Ágnes Dörnyei

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.

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.

Effect of electrolyte pH on CIEF with narrow pH range ampholytes

CIEF of components following sequential injection of ampholytes and the sample zone offers unique advantages for analysis. The most important one of these is the efficient separation of amphoteric compounds having pIs outside the pH range of the ampholytes applied, but the resolution of the components can be increased by an adequate setup in the injection protocol. In this study, the effect of the pH of the anolyte and catholyte on the selectivity and speed of the isoelectric focusing was investigated. Changes in the pH values significantly influenced the resolution and the length of the pH gradient, while changes in the charge state of components were also observed. Three ampholyte solutions (from different suppliers) covering only two pH units were used for the analyses of substituted nitrophenol dyes in uncoated capillary. With appropriate setup, the components, with pIs not covered by the ampholyte pH range, migrated in charged state outside the pH gradient. This phenomenon is preferable for coupling isoelectric focusing to MS detection, by evading the undesirable ion suppression effect of ampholytes.

Sequential injection setup for capillary isoelectric focusing combined with MS detection

Capillary isoelectric focusing in the presence of electroosmosis with sequential injection of carrier ampholytes and sample was found to be suitable for MS detection. The separate injection of the sample and the ampholytes provides good condition to suppress and overcome the undesirable effect of the presence of ampholytes in MS. By the appropriate selection of ampholyte solutions, whose pH range not necessarily covers the pI values of the analytes, the migration of the components can be controlled, and the impact of the ampholytes on MS detection is decreased. The unique applicability of this setup is shown by testing several parameters, such as the application of volatile electrolyte solutions, the type of the ampholytes, the order and the number of the ampholyte and sample zones. Broad and narrow pH range ampholytes were applied in experiments using uncoated capillaries with different lengths for the analyses of substituted nitrophenol dyes to achieve optimal conditions for the MS detection. Although the sample components are not leaving the pH gradient, due to the decrease in the ampholyte concentration at the position of the components, and because the sample components migrate in charged state, the ionisation is more effective for MS detection.

Characterization of complex, heterogeneous lipid A samples using HPLC–MS/MS technique II. Structural elucidation of non-phosphorylated lipid A by negative-ion mode tandem mass spectrometry

Non-phosphorylated lipid A species confer reduced inflammatory potential for the bacteria. Knowledge on their chemical structure and presence in bacterial pathogens may contribute to the understanding of bacterial resistance and activation of the host innate immune system. In this study, we report the fragmentation pathways of negatively charged, non-phosphorylated lipid A species under low-energy collision-induced dissociation conditions of an electrospray ionization quadrupole time-of-flight instrument. Charge-promoted consecutive and competitive eliminations of the acyl chains and cross-ring cleavages of the sugar residues were observed. The A-type fragment ion series and the complementary X-type fragment(s) with corresponding deprotonated carboxamide(s) were diagnostic for the distribution of the primary and secondary acyl residues on the non-reducing and the reducing ends, respectively, of the non-phosphorylated lipid A backbone. Reversed-phase liquid chromatography in combination with negative-ion electrospray ionization quadrupole time-of-flight tandem mass spectrometry could provide sufficient information on the primary and secondary acyl residues of a non-phosphorylated lipid A. As a standard, the hexa-acylated ion at m/z 1636 with the Escherichia coli-type acyl distribution (from E. coli O111) was used. The method was tested and refined with the analysis of other non-phosphorylated hexa- and several hepta-, penta-, and tetra-acylated lipid A species detected in crude lipid A fractions from E. coli O111 and Proteus morganii O34 bacteria. Copyright

Structural background for serological cross‐reactivity between bacteria of different enterobacterial serotypes

The structure of the oligosaccharide repeating units of endotoxins from Gram‐negative bacteria is characteristic for the different serogroups and serotypes of bacteria. Detailed examination of the cross‐reactions of three enterobacterial serotypes, Proteus morganii O34, Escherichia coli O111, and Salmonella enterica sv. Adelaide O35, was performed using sensitive tests (ELISA, immunoblotting). Fine differences between the endotoxins of the bacteria were detected using silver staining of SDS‐PAGE gels and chip‐technology for the intact lipopolysaccharides (LPSs). The compositions of the O‐specific polysaccharides of LPSs extracted from the bacteria were studied, and it was proven that the three cross‐reacting bacteria contain O‐antigens built from the same monosaccharides, namely colitoses linked to glucose, galactose, and N‐acetyl‐galactosamine. The NMR and GC‐MS studies revealed that the most probable component for the cross‐reaction is the rare sugar, colitose.

