Biomedical subjects

Ernst Kenndler

Publications and source records attributed to Ernst Kenndler.

At least 19 recordsLinked to original sources

PubMed2007

Sensitive and selective determination of glutathione in probiotic bacteria by capillary electrophoresis-laser induced fluorescence.

Glutathione (GSH) is a thiol with an important function in protecting tissue against the oxidative stress which has been related to carcinogenesis in the colon. For this reason the development of probiotic species producing glutathione could be of great interest. To determine the glutathione content of some probiotic bacteria of the Bifidobacterium and Lactococcus genera, a very sensitive and selective analytical method based on capillary electrophoresis coupled to laser-induced fluorescence detection has been developed. Pretreatment of cell-lysate samples is very simple--precipitation of protein with acetonitrile in 1:2 volume ratio. The fluorophore 5-iodoacetamidofluorescein (5-IAF) was chosen for glutathione derivatisation; it reacts with thiols at pH 12.5, forming a fluorescent adduct which is excited by a laser at 488 nm for detection. The reaction conditions optimised were temperature, time, and 5-IAF/GSH molar ratio. Electrophoresis was performed with a carbonate buffer (25 mmol L(-1), pH 9.8) as background electrolyte and a voltage of 30 kV; an electrophoretic run was complete in less than 7 min. There was a good linear relationship between concentration and response in the range 2.5-500 ng mL(-1) and the LOD was 0.5 ng mL(-1). The glutathione content of probiotic cells was determined by using the standard additions method to reduce matrix effects. The method was fully validated and shown to be of suitable sensitivity and selectivity for determination of GSH in probiotic cell lysates.

Acetonitriles↗

PubMed2006

Possible levomepromazine-clozapine interaction: two case reports.

OBJECTIVE: To report and comment upon two cases of suspected pharmacological interaction between the "classical" neuroleptic levomepromazine (LMP) and the "atypical" antipsychotic clozapine (CLZ). CASE SUMMARY: The patients who simultaneously took the two drugs had unusually low plasma levels of CLZ and its metabolites, even though they took high doses of CLZ. The patients were considered "non-responders" to the treatment by the psychiatrist. When LMP was withdrawn from the therapy, plasma concentrations of CLZ returned to therapeutic levels and one of the two patients experienced the anticipated therapeutic response. DISCUSSION: No information can be found in the literature regarding possible interactions between LMP and CLZ. However, the results of the two cases reported herein point out to a possible lack of therapeutic efficacy of CLZ therapy during this kind of polypharmacy. CONCLUSION: While two cases are too few to draw any conclusion, it would be prudent on the part of psychiatrists to consider a possible drug interaction in patients receiving both CLZ and LMP who fail to respond to the therapy.

Adolescent↗

PubMed2006

Temperature dependence of acidity constants, a tool to affect separation selectivity in capillary electrophoresis.

The mathematical models of migration and dispersion in capillary zone electrophoresis of small molecules form a sound basis for separation strategies of complex mixtures. It turned out that the key property is the effective mobility of the sample ions. To tune resolution parameters such as pH, complexation constants and ionic strength are widely used; temperature however is not although mobilities and pK(a) values depend in a more or less degree on temperature. From the temperature dependences of pK(a) values of a number of compounds listed in the literature a general rule can be derived: for carboxylic and inorganic acids dpK(a)/dT values are very small and the pK(a) values change less than +/-0.05 units/10K. Thermodynamically speaking, these compounds exhibit dissociation enthalpies close to zero. Phenols and amines, on the other hand, have systematically larger dpK(a)/dT values of about -0.1 to -0.2 units per 10K (the results of dissociation enthalpies of 20-70 kJ/mole). Based on this classification, a distinction can be made between different situations in capillary electrophoresis: (i) selectivity changes with temperature are largely due to the temperature dependence of the pK(a) of the buffering compound in the background electrolyte, (ii) selectivity changes mainly result from the temperature dependence of the pK(a) of the sample ions, and (iii) temperature effects on the pK(a) values of both, sample and buffer play a role. This work demonstrates such effects on selectivity in capillary electrophoresis highlighting the fact that in some instances temperature can be used to fine-tune separations.

