Ferenc Kilár

Determination of polyphenolic compounds by liquid chromatography-mass spectrometry in Thymus species

Polyphenolic compounds represent a wide group of phytochemicals, including well-known subgroups of phenolic acids, flavonoids, natural dyes, lignans etc., which are produced by plants. These natural bioactive compounds possess a variety of beneficial effects including antioxidant and anticarcinogenic activities, protection against coronary diseases as well as antimicrobial properties. Thymus species have already been reported as sources of different phenolic acids and flavonoids. Moreover, the composition and content of flavonoids in Thymus species play important role as taxonomic markers providing distinction of species. High-performance liquid chromatography (HPLC) coupled with diode array detector (DAD) and on-line mass spectrometry (ESI-MS) method was used for analysis. The method was evaluated for a number of validation characteristics (repeatability and intermediate precision, LOD, LOQ, calibration range, and recovery). The polyphenolic pattern of five native Hungarian Thymus species (T. glabrescens Willd., T. pannonicus All., T. praecox Opiz, T. pulegioides L., and T. serpyllum L.) was characterized. The dominant compound was rosmarinic acid, which ranged between 83.49 μg g(-1) and 1.436 mg g(-1). Other phenolic acids (ferulic acid, caffeic acid and its other derivatives, chlorogenic acid and p-coumaric acids) were present in every examined Thymus species, as well as flavanones: naringenin, eriodictyol and dihydroquercetin; flavones: apigenin and apigenin-7-glucoside, flavonols: quercetin and rutin. The polyphenolic pattern was found to be a useful additional chemotaxonomic tool for classification purposes and determination of the locality of origin.

Determination of pI by measuring the current in the mobilization step of high-performance capillary isoelectric focusing : Analysis of transferrin forms

Abstract In high-performance isoelectric focusing in capillaries, the focusing pattern of a sample is obtained during the mobilization step. A current parameter can be assigned to each peak in this pattern. Keeping the experimental conditions unchanged, these current parameters characterize the positions of the respective substances in the pH gradient and therefore they can be used for the determination of the isoelectric points of the substances.

Determination of tropane alkaloids atropine and scopolamine by liquid chromatography-mass spectrometry in plant organs of Datura species

Hyoscyamine (atropine) and scopolamine are the predominant tropane alkaloids in the Datura genus, occurring in all plant organs. The assessment of the alkaloid content of various plant parts is essential from the viewpoint of medical use, but also as a potential risk of toxicity for humans and animals. Therefore, a reliable method for the determination of tropane alkaloid content is of high importance. The present work aimed at the elaboration of a rapid method for determination of the most abundant Datura alkaloids by LC-MS technique using a new generation of core-shell particle packed column. Tropane alkaloid content was investigated in various plant organs of four Datura taxa (D. innoxia, D. metel, D. stramonium, and D. stramonium var. tatula), grown under the same conditions, in two developmental stages. We have developed a rapid LC-MS method for the quantitative determination of atropine and scopolamine, which was successfully applied to quantify the alkaloids in different plant organs (leaves, flowers, stems, seeds) of thorn apples after a simple sample preparation step. Elaboration and validation of the method and analysis of plant extracts were done by UFLC-MS technique, employing an Ascentis Express C18 column. Detection was done in positive ionization mode (ESI+) and the method suitability was evaluated by several validation characteristics. Quantitation limits are 333 and 167 pgmL(-1) for scopolamine and atropine, respectively, and the method shows very good repeatability. The analysis of Datura extracts revealed significant differences depending on the species, the organ and the sampling period. Atropine was found to be dominant over scopolamine in three out of the four taxa investigated. D. innoxia showed the highest concentrations of scopolamine in all organs examined, whereas D. metel accumulated the lowest scopolamine levels. Hyoscyamine, measured as atropine, was the highest in D. stramonium var. tatula, and the lowest in D. innoxia. Samples collected in summer had higher scopolamine levels than autumn samples, concerning both stems and leaves.

Bacterial outer membrane protein analysis by electrophoresis and microchip technology

Outer membrane proteins are indispensable components of bacterial cells and participate in several relevant functions of the microorganisms. Changes in the outer membrane protein composition might alter antibiotic sensitivity and pathogenicity. Furthermore, the effects of various factors on outer membrane protein expression, such as antibiotic treatment, mutation, changes in the environment, lipopolysaccharide modification and biofilm formation, have been analyzed. Traditionally, the outer membrane protein profile determination was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Converting this technique to capillary electrophoresis format resulted in faster separation, lower sample consumption and automation. Coupling capillary electrophoresis with mass spectrometry enabled the fast identification of bacterial proteins, while immediate quantitative analysis permitted the determination of up- and downregulation of certain outer membrane proteins. Adapting capillary electrophoresis to microchip format ensured a further ten- to 100-fold decrease in separation time. Application of different separation techniques combined with various sensitive detector systems has ensured further opportunities in the field of high-throughput bacterial protein analysis. This review provides an overview using selected examples of outer membrane proteins and the development and application of the electrophoretic and microchip technologies for the analysis of these proteins.

Twelve receptor molecules attach per viral particle of human rhinovirus serotype 2 via multiple modules

The crystallographic T=1 (pseudo T=3) icosahedral symmetry of the human rhinovirus capsid dictates the presence of 60 identical, symmetry related surface structures that are available for antibody and receptor binding. X‐ray crystallography has shown that 60 individual very‐low density lipoprotein receptor (VLDLR) modules bind to HRV2. Their arrangement around the fivefold axes of the virion suggested that tandem oligomers of such modules could attach simultaneously to symmetry‐related sites. By resolving virus particles carrying various numbers of artificial recombinant concatemers of VLDLR repeat 3 (V33333) by capillary electrophoresis and extrapolation of the measured mobilities to that at saturation of all binding sites, we present evidence for up to 12 molecules of the concatemer to bind one single virion.

