Biomedical subjects

Michał J Markuszewski

Publications and source records attributed to Michał J Markuszewski.

8 recordsLinked to original sources

PubMed2005

Human red blood cells targeted metabolome analysis of glycolysis cycle metabolites by capillary electrophoresis using an indirect photometric detection method.

A capillary electrophoretic (CE) method with UV indirect spectrophotometric detection for determination of the main compounds of glycolysis in human erythrocytes has been elaborated. Blood samples for electrophoretic analysis were prepared by isolation of erythrocytes, lysis of the cells by heating in double-distilled water and subsequent ultrafiltration with a centrifuge equipped with filter devices: Mr cut off 5000. Using 20 mM 2,6-pyridinedicarboxylic acid (PDC) as a highly UV absorbing carrier electrolyte and 4 mM cetyltrimetylammonium bromide (CTAB), at the resulting pH 12.3, nine of the glycolysis intermediates were separated and characterized. The repeatability and linearity of the method was assessed with percent relative standard deviation (%R.S.D.) for migration time ranging from 0.3 to 1.9% and correlation coefficients of 0.991-0.999 for the studied concentration range. Limits of detection (LOD) for the analyzed metabolites were in the range of 6.25 x 10(-6) to 5.0 x 10(-5) M. The optimized CE method was used to compare metabolome content of red blood cells of 22 healthy volunteers. Mean metabolite concentrations in erythrocytes ranged from 49.6 microM for fructose-6-phosphate (F-6-P) to 3.1 mM for 2,3-diphospho-D-glyceric acid (2,3-DPG). The method can be readily applied in clinical, pathophysiological and epidemiological studies.

2,3-Diphosphoglycerate↗

PubMed2005

Combined pH/organic solvent gradient HPLC in analysis of forensic material.

A combined pH/organic solvent linear gradient mode in high performance liquid chromatography (HPLC) is presented as a new approach to determination of low concentrations of ionogenic analytes in biological material. The approach consists in simultaneous development of linear gradients of pH and organic modifier in the mobile phase. Advantages of the method are illustrated in postmortem analysis of opipramol in material from suicide victims. Very narrow peaks without tailing were obtained and several times lower limits of analyte quantitation were achieved using ultraviolet detection as compared to a standard isocratic method. The double gradient HPLC method seems to be especially valuable in case of ionogenic analytes dispersed in complex biological matrices. That is due to a high selectivity of the double gradient method and the lack of peak tailing, which is commonly observed for basic analytes chromatographed at isocratic conditions.

Chromatography, High Pressure Liquid↗

PubMed2005

pH/organic solvent double-gradient reversed-phase HPLC.

A new reversed-phase high-performance liquid chromatographic (RP HPLC) procedure has been theoretically and experimentally established. The approach consists of the simultaneous development of a gradient of pH and of the organic modifier in the mobile phase. The proposed theoretical model of the pH/organic solvent double-gradient RP HPLC allows determination of both pK(a) and the lipophilicity parameter of the ionized and the nonionized form of the analyte and prediction of the retention times at specific separation conditions as well as bandwidth for all analytes. The model provides a rational basis for optimization of separation of ionizable analytes at any given chromatographic mode and analysis conditions. In addition, in the case of pH/organic solvent double-gradient RP HPLC, a compression of analyte peak and its reduced tailing can be expected.

Chromatography, High Pressure Liquid↗

PubMed2004

pH gradient high-performance liquid chromatography: theory and applications.

pH gradient high-performance liquid chromatography (HPLC) is a method of reversed-phase high-performance liquid chromatography suitable for ionogenic substances. It consists in programmed increase during the chromatographic process of the eluting strength of eluent with respect to the analytes separated. On the analogy of the conventional organic modifier gradient reversed-phase HPLC, in the pH gradient approach the eluting strength of the mobile phase increases due to its changing pH: increasing in case of acids or decreasing in case of bases. At the same time the content of organic modifier remains constant. A theory of the pH gradient HPLC has been elaborated. The resulting mathematical model is easily manageable. Its ability to predict changes in retention and separation of analytes following the changes in chromatographic conditions is demonstrated. The pH gradient method is uniquely suitable to determine pKa values of analytes. An equation is presented allowing to calculate pKa values basing on appropriate retention data. The effects on pKa are discussed of the concentration of methanol in the mobile phase. The RP HPLC-derived pKa data correlate to the reference pKa values (w(w)pKa) but are not identical. That may be explained by the effects on the chromatographically determined pKa of the specific interactions of analytes with stationary phases. The proposed pH gradient RP HPLC procedure offers a fast and convenient means to get comparable acidity parameters for larger series of compounds, like drug candidates, also when the analytes are available only in minute amounts and/or as complex mixtures.

