Biomedical subjects

Ernst Kenndler

Publications and source records attributed to Ernst Kenndler.

At least 37 records · Page 2Linked to original sources

PubMed2005

Analyte and system eigenpeaks in nonaqueous capillary zone electrophoresis: theoretical description and experimental confirmation with methanol as solvent.

A mathematical model developed for aqueous solutions and adapted to methanol as solvent was applied to predict the electromigration characteristics of analytes and background electrolytes in capillary zone electrophoresis. These characteristics are the effective mobility, and the tendency of the analyte to undergo peak-broadening due to electromigration dispersion. The input parameters for calculation like limiting mobilities and dissociation constants were experimentally determined or taken from the literature. By the aid of the model, the molar response for conductivity detection was calculated as well as the transfer ratio when indirect UV detection was used. They allow depicting the electropherogram by computer simulation. An additional important program output is the prediction of the occurrence of system- or eigenpeaks that mimic peaks of analytes or electroosmotic flow markers. The measured electropherograms were in agreement with those theoretically predicted. Deviations were attributed to ion pairing in methanolic solutions, which was not implemented in the model.

Cations↗

PubMed2005

Characterization of proteins from Spirulina platensis microalga using capillary electrophoresis-ion trap-mass spectrometry and capillary electrophoresis-time of flight-mass spectrometry.

In this work, a new capillary electrophoresis-mass spectrometry (CE-MS) procedure is developed to analyze proteins in Spirulina platensis microalgae. It is demonstrated that a fine optimization of several separation parameters is essential in order to achieve suitable CE-MS analysis of these proteins in natural extracts from microalgae. Namely, optimization of the composition of the separation buffer, electrospray conditions, and washing routine between runs are required in order to obtain reliable and reproducible CE-MS analyses of the main proteins found in this microalga (namely, allophycocyanin-alpha chain, allophycocyanin-beta, c-phycocyanin-alpha, and c-phycocyanin-beta). The relative molecular mass of these biopolymers is determined using two different MS instruments coupled to CE, i.e., CE-ion trap-MS and CE-time of flight-MS (CE-TOF-MS). A comparison between the results obtained with both instruments is carried out. The high resolution of the TOF-MS enables the distinction of small modifications in proteins and, thus, a more accurate mass determination. Interestingly, molecular mass values obtained by both CE-MS procedures agree very well while these experimental values are only in partial agreement with those theoretically expected (i.e., genetically derived masses). Some protein modifications due to amino acids exchange induced by nucleotide codon mutations are proposed to explain this difference.

Bacterial Proteins↗

PubMed2005

Comparison of methanol and acetonitrile as solvents for the separation of sertindole and its major metabolites by capillary zone electrophoresis.

Sertindole (1-[2-[4-[5-chloro-1-(4-fluorophenyl)-1H-indol-3-yl]-1-piperidinyl]ethyl]-2-imidazolidinone), an atypical antipsychotic drug, was separated by capillary electrophoresis from its two main metabolites norsertindole and dehydrosertindole. The low solubility of the analytes in water (octanol-water partition coefficient is about 10(5)) is overcome by the use of methanol (MeOH) and acetonitrile (ACN) as solvents for the background electrolyte (BGE). Mobilities were measured in BGEs with defined pH in a broad range. It was found that in MeOH the mobility of the analytes is mainly governed by acid-base equilibria, whereas in ACN other reactions like ion pairing and homo-conjugation play a pronounced role and lead to a complex pattern of the mobility as function of the pH. However, separation can be obtained in less than 10 min in both solvent systems.

Acetonitriles↗

PubMed2005

Capillary electrophoresis in N,N-dimethylformamide.