Volatile Composition of Macedonian and Hungarian Wines Assessed by GC/MS

The volatile composition of eight varietal wines Vranec, Merlot, Cabernet Sauvignon, Temjanika and Chardonnay from the Republic of Macedonia, and Portugieser, Kékfrankos and Tokaji Aszú from Hungary has been characterized by means of gas chromatography/mass spectrometry technique. The wine volatile compounds were extracted in dichloromethane, and the extracts were concentrated under nitrogen. Forty-four volatile compounds have been identified mainly using the NIST mass spectral library and by comparison with the available standards used for quantification as well. Differences between the wines were noted for a number of compounds, such as a higher concentration of 1-pentanol and 2-phenyl ethanol in the red wines. Monoterpenes, linalool and terpineol were detected only in the white wines, Chardonnay and Tokaji. Macedonian red wines were characterized by a higher level of alcohols, while the Hungarian wines contained a higher amount of esters, fatty acids and lactones. A statistical treatment including one-way ANOVA, followed by a Tukey’s test has been performed in order to ascertain possible significant differences between the wines studied, and principal component analysis to study the possible grouping of the wines.

COMPARATIVE STUDY OF THE CHIRAL RESOLUTION OF β-BLOCKERS ON CELLULOSE TRIS (3,5-DIMETHYL-PHENYLCARBAMATE) PHASES IN NORMAL AND REVERSED PHASE MODES

The chiral separation of β-blockers on cellulose tris (3,5-dimethylphenylcarbamate) columns is described. The separation power of a Chiracel OD normal phase column, a Chiracel OD-RH reversed phase column, and a Chiracel ODRH reversed phase narrow-bore column were compared. Hexane–2-propanol–diethylamine mixtures were used as mobile phase in the normal phase mode and sodium perchlorate buffer containing different amounts of acetonitrile in the reversed phase mode. Sixteen μ-blockers were resolved under normal phase conditions and eleven under reversed phase conditions. The values of α and Rs on normal phase column ranged from 4.46 to 1.10 and 10.04 to 0.97, respectively. The values of α and Rs on reversed phase columns varied from 4.04 to 1.04 and 3.98 to 0.40(on normal bore column) and 4.36 to 1.06 and 2.26 to 0.40(on narrow bore column).

Antifungal unsaturated cyclic Mannich ketones and amino alcohols : Study of mechanism of action

The antifungal activity of some known unsaturated Mannich ketones and their amino alcohols has been reported and the mechanism of antifungal action has been studied. The inhibition of the fungal ergosterol, chitin, protein synthesis and pseudohypha formation was investigated. According to our results, Mannich ketones can influence the development of pseudohyphae of Candida albicans strains. In addition, they are able to induce significant changes in the protein composition of this fungal strain. Some of our Mannich ketones have shown inhibitory effect on the fungal chitin synthase enzyme. QSAR studies were also carried out.

Effect of Antibiotics on Cell Surface Hydrophobicity of Bacteria Causing Orthopedic Wound Infections

Background: Despite antibiotic prophylaxis and treatment, the incidence of wound infections in orthopedic surgery is significant. Postoperative wound infection is a multifactorial process, which can be modified by several bacterial factors. Cell surface hydrophobicity of bacteria is a very important physicochemical feature, which has a great influence on the ability of bacteria to adhere to the surface of host cells or medical implants. Methods: In this study, the hydrophobic properties of thirteen bacterial strains (coagulase-negative staphylococci, Staphylococcus aureus and Pseudomonas aeruginosa) isolated from patients with postoperative deep wound infections following orthopedic procedures were determined by the salt aggregation test. Results were compared to the hydrophobicity of three Hungarian standard bacterial strains. The modifying effect of four antibiotics (cefuroxime, cefotaxime, amoxicillin combined with clavulanic acid and amikacin) – applied most often in our Department for prophylaxis and treatment of patients – were analyzed. Results: The cell surface hydrophobicity of certain strains showed considerable changes after antibiotic treatment. These alterations indicated the decrease in hydrophobicity. Supra-inhibitory concentrations (2× minimum inhibitory concentrations, MIC) of the antibiotics were able to induce more frequent alterations in hydrophobicity than sub-inhibitory (0.5× MIC) levels. Conclusions: Alterations in cell surface hydrophobicity caused by antibiotics can modify the adhesion process and thus the pathogenicity of bacterial strains. These changes should be taken into consideration in the management of proper antibiotic prophylaxis and in the treatment of orthopedic patients.

Structural variability of endotoxins from R-type isogenic mutants of Shigella sonnei†

The structural variations in the rough-type endotoxins [lipopolysaccharides (LPSs)] of Shigella sonnei mutant strains (S. sonnei phase II-4303, R41, 562H and 4350) were investigated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem MS. A series of S. sonnei mutants had previously been the subject of analytical studies on the biosynthesis of heptose components in the core oligosaccharide region of LPSs. This study gives a complete overview on the structures of the full core and lipid A of S. sonnei mutant strains by MS. We found that the LPSs of the isogenic rough mutants were formed in a step-like manner containing 0:1:2:3 heptose in the deep core region of 4350, 562H, R41 and 4303, respectively, and the longest LPS from the mutant S. sonnei 4303 contained also five hexoses. The structural variations in the lipid A moiety and in the oligosaccharide part of the intact LPS were followed by MALDI-TOF-MS/MS. For the dissolution and the ionization of the samples, 2,5-dihydroxybenzoic acid in citric acid solution was applied as matrix. The detailed evaluation of the mass spectra indicates heterogeneity in the lipid part due to the differences in the phosphate and fatty acid composition.