poly(ethylene glycols). This is evidence of the central fission of alkylphenol ethoxylates (APE). Simultaneously, the APE having omega-carboxylated oxyethylene chains were identified. This is the evidence that apart from central fission, the omega-oxidation oxyethylene chain pathway also occurs.

Profiling changes in metabolism of isoflavonoids and their conjugates in Lupinus albus treated with biotic elicitor

Liquid chromatography with ultraviolet and mass spectrometric detection was applied to monitor changes in profiles of isoflavonoid glycosides and free isoflavonoid aglycones in Lupinus albus L. Four isoflavonoid aglycones, fourteen isoflavonoid glycosides, four flavonol glycosides and flavone glycoside were identified in lupin tissue after LC/ESI/MS analyses. An elicitor preparation from purified yeast cell wall was used to inject the shoots of 3-week old seedlings or to infiltrate the cut lupin leaves. Qualitative and quantitative changes of isoflavonoids were measured at different time points after elicitation. In elicited lupin seedlings increased amounts of prenylated isoflavone aglycones were identified. The concentrations of glycosidic conjugates of isoflavones present in plant tissue were less affected.

Identification of flavonoid diglycosides in yellow lupin (Lupinus luteus l.) with mass spectrometric techniques

Various mass spectrometric techniques were used for the structural elucidation of flavonoid glycosides isolated from green parts of yellow lupin plants (Lupinus luteus). A methodological approach is proposed consisting of (1) extraction and purification of flavonoid glycosides from plant tissues, (2) registration of liquid secondary ion mass spectra of intact compounds and (3) gas chromatographic/mass spectrometric analyses of those compounds subjected to several types of chemical modification. On the basis of these data, it was possible to define the molecular mass of the glycosides, the structure of the aglycones, the configuration of sugar moieties and the position of glycosidic linkages between sugars in diglycosides or between aglycone and sugar moieties. Analysis of the mass spectrometric data permitted structural identification of four flavonoid diglycosides, where the aglycones had flavone, isoflavone and flavonol structures. They were recognized as: apigenin 7-O-(2″-O -rhamnopyranosyl)glucopyranoside (1), genistein 4′,7-O-diglucopyranoside (2), kaempferol 3-O -(6″-rhamnopyranosyl)glucopyranoside (3) and 3′- or 4′-methylquercetin 3-O-(6″-O -rhamnopyranosyl)glucopyranoside (4). Complete structural evaluation of flavonoid glycosides was possible with only submilligram quantities of samples needed to register various mass spectra. It was not possible, however, to define the anomeric configuration of the C-1 carbons in investigated glycosides. Copyright

Profiling of Flavonoid Conjugates in Lupinus albus and Lupinus angustifolius Responding to Biotic and Abiotic Stimuli

Qualitative and quantitative composition of flavonoid and isoflavo- noid glycosides as well as free aglycones in lupin seedlings (roots and aerial parts) grown under different light conditions or responding to infection with Pleiochaeta setosa, a fungus causing brown leaf spot, were monitored by liquid chromatography with UV and/or mass spectrometric detection. Both physical and biotic factors affected flavonoid and isoflavonoid levels in lupin tissues. Fungal infection evoked significant increase in the amounts of genistein, 2′-hydroxygenistein, and their prenylated derivatives that are thought to function as lupin phytoalexins. Effect on quantitative changes of glycosylated flavonoids and isoflavonoids in the roots and aerial parts was less significant. Moreover, different light conditions applied during seedling growth caused relative changes of flavonoid and isoflavonoid conjugates composition, especially in the leaves of white lupin plants. The chemical structures of flavonoid and isoflavonoid conjugates present in Lupinus angustifolius were elucidated. In addition to genistein and2′-hydroxygenistein glycosides, flavonol conjugates were identified in leaves, while the composition of root isoflavonoids was similar to that of L. albus reported earlier.

Identification of photodegradation products of nilvadipine using GC-MS.

Nilvadipine (NV) photodegradation products have been analysed with gas chromatography-mass spectrometry (GC-MS). The photodegradation was carried out in the conditions recommended in the first version of the document issued by the International Conference on Harmonization (ICH), currently in force in the studies of photochemical stability of drugs and therapeutic substances. Methanol solutions of NV were irradiated with a high-pressure UV lamp - type HBO 200. The maximum intensity at the wavelength lambda = 365 nm was achieved by applying the interference filter and Woods filter. Using the Reinecke salt as a chemical actinometer, apparent quantum yields of photodegradation were obtained, which after extrapolation to the zero time of irradiation gave the actual quantum yield ((phi = 7.58 x 10(-5). The structure of three nilvadipine photodegradation products was established, after mass spectra analysis of compounds registered during GC-MS carried out of irradiated nilvadipine solutions. The quantitative results of GC-MS analyses enabled to determination of the kinetic parameters of NV photodegradation, calculated from the dependence In c =f(t). Quantitatively the process was described with the calculated rate constant of decomposition (k), decomposition time of 50% of the compound (t0.5) and decomposition time of 10% of the compound (t0.1). The exposure of nilvadipine to UV light was found to lead to aromatization of the DHP ring and elimination of the HCN molecule.

