Saccharides form one of the major constituents of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response and receptor ...activation are regulated by glycosylation. In this work, we optimized a capillary electrophoresis method with capacitively coupled contactless conductivity detection for the separation of eight monosaccharides commonly found in glycoproteins, namely D‐glucose, D‐galactose, D‐mannose, N‐acetyl‐D‐glucosamine, N‐acetyl‐D‐galactosamine, D‐fucose, N‐acetylneuraminic acid, and D‐xylose. A highly alkaline solution of 50 mM sodium hydroxide, 22.5 mM disodium phosphate, and 0.2 mM CTAB (pH 12.4) was used as a background electrolyte in a 10 µm id capillary. To achieve baseline separation of all analytes, a counter‐directional pressure of –270 kPa was applied during the separation. The limits of detection of our method were below 7 µg/ml (i.e., 1.5 pg or 1 mg/g protein) and the limits of quantification were below 22 µg/ml (i.e., 5 pg or 3 mg/g protein). As a proof of concept of our methodology, we performed an analysis of monosaccharides released from fetuin glycoprotein by acid hydrolysis. The results show that, when combined with an appropriate pre‐concentration technique, the developed method can be used as a monosaccharide profiling tool in glycoproteomics and complement the routinely used LC‐MS/MS analysis.
A method for purity control of newly synthesized lactic acid–based liquid crystals has been developed. The electrokinetic chromatography proved to be suitable for the separation of these ...electroneutral substances from their impurities. The separations were performed in an acidic acetonitrile‐based background electrolyte (BGE) with a pseudostationary phase formed by a cationic surfactant. During the optimization step, appropriate concentrations of cetyltrimethylammonium bromide, acetic acid, and water were seeked. In the optimized method, separations were carried out in acetonitrile with 1‐mol/L acetic acid, 80‐mmol/L cetyltrimethylammonium bromide, and 6% (v/v) water. Interesting positive effects of a small water content in the BGE on electroosmotic flow and resolution of liquid crystal substances from their impurities were observed and discussed. Samples of five liquid crystal substances, both pure and containing impurities from synthesis, were analyzed. The identification of analytes was based on a comparison of relative migration times related to the migration time of mesityl oxide.
For all five samples, impurities were separated from the liquid crystals and the method thus showed its viability. To the best of our knowledge, this method is used for the first time for the purity control of newly synthesized liquid crystals. This method can be used to confirm or complement the results obtained by commonly used high‐performance liquid chromatography and supercritical fluid chromatography methods. Furthermore, the electrokinetic chromatography method requires very small amounts of sample, solvents, and buffer constituents. Overall, its operational costs are significantly lower.
As the demand for medical cannabis preparations increases, so does the use of the common organic solvents that are used in the extraction and quantification of phytocannabinoids. Since common organic ...solvents are typically hazardous to the environment and to human health, it is vital to identify safer, greener, and more efficient alternatives. The aim of the present research was to develop a series of hydrophobic deep eutectic solvents (DESs) based on terpenes and natural organic acids and to establish whether these might be potential substitutes for the extraction of phytocannabinoids (tetrahydrocannabinol, cannabidiol, and their carboxylated homologues) from raw cannabis plant material.
Data were obtained using capillary electrophoresis with diode array detection (DAD). Initial screening showed that the DES composed of a menthol: acetic acid (1:1 M ratio) mixture showed the greatest extraction efficiency (of all the DESs that were tested), with yields ranging from 118.6% to 132.6% (compared to a methanol: chloroform mixture). In conclusion, menthol: acetic acid DES extraction is efficient, as well as non-toxic and biodegradable. As such it has applications within the pharmaceutical industry and represents a greener alternative organic solvent for the extraction of phytocannabinoids.
•A series of menthol-based deep eutectic solvents (DESs) were prepared.•Hydrophobic DESs were used to extract phytocannabinoids from plant material.•Menthol:acetic acid DES showed superior extraction yields to conventional organic solvents.•Menthol-based DESs are greener alternatives for phytocannabinoid extraction.