Chromatography, High Pressure Liquid↗

PubMed2004

Determination of pKa by pH gradient reversed-phase HPLC.

pH gradient reversed-phase HPLC consists of a programmed increase during the chromatographic run of the eluting power of the mobile phase with regard to ionizable analytes. On the analogy of the conventional organic modifier gradient RP HPLC, in the pH gradient mode, the eluting strength of the mobile phase increases due to its increasing (with acid analytes) or decreasing (with basic analytes) pH, whereas the content of organic modifier is kept constant. We have shown previously that the pH gradient separations are technically possible using standard chromatographic equipment. Here we demonstrate that the method is uniquely suitable to determine pK(a) values of analytes. A strict theoretical model is proposed to determine pK(a) values based on the retention data from a pH gradient RP HPLC run. The pK(a) data so obtained are discussed in relation to the concentration of methanol in the mobile phase, the type of stationary phase, and the duration of the gradient. The pK(a) values determined by the pH gradient method are related to the respective data obtained conventionally in a series of isocratic experiments. A close similarity of the two types of chromatographically determined pK(a) data is demonstrated. The HPLC-derived pK(a) parameters correlate to the literature pK(a) values determined by titrations in water. The chromatographically derived and the reference pK(a) values are not identical, however. That is probably due to the effects on the chromatographic pK(a) of the specific sites of interactions with analytes on the surfaces of the HPLC stationary phases. Nonetheless, the proposed pH gradient HPLC method may supply in a fast and convenient manner comparable acidity parameters for larger series of drug candidates, including those available in only minute amounts, without need of their purification, and also when the compounds are provided as complex mixtures, like those produced by combinatorial chemistry.

Journal Article↗

PubMed2004

pH gradient reversed-phase HPLC.

pH gradient HPLC is reported, which is a new original mode of reversed-phase high-performance liquid chromatography applicable to ionogenic analytes. The method consists of programmed increase during the chromatographic run of the eluting strength of the mobile phase with respect to the acid/base analytes separated. Unlike the well-established conventional gradient HPLC, where the eluting power of the mobile phase is increased with time due to the increasing content of organic modifier, in the pH gradient HPLC that is realized by linearly increasing (in the case of acids) or decreasing (in the case of bases) the pH of the eluent of a fixed organic modifier content, thus providing functional increase in the degree of analyte dissociation and, hence, a decrease in its retention. The pH gradient mode has typical features of gradient HPLC, such as reduced peak width and minimized peak-tailing due to peak compression, which is especially advantageous in the case of organic base analytes. It may be of special value for separation of those analytes which are susceptible to the higher concentrations of organic solvents, as many bioanalytes are. A theory of the pH gradient HPLC has been elaborated, and its full mathematical formalistic is presented step by step in a comprehensive manner. Although fundamental relationships at the basis of pH gradient HPLC are more complex than in the case of the organic gradient variant, the resulting mathematical model is easily manageable. Its applicability to predict changes in retention and separation of test mixtures of analytes accompanying the changes in chromatographic conditions has been demonstrated experimentally in both gradient and isocratic HPLC. The proposed model supplies a rational basis for modifications of eluent pH aimed at optimization of separations and for convenient assessment of chromatographically relevant physicochemical parameters of analytes, such as pK(a).

Journal Article↗

PubMed2004

Capillary electrophoretic separation of cationic constituents of imidazolium ionic liquids.

A capillary electrophoretic method for resolving selected imidazolium ionic liquid cations is reported. The method, in which citric buffer is used as the running electrolyte, is simple and reproducible. The separation of a standard mixture is in linear accordance with the relative molecular mass (M(r)) of solutes regardless of the type of substitution (alkyl or aryl). The theoretical prediction of compounds as yet not analyzed is therefore possible; however, cations with identical molecular masses are inseparable with this method. Nevertheless, the method's quantitative analytical performance was excellent. The paper also discusses the applicability of a method for tracking the photodegradation kinetics of an exemplary ionic liquid.

Cations↗

PubMed2003

Analysis of carboxylic acid metabolites from the tricarboxylic acid cycle in Bacillus subtilis cell extract by capillary electrophoresis using an indirect photometric detection method.

With a growing interest in metabolome analysis, there is a need for developing robust methods for analysis of intracellular metabolites profiles in real samples like e.g., bacteria cell. Due to their weak absorbance properties, tri- and dicarboxylic acids from TCA cycle (citric, isocitric, 2-oxoglutaric, succinic, fumaric, malic) as well as carboxylic acid metabolites from glycolysis pathway, urea cycle and metabolism of amino compounds (formic, pyruvic, lactic, acetic, glutamic) were analyzed by capillary electrophoresis (CE) with indirect UV detection. Using 4 mM 2,6-pyridinedicarboxylic acid as a highly UV absorbing carrier electrolyte, 0.2 mM cetyltrimethylammonium bromide, 10% ethylene glycol and 10% acetonitrile, pH 3.5, carboxylic acids metabolites were analyzed in Bacillus subtilis cell extract from two different cultures: glucose and malate. CE with an electrokinetic injection mode achieved limits of detection in the range of 13-54 ppb (1.12-10(-7) - 5.96-10(-7) M). The reproducibility and linearity of method was investigated with RSD for migration time less than 1.3% and acceptable correlation coefficients. The optimized CE method was used to compare metabolome content of cell extract derived from two different culture media containing either glucose or malate as a carbon source. The changes in carboxylic acid metabolites profile were observed depending from used culture medium. Carboxylic acid concentrations ranged: in cell extract from malate culture from 59 to 0.5 microM for lactate and citrate, respectively, and in cell extract from glucose culture from 133 to 0.5 microM for glutamate and citrate, respectively. Appropriate concentrations of carboxylic acid in the single bacterium cell were estimated at mM and sub-mM levels.

Bacillus subtilis↗