Capillary electrophoretic analysis of flavonoids.

Combining the effects of electrophoresis and electroendosmosis, flavonoids were separated in less than ten minutes in a fused silica capillary tube with a borate buffer adjusted to pH 10. An increase in the concentration of borate from 0.1 to 0.2 M resulted in longer migration times due to a decrease in electroosmotic flow, but also in improved selectivity and higher resolution of flavonoids. The calibration curve of rutin showed a detection limit of 0.02 mg/mL and linearity over its pharmaceutical concentration range. Using an internal standard of known concentration, the content of rutin in a methanolic extract of Sambuci flos could be determined with a coefficient of variation as small as 3.8% by the molar ratio-peak area ratio method.     

Rapid electrochemical assay for theophylline in whole blood based on the inhibition of bovine liver alkaline phosphatase

A disposable electrochemical test strip for determining clinically relevant concentrations of theophylline (0–300 μM) in whole blood is described, based on the generation of p-aminophenol from p-aminophenyl phosphate by the action of bovine liver alkaline phosphatase. Theophylline is an uncompetitive inhibitor of alkaline phosphatase and thus inhibits this process. The test strip consists of a screen-printed, carbon-based electrode system containing the enzyme and substrate in separate layers. Application of a 20-μl blood sample to the strip initiates the enzymic reaction, which will proceed to an extent that is inversely dependent on the amount of theophylline in the sample. After a 2-min incubation, the p-aminophenol generated is quantified by its electrochemical oxidation at + 150 mV (vs. Ag/AgCl) on the underlying carbon electrode. Caffeine and theobromine (0–1 mM), phenylalanine (< mM) and endogenous alkaline phosphatase (<2 U ml ^?1) do not interfere.     

Capillary electrophoretic analysis of hydroxyl radicals produced by respiring mitochondria

Here, we report the use of a capillary electrophoretic method with laser-induced fluorescence detection to evaluate hydroxyl radicals produced by respiring mitochondria. The probe, hydroxyphenylfluorescein (HPF), is separated from the product, fluorescein, in under 5 min with zeptomole and attomole limits of detection for fluorescein and HPF, respectively. Purification of the probe with a C-18 SPE column is necessary to reduce the fluorescein impurity in the probe stock solution from 0.4 % to less than 0.001 %. HPF was responsive to hydroxyl radicals produced by isolated mitochondria from L6 cells, and this signal was blunted when DMSO was added to scavenge hydroxyl radicals and when carbonyl cyanide m-chlorophenylhydrazone was added to depolarize the mitochondria. The method was used to compare hydroxyl radical levels in mitochondria isolated from brown adipose tissue of lean and obese mice. Mitochondria from obese mice produced significantly more hydroxyl radicals than those from lean mice.

Capillary zone electrophoretic analysis of carbohydrates by direct and indirect UV detection

Summary The analysis of carbohydrates has been always hampered by their lack of UV absorbance above 200 nm, which is an especially challenging problem in capillary electrophoresis due to the very small (nl) sample volumes injected. The introduction of 2-aminopyridine as derivatizing agent allows sensitive direct UV detection of saccharides in the fmol range. However, due to the requirement of the presence of a free aldehyde group only aldoses and uronic acids can be determined. This limitation was recently overcome by means of precolumn derivatization withp-aminobenzoic acid or ethylp-aminobenzoate, which permits the analysis of fructose with a lower mass detection limit of 0.3 and 0.14 pmol, respectively. The detection limits for aldoses were even as low as 15 and 7 fmol. A more universal approach is the use of indirect UV detection, which permits the analysis of carbohydrates, including (1–2)-linked disaccharides and aldonic acids, at the lower pmol level without the need for derivatization.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

Capillary electrophoretic separation of nanoparticles

In the present work, CdSe nanocrystals (NCs) synthesized with a trioctylphosphine surface passivation layer were modified using amphiphilic molecules to form a surface bilayer capable of providing stable NCs aqueous solutions. Such modified nanocrystals were used as a test solute in order to analyze new electrophoretic phenomena, by applying a micellar plug as a separation tool for discriminating nanocrystals between micellar and micelle-free zones during electrophoresis. The distribution of NCs between both zones depended on the affinity of nanocrystals towards the micellar zone, and this relies on the kind of surface ligands attached to the NCs, as well as electrophoretic conditions applied. In this case, the NCs that migrated within a micellar zone can be focused using a preconcentration mechanism. By modifying electrophoretic conditions, NCs were forced to migrate outside the micellar zone in the form of a typical CZE peak. In this situation, a two-order difference in separation efficiencies, in terms of theoretical plates, was observed between focused NCs (N ~ 10⁷) and a typical CZE peak for NCs (N ~ 10⁵). By applying the amino-functionalized NCs the preconcentration of NCs, using a micellar plug, was examined, with the conclusion that preconcentration efficiency, in terms of the enhancement factor for peak height (SEF_height) can be, at least 20. The distribution effect was applied to separate CdSe/ZnS NCs encapsulated in silica, as well as surface-modified with DNA, which allows the estimation of the yield of conjugation of biologically active molecules to a particle surface.     