Anion-π interactions—interactions between benzo-crown ether metal cation complexes and counter ions

The loss of X· radical from [M + Cu + X]+ ions (copper reduction) has been studied by the so called in-source fragmentation at higher cone voltage (M = crown ether molecule, X− = counter ion, ClO4−, NO3−, Cl−). The loss of X· has been found to be affected by the presence/lack of aromatic ring poor/rich in electrons. Namely, the loss of X· occurs with lower efficiency for the [NO2-B15C5 + Cu + X]+ ions than for the [B15C5 + Cu + X]+ ions, where NO2-B15C5 = 3-nitro-benzo-15-crown-5, B15C5 = benzo-15-crown-5. A reasonable explanation is that Anion-π interactions prevent the loss of X· from the [NO2-B15C5 + Cu + X]+ ions. The presence of the electron-withdrawing NO2 group causes the aromatic ring to be poor in electrons and thus its enhances its interactions with anions. For the ion containing the aromatic ring enriched in electrons, namely [NH2-B15C5 + Cu + ClO4]+ where NH2-B15C5 = 3-amino-benzo-15-crown-5, the opposite situation has been observed. Because of Anion-π repulsion the loss of X· radical proceeds more readily for [NH2-B15C5 + Cu + X]+ than for [B15C5 + Cu + X]+. Iron reduction has also been found to be affected by Anion-π interactions. Namely, the loss of CH3O· radical from the ion [B15C5 + Fe + NO3 + CH3O]+ proceeds more readily than from [NO2−B15C5 + Fe + NO3 + CH3O]+.

Sulphated flavonoid glycosides from leaves of Atriplex hortensis

Two flavonoid sulphates, i.e. quercetin 3-O-sulphate-7-O-α-arabinopyranoside and kaempferol 3-O-sulphate-7-O-α-arabinopyranoside, were isolated from leaves of Atriplex hortensis L. The structures of these compounds were established by UV, 1H and 13C NMR, 2D NMR and MS spectra. The compounds were isolated for the first time from plant material.

Self-assembly of quaterpyridine ligands and Cu + cation into helical complexes of 2:2 stoichiometry under ESI condition

Solutions containing the quaterpyridine ligand 1 and Cu2+ cation were analysed by electrospray ionisation mass spectrometry (ESI-MS). It was found that copper reduction under ESI conditions and self-assembly of 1 and Cu(I) led to the formation of the 2:2 stoichiometry complex. Such stoichiometry is characteristic of the helical complex of a quaterpyridine ligand with Cu(I). The isotope pattern characteristic of the [Cu212]2+ ion, which is different from that of [Cu1]+ ion, is observed. When using methanol as the solvent, only [Cu212]2+ ions are observed in the mass spectrum obtained at low cone voltage. At higher cone voltage, the [Cu212]2+ ion easily dissociates, producing a [Cu1]+ ion. When using other solvents studied, water and acetonitrile, the 2:2 stoichiometry complex is formed, but along with complexes of other stoichiometries. The helical complex was not observed for the silver cation for which only a 1:1 stoichiometry complex was observed (ion [Ag1]+).

Unusual ion UO4 − formed upon collision induced dissociation of [UO2(NO3)3]−, [UO2(ClO4)3]−, [UO2(CH3COO)3]− ions

The following ions [UO2(NO3)3]−, [UO2(ClO4)3]−, [UO2(CH3COO)3]− were generated from respective salts (UO2(NO3)2, UO2(ClO4)3, UO2(CH3COO)2) by laser desorption/ionization (LDI). Collision induced dissociation of the ions has led, among others, to the formation of UO4− ion (m/z 302). The undertaken quantum mechanical calculations showed this ion is most likely to possess square planar geometry as suggested by MP2 results or strongly deformed geometry in between tetrahedral and square planar as indicated by DFT results. Interestingly, geometrical parameters and analysis of electron density suggest it is an UVI compound, in which oxygen atoms bear unpaired electron and negative charge.