N,N-Dimethylformamide (DMF) is a dipolar protophilic solvent with physicochemical properties that makes it suitable as solvent for capillary electrophoresis (CE). It is prerequisite for the proper application of CE to adjust and to change the pH of the background electrolyte (BGE) in a defined manner. This was done in the present work using benzoic acid-benzoate by selecting different concentration ratios of acid and salt, and calculating the theoretical pH from the activity-corrected Henderson-Hasselbalch equation. The mobilities of the analytes (chloro- and nitro-substituted phenolates) were found to follow reasonably well the typical sigmoid mobility versus pH curve as predicted by theory. The actual mobilities and pK(a) values (at 25 degrees C) of the analytes were derived from these curves. pK(a) values were in the range of 11.1-11.7, being thus 3-4.4 units higher than in water. This pK(a) shift is caused by the destabilization of the analyte anion and the better stability (solubility) of the molecular analyte acid in DMF, which overcome the higher basicity of DMF compared to water. Absolute mobilities were calculated from the actual mobilities; they were between 32x10(-9) and 42x10(-9) m(2)/Vxs. Slight deviations of the measured mobilities from the theoretical mobility versus pH curve were discussed on the bases of ion pairing and heteroconjugation and homoconjugation of either buffer components or buffer components and analytes. Heteroconjugation was used as a mechanism for the electrically driven separation of neutral analyte molecules in a BGE where salicylate acted as complex forming ion. Rough estimation of the complexation constants for the phenolic analytes gave values in the range of 100-200 L/mol. Addition of water to the solvent decreased the effect of heteroconjugation, but it was still present up to the surprisingly high concentration of 20% water. Electrophoretically relevant parameters like ionic mobilities and pK(a) values, and conjugation and ion pairing are dependent on the water content of the solvent. The water uptake of DMF was measured when exposed to humidity of ambient air. The resulted behavior of the water uptake was found rather similar to that for acetonitrile and methanol.

Dimethylformamide↗

PubMed2005

Are the asserted advantages of organic solvents in capillary electrophoresis real? A critical discussion.

Background electrolytes (BGEs) prepared in pure organic solvents are common alternatives to aqueous BGEs in capillary electrophoresis. Several general advantages of organic solvents over water have been asserted in the literature, namely (i) organic solvents increase the separation selectivity; (ii) organic solvents increase the separation efficiency; (iii) high separation voltages and/or high BGE ionic strengths can be used in organic solvents due to lower electric current compared to water. Related assumptions are that (iv) due to higher field strengths applicable in organic solvents the analysis time is shorter than in aqueous BGEs, and (v) the solubility and/or stability of components (either analytes or BGE chemicals) is higher/better in organic solvents. In the present work, these asserted advantages were critically evaluated based on the physical principles of ion transport and zone dispersion in solution. The result was that many of the above-mentioned general advantages are overestimated or even inexistent; often they have no fundamental basis.

Adsorption↗

PubMed2004

Labeling of capsid proteins and genomic RNA of human rhinovirus with two different fluorescent dyes for selective detection by capillary electrophoresis.

During uncoating of human rhinoviruses, the innermost capsid protein VP4 and the genomic RNA are released from the viral protein shell. This process gives rise to subviral particles that are composed of the remaining three capsid proteins VP1, VP2, and VP3. The process is believed to take place in a sequential manner in that first VP4 is expelled resulting in A-particles sedimenting at 135S followed by the RNA resulting in B-particles sedimenting at 80S. Aiming at ultimately analyzing this process in vivo, we introduced two different fluorophores into the RNA and the viral capsid proteins, respectively. Incubation of the virus with RiboGreen resulted in formation of a RNA-dye complex with lambda(ex)/lambda(em) = 500/525 nm, whereas subsequent derivatization of the viral protein shell in the same sample with AMCA-S introduced a label with lambda(ex)/lambda(em) = 345-350/440-460 nm. In this way, both viral components could be selectively detected via fluorescence in a capillary electrophoresis system. The intact virus delivers two superimposed signals in the electropherogram. Derivatization of the free amino groups of the capsid proteins partially preserved the bioaffinity of the virus toward a synthetic receptor fragment, an artificial recombinant concatemer of repeat number 3 of the very low density lipoprotein receptor. Between 10 and 20% of the infectivity were recovered after labeling when compared to native virus. In addition to analysis of factors influencing the stability of the virus by CE, double-labeled virions might be useful for the investigation of the uncoating process by real-time confocal fluorescence microscopy.

Amines↗

PubMed2004

Fluorescence labeling of human rhinovirus capsid and analysis by capillary electrophoresis.