In the present study, we characterize the binding of divalent cations to insulin in aqueous salt solutions by means of capillary electrophoresis and molecular dynamics simulations. The results show a ...strong pH dependence. At low pH, at which all the carboxylate groups are protonated and the protein has an overall positive charge, all the cations exhibit only weak and rather unspecific interactions with insulin. In contrast, at close to neutral pH, when all the carboxylate groups are deprotonated and negatively charged, the charge-neutralizing effect of magnesium, calcium, and zinc, in particular, on the electrophoretic mobility of insulin is significant. This is also reflected in the results of molecular dynamics simulations showing accumulation of cations at the protein surface, which becomes smaller in magnitude upon effective inclusion of electronic polarization via charge rescaling.
The fungus Amanita muscaria is universally recognizable for its iconic appearance; it is also widely regarded as poisonous, inedible, and even deadly. In spite of that, there have been documented ...cases of use of A. muscaria-containing preparations against various diseases, including cancer, to no apparent ill effect. The search for compounds that can be used to treat cancer among various plants and fungi has been intensifying in recent years. In light of this, we describe an HPLC HILIC analytical method for the evaluation of the content of the anticancer compound ergosterol (ERG) and the neuroactive alkaloids ibotenic acid (IBO) and muscimol (MUS) that contribute significantly to the unpleasant physiological syndrome associated with A. muscaria consumption. A ‘homemade’ A. muscaria tincture made using 80-proof rye vodka as the solvent, an A. muscaria extract made with a standardized water–ethanol solution as the solvent, and fractions obtained from the second extract via liquid–liquid extraction with nonpolar solvents were analyzed. The study also presents the results of capillary zone electrophoresis with contactless conductivity detection and UHPLC-MS/MS analyses of the IBO and MUS content of the two native A. muscaria extracts and an evaluation of the standardized extract’s cytotoxic effect against a small panel of lung cell cultures in vitro. Our results show that the standardized extract has a significant cytotoxic effect and does not contain the compounds of interest in any significant quantity.
Electroosmotic flow significantly impacts the resolution of separations in capillary electrophoresis and its modification is often necessary. Coating of the inner capillary surface either dynamically ...or permanently offers a way to alter the electroosmotic flow, potentially reducing the adsorption of analytes, in our case liposomes, to the capillary wall. At first, we measured fluorescently labeled liposomes in an uncoated capillary by capillary electrophoresis with laser-induced fluorescence detection. We used a special procedure of the electrophoretic experiment allowing us to observe the development of peak shape at the early stages of migration. We proved that the liposomes were adsorbed to the capillary wall, which led to a very quick and severe dispersion of their peak during their electromigration. For this reason, we used a commercially coated capillary with polyvinyl alcohol, where at the same separation conditions, we observed the peak of the liposome with a stable shape during the migration. However, this capillary is costly, thus four simple dynamic coating methods were tested for four polymers, namely Pluronic F-127, polyvinyl pyrrolidone K30, polyethylene glycol, and polydiallyldimethylammonium chloride. Among them, we chose a method where we flushed the capillary with a 10% solution of polyvinyl pyrrolidone K30 before the first measurement. In addition, in-house made permanent coating with linear polyacrylamide was investigated, which has led to effective suppression of the electroosmotic flow and stable liposome peak, not dispersed during its migration. Liposome separation using this coating fully confirmed that the studied liposomes are negatively charged and migrate in the anodic direction.