Application of a Novel Small-Scale Sample Cleanup Procedure Prior to MALDI-TOF-MS for Rapid Pigment Fingerprinting of Red Wines

This study evaluates the anthocyanin and derived pigment composition of Vitis vinifera red wines of Vranec, Merlot, and Cabernet Sauvignon produced in 2006, 2007, and 2008 vintages from the Tikveš wine region in the Republic of Macedonia. Their profile was established using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technique. A total of 22 anthocyanins and derived pigments have been identified in the samples including 10 anthocyanins, 1 ethyl-bridged flavanol–anthocyanin adduct, and 11 pyranoanthocyanins. MALDI-TOF-MS analysis was performed after solid-phase extraction of the wines by using, for the first time, the Zip-Tip® C18 stationary phase, introducing a novel small-scale sample cleanup procedure prior to the rapid MALDI-TOF-MS fingerprinting of wine samples. 2′,4′,6′-Trihydroxyacetophenone (dissolved in acetonitrile/water 1:1, v/v) was used as a matrix. The qualitative screening of anthocyanins and derived pigments with MALDI-TOF-MS confirmed the presence of glucoside, acetylglucoside, and p-coumaroylglucoside derivatives of anthocyanins in the wine samples. Furthermore, pyranoanthocyanins formed by reactions of anthocyanins with pyruvic acid and acetaldehyde, as well as flavanol–pyranoanthocyanins and ethyl-bridged flavan-3-ol-anthocyanin adduct pigments have been detected in the samples.

Phosphoglycolipid Profiling of Bacterial Endotoxins

Abstract Much interest is at present focused on bacterial endotoxins, also known as lipopolysaccharides (LPS), as they are responsible for the development of clinical symptoms of Gram-negative sepsis which is the leading cause of death in intensive care units. Endotoxicity is associated with the special phosphoglycolipid part of LPS, termed lipid A. Main challenges in the structural elucidation of lipid A arise from its amphiphilic character and inherent heterogeneity. A mass spectrometrybased de novo method combined with reversed-phase liquid chromatography for the detailed structural characterization of complex lipid A mixtures (obtained by mild acid hydrolysis of LPS) from different bacterial sources has been developed. Tandem mass spectrometric measurements were performed with an electrospray-ionisation quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer in both negative- and positive-ionization modes in order to explore fragmentation pathways. It was found that characteristic product ions in the positive-ion mode could be used for the unambiguous assignment of the phosphorylation site, whereas the use of both ionization modes provided consistent and/or complementary information about the fatty acyl distribution between the two glucosamine moieties of lipid A. Since the immunostimulatory (advantageous) vs. proinflammatory (endotoxic) effect of the lipid A is closely related to the fine chemical structure, our relatively simple structural elucidation strategy could offer great potential in the bioanalysis of native lipid A samples and lipid A-based vaccines

Mass Spectrometry for Profiling LOS and Lipid A Structures from Whole-Cell Lysates: Directly from a Few Bacterial Colonies or from Liquid Broth Cultures

Lipopolysaccharides (LPSs, endotoxins) are components of the outer cell membrane of most Gram-negative bacteria and can play an important role in a number of diseases of bacteria, including Gram-negative sepsis. The hydrophilic carbohydrate part of LPSs consists of a core oligosaccharide (in the case of an R-type LPS or lipooligosaccharide, LOS) linked to an O-polysaccharide chain (in the case of an S-type LPS), which is responsible for O-specific immunogenicity. The hydrophobic lipid A anchor is composed of a phosphorylated diglucosamine backbone to which varying numbers of ester- and amide-linked fatty acids are attached and this part of the LPSs is associated with endotoxicity. The detailed chemical characterization of endotoxins requires long-lasting large-scale isolation procedures, by which high-purity LPSs can be obtained. However, when a large number of bacterial samples and their LPS content are to be compared prompt, small-scale isolation methods are used for the preparation of endotoxins directly from bacterial cell cultures. The purity of the endotoxins extracted by these methods may not be high, but it is sufficient for analysis.Here, we describe a fast and easy micromethod suitable for extracting small quantities of LOS and a slightly modified micromethod for the detection of the lipid A constituents of the LPSs from bacteria grown in different culture media and evaluate the structures with mass spectrometry. The cellular LOS and lipid A were obtained from crude isolates of heat-killed cells, which were then subjected to matrix-assisted laser desorption/ionization mass spectrometry analysis. The observed ions in the 10-colony samples were similar to those detected for purified samples. The total time for the sample preparation and the MS analysis is less than 3 h.