Buffers↗

PubMed2006

Simultaneous determination of the antipsychotic drugs levomepromazine and clozapine and their main metabolites in human plasma by a HPLC-UV method with solid-phase extraction.

A HPLC method with UV detection has been developed for the simultaneous determination of levomepromazine, clozapine and their main metabolites: N-desmethyl-levomepromazine, levomepromazine sulphoxide, O-desmethyl-levomepromazine, N-desmethylclozapine and clozapine N-oxide. The analytes were separated on a C8 reversed-phase column using a mobile phase composed of acetonitrile and a pH 2.0, 34 mM phosphate buffer containing 0.3% triethylamine (29:71, v/v). Loxapine was used as the internal standard. A reliable biological sample pre-treatment procedure by means of solid-phase extraction on C1 cartridges was implemented, which allows to obtain good extraction yields (>91%) for all analytes and appropriate sample purification from endogenous interference. The method was validated in terms of extraction yield, precision and accuracy. These assays gave RSD% values for precision always lower than 4.9% and mean accuracy values higher than 92%. The method is suitable for the therapeutic drug monitoring (TDM) of patients undergoing polypharmacy with levomepromazine and clozapine.

Antipsychotic Agents↗

PubMed2006

Analysis of liposomes by capillary electrophoresis and their use as carrier in electrokinetic chromatography.

This contribution reviews work about liposomes in the context of electrically driven separation methods in the capillary format. The discussion covers four topics. The one broaches the application of liposomes as pseudo-stationary phases or carriers in vesicle or liposome electrokinetic chromatography (EKC) in the way as microemulsions and micelles are used; it includes the chromatographic use of liposomal bilayers as stationary phases attached to the wall for capillary electrochromatography (CEC). The second topic is the characterization and separation of liposomes as analytes by capillary electrophoresis (CE). Then the determination of distribution coefficients and binding constants between liposomes and ligands is discussed, and finally work dealing with peptides and proteins are reviewed with lipid bilayers as constituents of the electrically driven separation system.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2006

Determination of Olanzapine in rat brain using liquid chromatography with coulometric detection and a rapid solid-phase extraction procedure.

A sensitive and selective method was developed for the determination of the antipsychotic drug Olanzapine levels in rat brain tissue, based on HPLC with electrochemical detection. The analyses were carried out on a C8 reversed phase column (150 mm x 4.6 mm, 5 microm), using a mobile phase composed of methanol and a phosphate buffer (44.0 mM, pH 3.5), containing triethylamine (21:79, v/v), flowing at 1.2 mL min(-1). A high sensitivity coulometric detection analytical cell containing two flow-through low volume working electrodes was used: electrode 1 was set at +0.350 V and electrode 2 at -0.200 V. Olanzapine, administered to rats in different doses or in different times, was extracted from tissue homogenate of either the whole brain or specific areas (cortex, hyppocampus, nucleus striatum) with a rapid solid phase extraction procedure (SPE) on Oasis HLB cartridges. The method provided a high extraction yield of Olanzapine and internal standard (2-methylolanzapine) from brain tissue homogenate with absolute recovery values higher than 90.0%. The detector response was linear over a concentration range of 0.2-100.0 ng mL(-1) of Olanzapine. The limit of quantification (LOQ) was 0.2 ng mL(-1). Precision results, expressed by the intra-day and the inter-day relative standard deviation values, were satisfactory, better than 4.6%. Accuracy was satisfactory as well. This method proved to be suitable for the analysis of Olanzapine in rat brain tissues and for the study of distribution and pharmacokinetics of Olanzapine in rat brain after a single treatment with the antipsychotic drug.

Animals↗

PubMed2006

HPLC analysis of the second-generation antidepressant sertraline and its main metabolite N-desmethylsertraline in human plasma.