Capillary electrophoretic analysis of inorganic anions in atmospheric hailstone samples

Micellar electrokinetic capillary chromatography (MEKC) coupled with sample stacking and polarity switching was investigated for the determination of Viagra (sildenafil citrate, SC) and its metabolite (UK-103,320, UK) in human serum in the concentration range of clinical interest. Human serum samples spiked with SC and UK were eluted with methanol from a C18 cartridge, the extract was evaporated and regenerated in a solution that contained 1 mM phosphate buffer (pH 12.3) and 20% methanol. The MEKC separation was performed using an injection time of 275 s, a polarity switching time of 93 s, a phosphate buffer, (pH 12.3, 15 mM) containing 25 mM sodium dodecyl sulfate as separation electrolyte and a fused-silica capillary. The analysis takes about 6 min and gives satisfactory inter-day precision with respect to migration times and linear responses over the 80-900 ng/ml concentration range investigated for SC and UK. Intra-day RSDs (n=4 graphs) for the slopes of the calibration graphs were 4.86% for SC and 3.50% for UK. Inter-day RSDs for the slopes were 4.37% for SC and 5.39% for UK. Detection limits (S/N=3) were about 17 ng/ml for both compounds in human serum. A 1-ml volume of blood serum was necessary to do this determination.     

Capillary electrophoretic separation of glycoproteins

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Capillary electrophoretic and mass spectrometric analysis of a polydisperse fluorosurfactant

A fluorosurfactant has been studied using capillary electrophoresis and mass spectrometry. The fluorosurfactant, FC134, can be used as a buffer additive in capillary electrophoresis in order to decrease wall adsorption of proteins and in micellar electrokinetic chromatography. However, it has been discovered that this fluorosurfactant is polydisperse, thus containing substances with different lengths and structures. In this work, the fluorosurfactant sample components were separated by capillary electrophoresis. An uncoated as well as a poly(vinyl alcohol)-coated capillary were used with running electrolytes containing methanol and acetic acid. Following the capillary electrophoretic separation, fractions were collected for further analysis by MALDI-MS. Non-fractionated samples were also analyzed both by MALDI-MS and by ESI-MS.     

Capillary electrophoretic analysis of cyclodextrins with dynamic fluorescence labeling and detection

The use of 8-anilinonaphthalene-1-sulfonic acid (8,1-ANS) as buffer additive in the capillary electrophoretic separation of cyclodextrins (CDs) was investigated. Better detection sensitivity was obtained for - and γ-CDs than with previously reported capillary electrophoretic methods. Increasing the concentration of 8,1-ANS improved resolution and sensitivities for -, β- and γ-CDs, while decreasing the pH of the background electrolyte can improve sensitivities. Detection limits for -, β- and γ-CDs were determined to be 60, 20 and 7 μM, respectively. The formation constants of CD–8,1-ANS complexes at pH 6 were also measured by capillary electrophoresis. Finally the specificity of amyloglucosidase to CDs was analyzed as a practical application of this method.     

Capillary electrophoretic analysis of dimethylsulfoniopropionate in sugarcane and marine algal extracts

A simple and high-resolution analytical method for the determination of dimethylsulfoniopropionate (DMSP) in sugarcane and marine algae is described. Effective extraction of DMSP from plant samples was also investigated using organic solvents, 5% perchloric acid or deionised water. To increase the sensitivity, DMSP in the extracts was first converted to a phenacyl ester, and the reaction mixture was applied directly to capillary electrophoresis without any pretreatment. Water extraction followed by esterification in a pH 4 reaction buffer was found most suitable for the measurement of alkaline-labile DMSP. This method was applied to the determination of DMSP levels in marine algae samples collected from the seashore of Nagasaki, Japan. An increase in DMSP content in Ulva pertusa in the winter period was observed.     

