Róbert Ohmacht

Enrichment of Amadori products derived from the nonenzymatic glycation of proteins using microscale boronate affinity chromatography

Amadori peptides were enriched using boronate affinity tips and measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). As demonstrated by electrochemical measurements, the tips show the highest binding efficiency for glucose at pH 8.2 employing ammonium chloride/ammonia buffer with ionic strength of 150 mM, exceeding taurine buffer at the same concentration. The bound constituents were released by sorbitol and formic acid. It was also demonstrated that elution with sorbitol at 1.2 M is superior to acidic media. Comparison of results was based on the numbers of detected peptides and their glycated sites. Using sorbitol for elution requires desalting prior to analysis. Therefore, three different sorbents were tested: fullerene-derivatized silica, ZipTip (C18), and C18 silica. Fullerene-derivatized silica and ZipTip showed the same performance regarding the numbers of glycated peptides, and sites were better than C18 silica. The elaborated off-line method was compared with liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements, by which considerable less modified peptides were detected. Affinity tips used under optimized conditions were tested for the analysis of human serum albumin (HSA) from sera of healthy and diabetic individuals. A peptide with a mass of 1783.9 Da could be detected only in samples of diabetic patients and, therefore, could be a very interesting biomarker candidate.

Alcohol-free red wine inhibits isoproterenol-induced cardiac remodeling in rats by the regulation of Akt1 and protein kinase C α/β II☆

There is increasing evidence that moderate consumption of red wine containing high amount of polyphenols and anthocyanins is associated with decreased incidence of cardiovascular morbidity and mortality. Therefore, we hypothesized that cardiac hypertrophy and fibrosis as well as Akt (protein kinase B, PKB) and protein kinase C (PKC) cascades can be beneficially influenced by an alcohol-free red wine (AFRW) extract rich in 14 types of polyphenols and 4 types of anthocyanins during cardiac remodeling. To test this assumption, rats were treated with isoproterenol (ISO) to induce postinfarction remodeling and were given tap water or AFRW ad libitum for 8 weeks. Control rats received vehicle instead of ISO. Heart mass/body mass and ventricle mass/body mass ratios, diameter of cardiomyocytes, phosphorylation of PKC alpha/beta II and protein kinase B/Akt, and deposition of collagen type III were determined from the hearts of all four groups of rats. All measured gravimetric parameters, myocyte diameters and the amount of collagen type III decreased, and the phosphorylation of PKC alpha/beta II was reduced in the ISO+AFRW group compared to the ISO group. AFRW induced activation of Akt, one of the best characterized cytoprotective pathways even without ISO treatment, and this activation was further increased in the ISO+AFRW group. These data suggest that AFRW treatment has a protective effect on hearts undergoing postinfarction remodeling by repressing hypertrophy-associated increased phosphorylation of PKC alpha/beta II and by activating Akt, providing a molecular mechanism for the cardioprotective effect of red wine polyphenols.

Use of fullerene‐, octadecyl‐, and triaconthyl silica for solid phase extraction of tryptic peptides obtained from unmodified and in vitro glycated human serum albumin and fibrinogen

SPE plays a crucial role in bioanalytical research. In the present work a novel fullerene(C60)-derivatised silica material is compared with octadecyl(C18) - and triaconthyl(C30)-silicas regarding recoveries of peptides and sequence coverage of HSA and fibrinogen digests. C30- and C60(30 nm)-SPE materials were found to be the two most prominent SPE materials. At low peptide concentrations C60-material prepared from a silica gel with a pore size of 30 nm has proven to be the best material with regards to recoveries. By increasing the amount of loaded peptides recoveries decrease due to its relative low binding capacity in contrast to C30-silica particles, showing no changes. The best sequence coverages of Aalpha- and Bbeta-chains of 20 pmol fibrinogen digest can also be achieved using these two SPE materials, C60 (30 nm) demonstrates an outstanding value of sequence coverage (62.15%) achieved for the gamma-chain. After nonenzymatic glycation the digests of fibrinogen and HSA were also separated. This makes the detection of a considerably higher number of glycated peptides possible compared to the unfractionated digests and the use of boronate affinity chromatography in the case of fibrinogen. For HSA, ten new sites of glycation at lysine and arginine residues have been explored. Using the detailed SPE/off-line MALDI method the glycation sites on fibrinogen are first described in this paper.

Olive Oil as a Biocompatible Solvent for Pristine C60

Abstract Olive oil is suggested a new solvent for C60 which offers the possibility to deliver in a biocompatible from this compound to biosystems and living organisms. This allows investigating for the first time the true biocompatibility and toxicity of C60.