A liquid chromatographic method with ultraviolet detection was developed for the analysis of the recent antidepressant sertraline and its main metabolite N-desmethylsertraline in human plasma. The analytes were separated on a C8 reversed phase column, using a mobile phase composed of acetonitrile and a 12.3 mM, pH 3.0 phosphate buffer containing 0.1% triethylamine (35:65, v/v). Clomipramine was used as the Internal Standard. Using a solid phase extraction procedure with C2 cartridges high extraction yields (>94%) and good purification from matrix interference were obtained. Good linearity was obtained in the 7.5-250.0 ng mL(-1) range for sertraline and in the 10-500 ng mL(-1) range for N-desmethylsertraline. The analytical method was validated in terms of precision, extraction yield and accuracy. These assays gave R.S.D.% values for precision always lower than 3.9% and mean accuracy higher than 90%. Thanks to its good selectivity, the method proved to be suitable for the analysis of plasma samples from patients treated with sertraline as either monotherapy or polypharmacy.

Chromatography, High Pressure Liquid↗

PubMed2006

Theoretical and empirical approaches to express the mobility of small ions in capillary electrophoresis.

A discussion is given about the concepts of the ion mobility, the analyte property which governs migration and thus separation selectivity in CE. It deals with small organic and inorganic ions, not with charged polymers or large particles like colloids. The discussion is directed to two main concepts. (i) The first is based on physico-chemistry of ion conductance in solution, and distinguishes three types of mobility. The absolute mobility is the limiting mobility at zero ionic strength; it depends on the solvent and the temperature. It is obtained by extrapolation of the actual mobilities, those of the fully charged particles at finite ion concentration. The observed reduction of the absolute mobility with ionic concentration is related to an ion cloud, and is formulated by the established theories of ion conductance. It explains the actual mobility as function of (beside other factors) the ionic strength, the viscosity and relative permittivity of the solvent, the temperature, the relaxation time of solvent polarisation and the distance of closest approach between ion and counterion. The effective mobility, finally, is the mobility when association and dissociation equilibria play a role. Most important are acid-base reactions, but complexation, ion pairing and homo- and heteroconjugation were considered as well. (ii) The second approach treats mobility data with different mathematical methods, and formulates their dependence on variables like solvent composition with appropriate algorithms. These empirical methods mainly include least squares and neural network-based methods. The least square methods ranges from the simplest model, which uses only the molecular weight of the analyte, to more complicated model requiring three-dimensional structural descriptors of the solutes. Neural networks have been applied to model the mobility using different input variables and various architectures. Work comparing the accuracy of least squares and neural network methods was discussed; the results showed that the neural network method leads to a more accurate mobility calculation. However, the least squares methods could give some information to the factors affecting the mobility of the analytes. The resulting methods allow the prediction of mobilities under different experimental conditions with certain accuracy. It has been shown that using such models, it is possible to predict mobility of analytes after training the models by a minimum number of data to speed up the method development stage.

Algorithms↗

PubMed2006

Influence of detergent additives on mobility of native and subviral rhinovirus particles in capillary electrophoresis.

The electrophoretic properties of two human rhinovirus (HRV) serotypes, HRV2 and HRV14, their subviral particles, and their capsid proteins were investigated by CE using borate buffer, pH 8.3, as BGE and three different detergents as additives. In addition, the influence of modification of the capsid with an amine reactive fluorescent dye, Cy3.5, on migration in the electric field was assessed. We found that the reproducibility of the electrophoretic results was decisively dependent on the presence of the detergents above their respective CMC. As compared to the strong ionic detergent SDS, the nonionic, mild detergent dodecylpoly(ethyleneglycol ether) (D-PEG) efficiently and reproducibly resolved both, native viruses as well as subviral particles. Most of the analytes behaved as expected except native HRV2; this serotype showed a dramatically higher anionic mobility in SDS than in D-PEG. Additionally, its mobility decreased when each positive charge contributed from a lysine at the capsid surface was substituted by four negative charges upon derivatization with Cy3.5. We discuss the possibility that this effect is caused by differences in number and in arrangement of exposed lysines in the two serotypes leading to differences in the amount of bound SDS micelles.

Buffers↗

PubMed2006

Capillary electrophoresis of affinity complexes between subviral 80S particles of human rhinovirus and monoclonal antibody 2G2.