The capsid of human rhinovirus serotype 2, consisting of four viral proteins, was fluorescence-labeled with fluorescein isothiocyanate and analyzed by capillary electrophoresis using UV and laser-induced fluorescence detection. Heat denaturation, proteolytic digestion, and receptor binding were applied for confirmation of the identity of the peak with the labeled virus. Incomplete derivatization with the fluorophore preserved the affinity of the virus for its receptor, indicating that its cell entry pathway is unperturbed by this chemical modification; indeed, an infectivity assay confirms that the labeled virus samples are infectious. The results show that fluorescence labeling of the viral capsid might lead to a valuable probe for studying infection processes in the living cell.

Capsid↗

PubMed2004

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.

Electrophoresis, Capillary↗

PubMed2004

Capillary zone electrophoresis in non-aqueous solutions: pH of the background electrolyte.

Although the establishment of a pH scale and the determination of the pH in water is not problematic, it is not a straightforward task in non-aqueous solvents. As capillary zone electrophoresis (CZE) in organic solvents has gained increasing interest, it seems to be valuable to re-discuss the concept of the pH in such media, especially pointing to those aspects, which make pH measurement uncertain in non-aqueous solvents. In this review, the relevant aspects when dealing with primary standard (PS) and secondary standard (SS) as recommended by the International Union of Pure and Applied Chemistry (IUPAC), and the usage of the operational pH are discussed with special emphasis to non-aqueous solvents. Here, different liquid junction potentials, incomplete dissociation of the electrolytes (especially in solvents with low or moderate relative permittivity) and the occurrence of homo- and heteroconjugation must be taken into account. Problems arising in capillary zone electrophoresis practice are addressed, e.g. when the background electrolyte (BGE) consists of organic solvents, but the measuring electrode (normally the glass electrode) is calibrated with aqueous buffers, and the liquid junction potentials between the solvents do not cancel each other. The alternative concept of establishing a certain pH is described, using mixtures of reference acids or bases with known pKa in the organic solvent, and their respective salts, at a certain concentration ratio, relying to the Henderson-Hasselbalch equation. Special discussion is directed to those organic solvents most common in capillary zone electrophoresis, methanol (MeOH) and acetonitrile (ACN), but other solvents are included as well. The potential significance of small amounts of water present in the organic solvent on changes in pKa values, and thus on the pH of the buffering components is pointed out.

Acetonitriles↗

PubMed2004

Separation of ethoxylated bisphenol A dimethacrylates in dental composite after derivatisation to ionisable amines by capillary zone electrophoresis.

Bisphenol A ethoxylate dimethacrylates (Bis-EMA) are transformed into ionisable amines by derivatisation in order to make the analytes applicable to capillary electrophoresis. For this goal, piperidine was added onto the C=C double bond of the alpha,beta-unsaturated ester group forming a tertiary amine with pKa values between 9 and 10. Formation of the derivatives was confirmed by electrospray ionisation MS. Commercial Bis-EMA is a mixture of homologues with different number of ethoxy groups; it is characterised by the average number of the ethoxy groups in the chains. These homologues were resolved by capillary zone electrophoresis at pH 4. It is shown for the product with an average of four ethoxy groups per Bis-EMA molecule that about seven homologues can be baseline separated when differing by only one ethoxy group. For Bis-EMA with 30 ethoxy groups in average, about 23 homologues could be differentiated. The high resolution power of capillary zone electrophoresis enables characterisation of commercial dental composite material concerning the Bis-EMA constituents.

Amines↗

PubMed2004

Binding of fluorescent dye to genomic RNA inside intact human rhinovirus after viral capsid penetration investigated by capillary electrophoresis.

RiboGreen is used for concentration measurements of RNA. Upon binding to the RNA, an approximately 1000-fold increase in sensitivity in comparison with the UV absorbance of the free polynucleotide is observed. In the present work, we demonstrate that this dye can penetrate in a time- and temperature-dependent manner the intact viral capsids of human rhinovirus serotypes 2 and 14, where it forms a fluorescent complex with the viral RNA. Capillary electrophoresis with laser-induced fluorescence detection of virus incubated with RiboGreen shows that the electrophoretic mobility of the viruses remained unchanged upon dye-binding. As shown for human rhinovirus serotype 2, its native conformation was conserved, since it still bound a recombinant soluble receptor fragment derived from the very low density lipoprotein receptor. The labeled RNA was released by heat-induced uncoating of the virus, and the RNA-dye complex could be directly detected if degradation was prevented with an RNase inhibitor. This in vitro labeling of viral RNA encased within a protein shell demonstrates the virion's dynamic nature that temporarily allows access of a low-molecular-mass compound to the otherwise protected RNA. It might be of great value for experiments requiring fluorescent viral particles with an unmodified surface, such as investigations of endocytosis and viral uncoating on the single molecule level.