Effect of pressure on retention factors in HPLC using a non-porous stationary phase

SummaryPressure and temperature have significant influence on retention in HPLC. This study investigates the effect of pressure and temperature on the retention behavior of aromatic hydrocarbons (toluene, ethyl benzene, butyl benzene, pentyl benzene) and polar, acidic and basic samples (phenol, acetophenone, N,N-dimethyl aniline, benzophenone) on a reversed phase column. The effect has been studied on non-porous, tetradecyl (C14) coated silica particles. We found that the adsorption-induced decrease of the partial molar volume of the solutes investigated was between ΔVm=5–15 cm3 mol−1. The increment of the decrease of the partial molar volume due to the addition of one CH2 group, for the homologous series of the aromatic hydrocarbons is approximately ΔVCH2=2.3 cm3mol−1.

Effect of pressure on solute capacity factor in HPLC using a non-porous stationary phase

SummaryThe pressure applied in liquid chromatography (LC) can influence the capacity factor of analytes. Using commercial equipment, column (33×4.6 mm) and 1.5 μm non-porous, tetradecyl (C14) coated silica particles, a moderate but significant change in the capacity factor was observed with increasing pressure. Depending on the experimental conditions the relationship was found to be either linear or non-linear. In the latter case the retention increased at first, but later tended to decrease at still higher pressure. The results direct attention to the role of the pressure (and hence of flow rate and heat of friction) in determining capacity factors.

Near infrared spectroscopy, cluster and multivariate analysis - Characterisation of silica materials for liquid chromatography

Near infrared (NIR) reflectance spectroscopy is a well established method within the analytical chemistry and a wide range of applications, for example in the field of food and pharmaceutical products, is covered. In the era of material science and nano technology, we brought together, in this feasibility study, silica chemistry with NIR. NIR, in the fibre-optics mode, is used for morphological studies. It enables differentiation between silica materials in a three-dimensional factor-plot depending on their functionalisation and physical properties such as particle diameter and pore diameter with Q-values of 0.95 and 0.99. The well established, common reference methods for the determination of surface area and porosity are the so called Brunnauer Emmett Teller method and size-exclusion chromatography. Both methods are time-consuming and require a lot of experience. The recently elaborated NIR method offers a physicochemical quantitative description at the nano-scale level of particle size, specific surface area, pore diameter, pore porosity, pore volume and total porosity with high linearity of R2 > 0.97 for calibration and r2 > 0.98 for validation and a bias of < 2.48 × 10−14. Compared to the reference method, which enables the determination of the individual parameters with a relative standard deviation between 6 and 28%, the NIR method shows RSD% between only 0.010 and 13.7%, which means a fundamental improvevement in precision. The measurement takes only a few seconds so high sample throughput is guaranteed. The suitability of the newly established method in material science is demonstrated, as an example, on the silica stationary phases for liquid chromatography.

HPLC separation of opium alkaloids on porous and non-porous stationary phases

SummaryA new reversed-phase (RP) HPLC method has been developed and validated for the separation of the main opium alkaloids morphine, codeine, thebaine, papaverine and noscapine on a non-porous (micropellicular) stationary phase. On this phase quantification of the compounds by internal standardization with brucine was achieved extremely rapidly, in ca 1.5 min, only. Thus, the analysis time for the opium alkaloids was approximately one tenth of that on porous stationary phases. Different opium samples were investigated using non-porous and porous packings. The correlation between the results was excellent.

Analysis of nonderivatized steroids by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using C70 fullerene as matrix

Neutral steroid hormones are currently analyzed by gas or liquid chromatography/mass spectrometry based methods. Most of the steroid compounds, however, lack volatility and do not contain polar groups, which results in inadequate chromatographic behavior and low ionization efficiency. Derivatization of the steroids to form more volatile, thermostable, and charged products solves this difficulty, but the derivatization of compounds with unknown chemical moieties is not an easy task. In this study, a rapid, high-throughput, sensitive matrix-assisted laser desorption/ionization time-of-flight mass spectrometry method is described using C70 fullerene as a matrix compound. The application of the method is demonstrated for five general sex steroids and for synthetic steroid compounds in both negative and positive ionization modes.

Quick and sensitive HPLC separations on non-porous reversed-phase packings

SummaryThe aim of this work was to evaluate reversed-phase chemically bonded non-porous (micropellicular)dp=1.5 μm stationary phases. On these modern phases the time for analysis of complex mixtures of solutes—whether monomeric or polymeric (e.g. drugs, vitamins, peptides, or protein)—is very short compared with that on porous phases. Different surface chemistries were elaborated for the separation of different types of sample. For the separation of small molecules a long-chain (C14) hydrocarbon-coated phase seems to be optimum; a short chain (C6) hydrocarbon bonded to the surface of the silica seems better for the separation of polymers.The efficiency, the low analysis times, and sensivities were demonstrated by separation of different proteins, peptides, drugs, alkaloids, and mixtures of water and fat-soluble vitamins.