Human rhinoviruses (HRVs), the main etiologic agents of the common cold, transform into subviral B- or 80S particles (they sediment at 80S upon sucrose density gradient centrifugation) during infection and, in vitro, upon exposure to a temperature between 50 and 56 degrees C. With respect to the native virion they lack the genomic RNA and the viral capsid protein VP4. 80S particles are unstable and easily disintegrate into their components, VP1, VP2, and VP3 in buffers containing SDS. However, this detergent was found to be a necessary constituent of the BGE for the analysis of these viruses and their complexes with receptors and antibodies by CE. We here demonstrate that dodecylpoly(ethyleneglycol ether) (D-PEG) a nonionic detergent, is suitable for analysis of subviral particles as it preserves their integrity, in contrast to SDS. Electrophoresis of the 80S particles in borate buffer (pH 8.3, 100 mM) containing 10 mM D-PEG resulted in a well-defined electrophoretic peak. The identity of the peak was confirmed, among other means, by complexation with mAb 2G2, which recognizes a structural epitope exclusively present on subviral particles but not on native virus. Upon incubation of the 80S particles with mAb 2G2 the peak disappeared, but a new peak, attributed to the antibody complex emerged. The separation system allowed following the time course of the transformation of intact HRV serotype 2 into 80S particles upon incubation at temperatures between 40 and 65 degrees C. We also demonstrate that subviral particles derived from HRV2 labeled with the fluorescence dyes FITC or Cy3.5 were stable in the separation system containing D-PEG. Dye-modified particles were still recognized by mAb 2G2, suggesting that the exposed lysines that are derivatized by the reagent do not form part of the epitope of the antibody.

Antibodies, Monoclonal↗

PubMed2006

Capillary electrophoresis of liposomes functionalized for protein binding.

CE enabled assessing the attachment of hexa-histidine-tagged proteins to functionalized phospholipid liposomes. The liposomes were made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, phosphatidyl-ethanolamine, cholesterol and distearoyl-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) in a molar ratio of 29:26:40:5. The unilamellar vesicles, which had an average diameter of 170 nm, were labelled by inclusion of FITC-dextran for fluorescence detection. CE was carried out in poly(vinyl alcohol) (PVA)-coated capillaries at 25 degrees C with a BGE consisting of Tris-HCl (50 mM, pH 8.0). For conjugation of the liposomes with the proteins (soluble synthetic receptor fragments with molecular mass of 60 and 70 kDa, respectively), Ni(2+) was implanted into the vesicle surface by an anchor lipid containing a nitrilotriacetate acid (NTA) group as complexation agent for the metal ions. The difference in surface charge enabled the separation of the different species of interest by CE: plain vesicles, vesicles functionalised with Ni-NTA, vesicle-protein complexes and the species formed upon removal of the Ni-ions by complexation with EDTA. Loss of the Ni-ions resulted in the release of the proteins and the reappearance of the plain Ni-free NTA-liposome species in the electropherograms.

Cholesterol↗

PubMed2005

Rhinovirus-stabilizing activity of artificial VLDL-receptor variants defines a new mechanism for virus neutralization by soluble receptors.

Members of the low-density lipoprotein receptor family possess various numbers of ligand binding repeats that non-equally contribute to binding of minor group human rhinoviruses. Using an artificial concatemer of five copies of repeat 3 of the human very-low density lipoprotein receptor, we demonstrate protection of HRV2 against low-pH mediated uncoating and inhibition of penetration of an RNA-specific fluorescent dye into the intact virion. This indicates that the recombinant receptor inhibits viral breathing and irreversible conformational modifications of the capsid that precede RNA release, providing a new mechanism for rhinovirus neutralization by soluble receptor molecules.

Anti-Retroviral Agents↗

PubMed2005

Pressurized liquid extraction-capillary electrophoresis-mass spectrometry for the analysis of polar antioxidants in rosemary extracts.