Capsid Proteins↗

PubMed2004

Analysis of drying oils used as binding media for objects of art by capillary electrophoresis with indirect UV and conductivity detection.

Capillary electrophoresis (CE) was applied to analyse the long-chain fatty acid composition of vegetable oils, and their degradation products formed upon ageing when drying oils are used as binding media. The analytes were detected with contactless conductivity detection (CCD) and indirect UV absorption, both detectors positioned on-line at the separation capillary. The long-chain fatty acids were resolved in a background electrolyte (BGE) consisting of phosphate buffer (pH = 6.86, 15 mM) containing 4 mM sodium dodecylbenzensulfonate, 10 mM Brij 35, 2% (v/v) 1-octanol and 45% (v/v) acetonitrile. As in this system dicarboxylic analytes, the products of oxidative degradation of unsaturated fatty acids, cannot be determined, a suitable background electrolyte was developed by the aid of computer simulation program PeakMaster. It makes use of a 10 mM salicylic acid, 20 mM histidine buffer, pH 5.85, which combines buffering ability with the optical properties obligatory for indirect UV detection. This buffer avoids system eigenpeaks, which are often impairing the separation efficiency of the system. Separation of the dicarboxylic analytes was further improved by a counter-directed electroosmotic flow (EOF), obtained by dynamically coating the capillary wall with 0.2 mM cetyltrimethylammonium bromide. Long-chain fatty acids and their decomposition products could be determined in recent and aged samples of drying oils, respectively, and in samples taken from two paintings of the 19th century.

Art↗

PubMed2004

High-performance liquid chromatographic determination of Levetiracetam in human plasma: comparison of different sample clean-up procedures.

An accurate and precise high-performance liquid chromatographic method using diode array detection for the determination of the novel antiepileptic, Levetiracetam, has been developed. Three clean-up procedures for the analysis of Levetiracetam in human plasma were implemented and evaluated, namely solid-phase extraction, deproteinization by addition of organic solvents and formation of insoluble salts. Adenosine was used as the internal standard for all three sample pretreatment procedures. Among the several cartridges used for solid-phase extraction, the hydrophilic-lypophilic balance (Oasis) HLB) phase provides the best extraction yield of Levetiracetam, together with high precision. With the two other clean-up procedures involving plasma deproteinization by addition of methanol or zinc sulphate, lower sensitivity and precision of the assays were obtained. However, they are cheaper and faster when compared with the solid-phase extraction procedure.

Anticonvulsants↗

PubMed2004

Determination of cationic mobilities and pKa values of 22 amino acids by capillary zone electrophoresis.

The effective mobilities of the cationic forms of common amino acids--mostly proteinogenic--were determined by capillary zone electrophoresis in acidic background electrolytes at pH between 2.0 and 3.2. The underivatized amino acids were detected by the double contactless conductivity detector. Experimentally measured effective mobilities were fitted with the suitable regression functions in dependence on pH of the background electrolyte. The parameters of the given regression function corresponded to the values of the actual mobilities and the mixed dissociation constants (combining activities and concentrations) of the compound related to the actual ionic strength. McInnes approximation and Onsager theory were used to obtain thermodynamic dissociation constants (pK(a)) and limiting (absolute) ionic mobilities.

Amino Acids↗

PubMed2004

Capillary electrophoresis analysis of fosfomycin in biological fluids for clinical pharmacokinetic studies.