Graphical abstract
The feasibility of on‐capillary derivatization of saccharides by aromatic amine‐based fluorescent labeling agents was tested. To avoid the problematic evolution of gaseous hydrogen cyanide, the ...Schiff base reduction by sodium cyanoborohydride, as the second step of the standard reductive amination protocol, was omitted. Glucose was used as a model analyte and 7‐amino‐1,3‐naphthalenedisulfonic acid as the labeling agent. Our experiments showed that the direct reaction of the saccharide with the labeling agent in 2.5‐M acetic acid yields a labeled product that is sufficiently stable to be separated from the labeling agent in 20‐mM phosphate buffer, pH 3.5, and detected using UV detection. The glucose and label zones were introduced separately into the capillary and mixed using a negative voltage. Mixing voltage, its duration, the concentration of acetic acid in the reaction zone, and the waiting time between mixing and separation were optimized. To show the applicability of the procedure to a broader range of analytes, a mixture of different types of saccharides, that is, xylose (pentose), fucose (hexose), glucose (hexose), N‐acetylglucosamine (N‐acetylaminosaccharide), and lactose (disaccharide), was subjected to derivatization and analysis under the optimal conditions. The linearity and repeatability of the process were evaluated as critical parameters for its analytical applications. Six‐point calibration dependences in the 1–50 mM range showed excellent determination coefficients of 0.9992 or higher for all five saccharides tested. The repeatability of the labeled saccharide peak areas was between 2.2% and 4.3%.
Dandelion-flavored syrup, also known as “dandelion honey”, is prepared and used as a substitute for bee honey. One of the major differences between dandelion syrup and bee honey seems to be the ...carbohydrate composition, as bee honey consists mainly of glucose and fructose while pure sucrose is used for the preparation of dandelion syrup. In this study, we show that lemon juice, added during the preparation of dandelion syrup, catalyzes the acid hydrolysis of sucrose to glucose and fructose. We show the crucial role of pH, temperature, and boiling time on the carbohydrate composition of the prepared syrup. We conclude that when a sufficient amount of lemon juice is added and the mixture is heated for a sufficiently long time, the carbohydrate composition of the resulting dandelion syrup can mimic the carbohydrate composition of bee honey, i.e., it contains glucose and fructose with an insignificant amount of sucrose.
Graphic abstract
Capillary electrophoresis methods for the separation of carbohydrates with four different detection techniques, namely direct UV, indirect UV, capacitively coupled conductivity, and laser-induced ...fluorescence detection, were tested and their performance was evaluated and compared in terms of linearity, limits of detection and quantitation, repeatability, recovery, analysis time, and sample treatment. The test set of analytes comprised sucrose, glucose, and fructose. The effect of using lactose as an internal standard on the individual methods was investigated, too. The results showed that laser-induced fluorescence detection is a technique of choice for applications requiring the detection of very low amounts of reducing carbohydrates. Contactless conductivity detection is favorable when detection sensitivity is not a crucial parameter but fast and reliable analysis is required. When only a UV detector is available as a standard part of capillary electrophoresis instruments, direct UV detection can be used when analysis time is not a critical parameter. For fast analysis with UV detectors, indirect UV detection is the technique of choice. Finally, to verify the applicability of the tested methods, samples of cola beverage, honey, and orange juice were analyzed and the results obtained by all four methods were compared.
Graphical abstract
The oligomeric state of the storage form of human insulin in the pancreas, which may be affected by several endogenous components of β-cell storage granules such as arginine, is not known. Here, the ...effect of arginine on insulin oligomerization is investigated independently by protein crystallography, molecular dynamics simulations, and capillary electrophoresis. The combined results point to a strong effect of ionic strength on insulin assembly. Molecular simulations and electrophoretic measurements at low/mM salt concentrations show no significant effect of arginine on insulin aggregation. In contrast, crystallographic data at high/molar ionic strength indicate inhibition of insulin hexamerization by arginine due to its binding at the site relevant for intermolecular contacts, which was also observed in MD simulations. Our results thus bracket the in vivo situation in pancreatic β-cell storage granules, where the ionic strength is estimated to be in the hundreds of millimolar to submolar range. The present findings add to a molecular understanding of in vivo insulin oligomerization and storage, with additional implications for insulin stability in arginine-rich injections.
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