A method based on capillary electrophoresis-electrospray-mass spectrometry (CE-ESI-MS) was developed to qualitatively characterize natural antioxidants from rosemary (Rosmarinus officinalis L.) in different fractions obtained by pressurized liquid extraction (PLE) using subcritical water. The parameters of CE-ESI-MS were adjusted allowing the separation and characterization of different compounds from rosemary in the PLE fractions. These parameters for CE are kind, pH and concentration of the separation buffer, parameters for ESI-MS are dry gas temperature and flow, nebulizing gas pressure, and make-up flow. The following analytical conditions were found most favorable: aqueous CE buffer (40 mM ammonium acetate/ammonium hydroxide, pH 9); sheath liquid containing 2-propanol-water (60:40, v/v) and 0.1% (v/v) triethylamine at a flow rate of 0.24 mL/h; drying gas flow rate equal to 7 L/min at 350 degrees C, nebulizing gas pressure of 13.8 kPa (2 psi), using a compound stability of 50%. Different antioxidant compounds (e.g., rosmarinic acid and carnosic acid) could be detected in the rosemary extracts by CE-ESI-MS without any additional treatment, enabling the determination of variations in the extract composition caused by the different PLE conditions (i.e., 60 and 100 degrees C). The results provide complementary information to HPLC analysis.

Abietanes↗

PubMed2005

Determination of fosfomycin in pus by capillary zone electrophoresis.

A method is described for the determination of fosfomycin in pus by capillary zone electrophoresis with reversed electroosmotic flow, and indirect UV absorbance detection. Sample pre-treatment is limited to removal of proteins and cell debris by adding the double volume of methanol, followed by vortexing for few seconds, and centrifugation at 15,000 x g for 2 min. The supernatant is directly injected into the instrument. Fosfomycin is separated from sample constituents with a background electrolyte at pH 7.25 (25 mM benzoate buffer with 0.5 mM hexadecyltrimethylammonium bromide added, adjusted to pH with tris(hydroxymethyl)-aminomethane (TRIS)). Separation is carried out in a capillary with 50 microm I.D., 64.5 cm total length, 56.0 cm to the detector, at 25 degrees C with -25 kV voltage applied. Due to the low absorbance of the analyte, indirect UV detection was performed at 254 nm using a bubble cell capillary. Sample was injected by pressure (450 mbar s). Repeatability for fosfomycin in spiked pus (from 8 or 10 consecutive injections of three different series at concentrations of 100 microg/mL of the antibiotic) was between 2.4 and 8.2% relative standard deviation (RSD). Accuracy (expressed as recovery of fosfomycin determined by three independent analysis at 10, 100 and 300 microg/mL fosfomycin added to plain pus) was between 75 and 102%. Intermediate reproducibility (n = 9 at three different days) was between 2 and 12% RSD. Limit of detection and limit of quantitation were 4.5 and 15 microg/mL, respectively. The concentration of fosfomycin in pus of patients treated with the antibiotic ranged up to 240 microg/mL. The concentration of other anionic pus constituents identified beside chloride (acetate, succinate, lactate, phosphate) ranged between 20 and 7800 microg/mL.

Abscess↗

PubMed2005

Enantioselective separation of rivastigmine by capillary electrophoresis with cyclodextrines.

Different beta-cyclodextrines (beta-cyclodextrin, heptakis (2,3,6-tri-O-methyl)-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, and sulfated beta-cyclodextrin) were investigated as additives for the enantioselective separation of the R-form from rivastigmine ((S)-N-ethyl-3-[(1-dimethylamino) ethyl]-N-methyl-phenyl carbamate), contained as impurity in this drug, which is used for the treatment of Alzheimer's disease. Electrophoresis was performed in an acidic background electrolyte (triethanolammonium phosphate, 75 mM, pH 2.5) with various concentrations of the additives. The electrophoretic mobilities measured are typical functions of the additive concentrations, with complex constants (obtained by fitting the appropriate binding curve on the data) ranging between about 180 and 770 M(-1). Best separation was obtained with 7.5 mM beta-cyclodextrin, with the R-enantiomer as impurity migrating before the main S-compound. Intra- and interday reproducibility (n = 6 and 18, respectively) of migration time and peak area was in the low percentage range, linearity of the calibration line for the quantitation of the impurity in the range between 2.3 and 50 microg/ml, expressed by the linear correlation coefficient, was 0.9998. The limits of detection and quantitation, respectively, were 0.7 and 2.3 microg/ml, corresponding to 0.05 and 0.15%, m/m of the R- relative to the S-compound. Analysis can be carried out at 18 degrees C in less than 19 min.

Electrophoresis, Capillary↗

PubMed2005

Animal glues in mixtures of natural binding media used in artistic and historic objects: identification by capillary zone electrophoresis.