A feasible capillary zone electrophoresis (CZE) method with indirect UV and contactless conductivity detection was developed for the determination of fosfomycin, an antibiotic, in human plasma and microdialysis samples. Samples were collected from test persons during a clinical trial. The background electrolytes used consisted of 25 mM benzoic acid and 0.5 mM hexadecyltrimethylammonium bromide, adjusted with tris(hydroxymethyl)aminomethane solution to pH 6.95 for plasma, and to pH 8.05 for microdialysis samples. CZE separations of the anionic analyte were carried out with reversed electroosmotic flow directed towards the anode. The limit of detection was between 0.6 and 2 microg/mL, depending on the matrix and the detection method. No sample preparation was needed for microdialysis samples; for plasma samples, proteins were precipitated with methanol (1+2, v+v), and the supernatant was analyzed. The yield determined with spiked samples was about 100%, the reproducibility of the entire method, expressed by the RSD% of three independent determinations of fosfomycin in triplicate after spiking Ringer's solutions and plasma samples, respectively, was better than 8%. The method is thus well-suited for clinical studies for the determination of the antibiotic in biological fluids.

Benzoic Acid↗

PubMed2004

Capillary electrophoresis of biological particles: viruses, bacteria, and eukaryotic cells.

A review about the application of electrophoretic methods in the capillary format for the investigation of large biological assemblies like viruses, bacteria, yeast or entire mammalian cells is given. These entities are of a size ranging between some nanometers and tens of micrometers. They can form colloidal solutions or dispersions and move under the influence of an electric field. They are separated by zone electrophoresis according to their different electrophoretic velocity, and characterized by the electrophoretic mobility, which is easily determinable in free solution in capillaries or in other microdevices. As the charge of these particles, when being amphoteric, is pH-dependent, isoelectric focusing can also be carried out and the capillary format is increasingly being employed for their separation and determination of pI values. Furthermore, interactions with ligands can be assessed by various modes of affinity capillary electrophoresis. Capillary zone electrophoresis has thus become a valuable tool for investigation of large macromolecular assemblies in the field of biochemistry, clinical chemistry, toxicology, and nutrition chemistry amongst many others.

Bacteria↗

PubMed2004

Formamide as solvent for capillary zone electrophoresis.

A comprehensive investigation of a number of aspects when using formamide as background electrolyte solvent in capillary zone electrophoresis was presented. It included (i) the change of the ion mobility with ionic strength, (ii) the influence of the ionic strength on diffusion coefficients, and (iii) on the separation efficiency expressed by the maximum reachable plate numbers (when only longitudinal diffusion contributed to zone broadening), (iv) the effect of the solvent on pKa values (taken from the literature) of neutral and cation acids, (v) the establishment of the a pH scale in formamide by dissolving acids with known pKa values and their salts at defined proportion (thus circumventing the problem of calibrating the pH meter), (vi) the agreement between the experimentally derived and the theoretical dependence of the effective mobility on pH, (vii) the uptake of water of this hygroscopic solvent from the humidity of the environment and its consequence to the ion mobilities, pKa values, and the chemical stability of the solvent (e.g., hydrolysis), and finally (viii) the use of conductivity and indirect UV absorption to enable detection of analytes below the optical cutoff of formamide.

Diffusion↗

PubMed2004

System zones in capillary zone electrophoresis.

When working with capillary zone electrophoresis (CZE), the analyst has to be aware that the separation system is not homogeneous anymore as soon as a sample is brought into the background electrolyte (BGE). Upon injection, the analyte creates a disturbance in the concentration of the BGE, and the system retains a kind of memory for this inhomogeneity, which is propagated with time and leads to so-called system zones (or system eigenzones) migrating in an electric field with a certain eigenmobility. If recordable by the detector, they appear in the electropherogram as system peaks (or system eigenpeaks). However, although their appearance can not be forecasted and explained easily, they are inherent for the separation system. The progress in the theory of electromigration (accompanied by development of computer software) allows to treat the phenomenon of system zones and system peaks now also in very complex BGE systems, consisting of several multivalent weak electrolytes, and at all pH ranges. It also allows to predict the existence of BGEs having no stationary injection zone (or water zone, EO zone, gap, dip). Our paper reviews the theoretical background of the origin of the system zones (system peaks, system eigenpeaks), discusses the validity of the Kohlrausch regulating function, and gives practical hints for preparing BGEs with good separation ability not deteriorated by the occurrence of system peaks and by excessive peak-broadening.

Electrophoresis, Capillary↗