Juho Jumppanen

Screening for diuretics in urine and blood serum by capillary zone electrophoresis

Diuretics are therapeutic agents used to promote the excretion of bodily fluids and salts. They are also misused by some athletes to decrease body mass or to mask the use of anabolic steroids and other drugs. We have developed a method that screens for diuretics in urine and blood serum. Two successive runs were required because of the heterogeneity of this group of compounds. Screening for diuretics that contained sulphonamide and/or carboxylic groups was done at pH 10.6 with 3-(cyclohexylamino)-1-propane-sulphonic acid (0.06 M) as buffer. Diuretics that contained primary, secondary or tertiary amine groups were investigated at pH 4.5 with an acetate (0.07 M)-betaine (0.5 M) buffer system. Hydrostatic injection mode for 5 s gave the best efficiency. Longer injection times were acceptable but efficiency was then somewhat reduced. Detection limits at the low femtomole level are achievable for most compounds with a UV-Vis detector operating at 220 and 215 nm. Temperature affected the separation, and 20 degrees C proved best. All compounds were separated in less than 30 mins. A confirmation analysis of all compounds was done by GC-MS.

Capillary electrophoresis of diuretics

The review surveys the application of capillary electrophoresis to the screening, identification and determination of diuretics and probenecid. The number of publications is still limited, but the studies already published clearly show that capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography are excellent alternatives for the investigation of diuretics. High accuracy identifications of diuretics and probenecid, even in urine samples, can be obtained when CZE is used with the marker techniques. This review paper has been written from the viewpoint of practical use and some hints are given for future CE studies on diuretics.

Effect of buffering and buffer replenishment on repeatability in capillary electrophoresis

Adequate buffering is of vital importance in capillary electrophoresis. Inadequate buffering may result in severe pH drift and erratic separation behavior. The aim of this work was to study the effects of buffering and buffer replenishment on the repeatability of absolute migration times and on the repeatability of electrophoretic mobilities of the analytes determined by the two-marker technique. Three buffers 3-(cyclohexylamino)-1-propane-sulfonic acid (CAPS), 3[(1,1-dimethyl-2-hydroxy-ethyl) amino]-2-hydroxypropane sulfonic acid (AMPSO), and phosphate) with differentpKa, values were applied at pH 10.6. Both the analytes and the marker compounds were aromatic mono- and dicarboxylic acids. o-Cresol was added to the analyte mixture to act as a pH-sensitive probe. CAPS, which had a pKa value (10.4) closest to the pH of the buffer, was clearly the best buffer at pH 10.6. Buffer replenishment and the use of marker compounds improved the repeatability in every case. From this work it is easy to conclude that when suitable buffer is used and the buffer is replenished after each run, highly repeatable results can be obtained especially when marker techniques are employed.

A Novel Chromatographic Production Scale Separation Process for L-Fucose

Abstract There is an increasing interest towards a special and very rare sugar, L-fucose, because of its functionalities, i.e., in the human body. There are only a few industrial production processes for L-fucose, and chromatographic separation of this valuable component from hemicellulose hydrolysates could be a feasible alternative. However, chromatographic separation of L-fucose with sufficient purity from other monosaccharides, especially deoxy sugars, has presented a problem in the state of the art. The aim of this work was to find a new process scale chromatographic purification method for L-fucose containing liquor. Of special interest was a process suitable for recovery of L-fucose from plant based biomass hydrolysate. This paper presents an industrial scale chromatographic separation method for the production of L-fucose. The use of a combination of strong and weak acid cation exchange resins increases L-fucose purity gradually, while the use of strong base anion exchange resin gives an excellent separation of L-fucose from other remaining sugar compounds.

Effect of the buffer solution on the elution order and separation of bis(amidinohydrazones) by micellar electrokinetic capillary chromatography

Abstract The effect of five different buffer solutions on the elution order and separation of bis(amidinohydrazones) by micellar electrokinetic capillary chromatography was studied at pH 7.0. The buffers were sodium phosphate, tris(hydroxymethyl)aminomethane, N-(2- hydroxyethyl)piperazine-N′-(2-ethanesulphonic acid), N,N-dimethylmethylenediamine and N,N-diethylethylenediamine. The factors affecting the elution order of the solutes were: (1) ion-pair formation between the solute and the buffer ion, (2) the cationic nature and structure of the solute, (3) reactions between ion-pair complexes and miceles and (4) the nature of the buffer solution. Sodium phosphate (0.05 M) with 1 mM N-cetyl-N,N,N-trimethylammonium bromide was the only buffer solution to fully separate eight aliphatic congeners of bis(amidinohydrazone).

Comparison of capillary electrophoresis systems for the reliable identification of some carboxylic acids using the marker technique

Many commercial capillary electrophoretic (CE) systems differing in degree of automation, type of injection and capillary cooling are available. The aim of this study was to investigate if the marker technique can be used for reliable peak identification of compounds separated with different CE instruments. In the marker technique, which was developed earlier in our laboratory, unknown compounds are identified through determination of their electrophoretic mobilities relative to the known mobilities of the marker compounds. Carboxylic acids were used as markers and model components and a phenolic compound was added to the sample mixture as a pH-sensitive probe. The repeatabilities and reproducibilities of both the migration times and the mobilities of the analytes were measured with five different CE instruments. With the electrophoretic mobilities, excellent repeatability and reproducibility were obtained.

Identification of nucleic acids and oligonucleotides by capillary zone electrophoresis with the four marker technique

Abstract The paper describes the improvement brought by the four marker technique to the repeatability of the separation of macromolecules [nicotinamide nucleotides (NADPH and NADH), one adenine compound (ATP), two oligonucleotides (135-oligomer and 84-primer containing 135 and 20 bases, respectively) and N2-5′-deoxyquanosine-benzo[a]pyrene-diolepoxide (dG5′BaDE)] by capillary zone electrophoresis with UV detection at the wavelength of 220 nm. Optimized conditions [3-(cyclohexylamino)-1-propanesulphonic acid as the organic electrolyte solution with pH of 11.2, and concentration of 50 mM] were used for the separation. The counter-ion in the system was sodium. The macromolecules could be separated within 20 minutes. The four marker compounds were (-)mandelic acid, meso-2,3-diphenylsuccinic acid, 1,2-phenylenediacetic acid and o-phthalic acid, which were used for the calculations of the mobilities for the analytes. The identification of the macromolecules was confirmed with the coefficients of identification...