Animal glues were often used in historic and artistic objects, e.g. as paint ground, as binders for pigments, or as adhesives. The sources were egg, casein, or different collagens. For restoration and conservation purposes it is important to know which kind of animal glue a museum object contains. Capillary electrophoresis can deliver such information, because it enables differentiation among the three proteinaceous glue classes according to their different amino acid patterns after hydrolysis. This work deals with the most relevant problem in practice, whether this identification is obstructed by the presence of other binders, with which they are mixed in many real samples; in particular, interference from plant gums and drying oils was investigated. Capillary electrophoresis of the hydrolysates (after reaction with 6 mol L(-1) HCl) was performed with an acidic background electrolyte consisting of chloroacetic acid (51.9 mmol L(-1)) adjusted with LiOH to pH 2.26. The underivatised analytes were detected with a contactless conductivity detector. It was found that the constituents of the plant gums (monosaccharides) or drying oils (long-chain fatty acids and short-chain dicarboxylic acids) never interfered with identification of the animal glues, as shown for artificial mixtures of the different binders even at tenfold excess over the animal glue, and for egg tempera samples. The method was used to identify the filling material from a statue from the eighteenth century.

Journal Article↗

PubMed2005

Nitromethane as solvent in capillary electrophoresis.

Nitromethane has several properties that make it an interesting solvent for capillary electrophoresis especially for lipophilic analytes that are not sufficiently soluble in water: freezing and boiling points are suitable for laboratory conditions, low viscosity leads to favourable electrophoretic mobilities, or an intermediate dielectric constant enables dissolution of electrolytes. In the present work we investigate the change of electrophoretically relevant analyte properties - mobilities and pKa values - in nitromethane in dependence on the most important experimental conditions determined by the background electrolyte: the ionic strength, I, and the pH. It was found that the mobility decreases with increasing ionic strength (by, e.g. up to 30% from I = 0 to 50 mmol/L) according to theory. An appropriate pH scale is established by the aid of applying different concentration ratios of a buffer acid with known pKa and its conjugate base. The mobility of the anionic analytes (from weak neutral acids) depends on the pH with the typical sigmoidal curve in accordance with theory. The pKa of neutral acids derived from these curves is shifted by as much as 14 pK units in nitromethane compared to water. Both findings confirm the agreement of the electrophoretic behaviour of the analytes with theories of electrolyte solutions. Separation of several neutral analytes was demonstrated upon formation of charged complexes due to heteroconjugation with chloride as ionic constituent of the background electrolyte.

Electrophoresis, Capillary↗

PubMed2005

Characterisation of natural polysaccharides (plant gums) used as binding media for artistic and historic works by capillary zone electrophoresis.

The monosaccharide constituents of plant gums were separated by capillary electrophoresis at pH 12.1 and detected with indirect UV absorbance. The plant gums investigated were gum arabic, gum acacia, gum tragacanth, cherry gum and locust bean gum (carob gum). The monosaccharides obtained after hydrolysis with 2M trifluoroacetic acid and lyophilisation of the hydrolysate were arabinose, galactose, mannose, rhamnose, xylose, fucose, and glucose, and the two sugar acids galacturonic and glucuronic acid, in accordance with the literature. They were separated in a background electrolyte consisting of NaOH to adjust the pH, 20 mM 2,6-pyridinedicarboxylic acid as chromophore for detection and 0.5 mM cetyltrimethylammonium bromide as additive to reverse the electroosmotic flow. Based on their electropherograms, the plant gums could be identified by their typical composition (depicted in a decision scheme) as follows: a peak of glucuronic acid, together with that of rhamnose, is indicative for gum arabic. Peaks of galacturonic acid and fucose point to gum tragacanth. Locust bean gum shows a major peak for mannose (with the concomitant galactose peak in ratio 4-1), whereas a glucuronic acid and a mannose peak together with a prominent arabinose peak indicates cherry gum. The method was applied to identify the plant gums in samples like watercolours and in several paint layers like gum tempera or those with egg white or drying oils as additives. Artificial aging experiments of thin layers of gum arabic on paper or glass carried out with UV-A radiation (366 nm) did not result in changes of the saccharide patterns, in contrast to the simultaneously conducted aging of a drying oil layer.

Art↗