Capillary electrophoretic analysis of the derivatives and isomers of benzoate and phthalate

A capillary electrophoretic method for the analysis of 12 commonly found derivatives and isomers of benzoate and phthalate, including p-toluic acid, p-acetamido and p-hydroxy derivatives of benzoic acid, salicylic acid and its acetyl ester, 2- and 4-isomers of carboxybenzaldehyde, meta-, para-, and ortho-isomers of phthalic acid, and monomethyl terephthalic acid was developed. Capillary electrophoresis (CE) was performed in the free zone electrophoresis mode. Performing CE in 10 mM phosphate buffer, pH 7.0 could separate most of the benzoic acid derivatives except the structural or positional isomers. The positional isomers of phthalic acids could be completely separated with co-addition of alpha- and beta-cyclodextrins. Addition of poly(ethylene glycol) 600 (4%) could further resolve some structural isomers. The CE method developed here is rapid, i.e. complete separation could be achieved in less than 8 min for the nine monoanionic benzoate derivatives and in less than 14 min for the three dianionic phthalate isomers. The new method has good precision and linearity and can be readily applied to real samples for quantitative analysis. It is sensitive and can detect sub-ppm (w/w) level of impurity in real terephthalic samples.     

Determination of serum alkaline phosphatase activity by electrochemical detection with flow injection analysis

Summary In this enzyme assay for kinetic measurement of alkaline phosphatase (EC 3.1.3.1) activity in human serum by flow injection analysis with electrochemical detection (FIA-EC), the substrate phenyl phosphate is enzymatically hydrolyzed and the phenol produced is quantified by FIA-EC. The standard curve is linear over the range 40 to 500 U/l, and the predicted linearity is up to 1000 U/l. The CV is 5% over the range of the assay. Correlation with the Sclavo Diagnostics nitrophenyl phosphate spectrophotometric technique gave an r of 0.98. This new technique requires as little as 1 l of serum. The FIA-EC method is not subject to interference by hemolysis, icteria, or lipemia or by electroactive substances such as urate, ascorbate, or acetaminophen. Sensitive measurements free from spectrophotometric interferences are thus possible.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday     

Capillary electrophoretic analysis of mu- and m-calpain using fluorescently labeled casein substrates

Calpains are unique calcium-dependent thiol proteases that have been proposed to participate in a number of physiological processes including signal transduction and protein turnover in skeletal muscle. Calpains exist in two major forms. Interestingly, the two forms of protease show no significant difference in their action on various substrates. The only demonstrable difference in their activity involves the concentration of calcium required for activation. Both mu- and m-calpains typically achieve half maximal activation at 50 microM and 0.7 mM calcium, respectively. The focus of this study was to examine the action of both forms of calpain on casein substrates and assess whether any differences could be observed in the resulting peptide finger print using capillary electrophoresis. Purified mu- and m-calpain were incubated for various lengths of time with Oregon Green labeled alphas- and beta-casein. The reactions were stopped with sodium dodecyl sulfate (SDS) and products separated by capillary electrophoresis in micellar electrokinetic capillary chromatography (MEKC) mode using laser-induced fluorescence (LIF) detection. Comparison of the electropherograms showed no difference in the peptide profile for either enzyme. However, it was found that beta-casein was hydrolyzed more extensively than alphas-casein, by both enzymes. Capillary electrophoresis was found to be a very sensitive technique for detection of calpain activity. Using beta-casein as substrate, the CE approach was able to detect 2-3 ng of calpain activity. The results also suggest that capillary electrophoresis is a useful tool for proteolytic investigations of protein structure.     

Capillary electrophoretic analysis of neutral carbohydrates using ionic liquids as background electrolytes

In this study, ionic liquids (ILs) as BGE additives were applied for the analysis of neutral carbohydrates in CE. The ILs served primarily as chromophores for indirect UV detection. The influence of imidazolium-based ionic liquids on the separation, detection limits and mobility of underivatized neutral carbohydrates was investigated. BGEs consisting of 10-50 mM of ILs at pH 12.4 without other additives provided fast separation of neutral sugars. This method was used to determine sucrose, glucose and fructose in certain vegetable juices.     

Capillary electrophoretic analysis of carbohydrates derivatized by in-capillary condensation with 1-phenyl-3-methyl-5-pyrazolone

Our previous papers on capillary electrophoresis (CE) have shown that samples can be derivatized in a capillary and the derivatives can be analyzed immediately after derivatization, provided that the derivatization reaction is so rapid as to complete in seconds. The present paper presents extended application of in-capillary derivatization to a much slower reaction such as the condensation of reducing carbohydrates with 1-phenyl-3-methyl-5-pyrazolone (PMP) which requires 30 min at 70 degrees C in pre-column derivatization by manual operation. It was necessary to first drive the introduced plugs of sample and reagent solutions to put them together at the entrance of the heated portion of a capillary, then to allow the superimposed plugs to react for a relevant period. We showed how to determine the introduction times of the sample and the reagent solutions as well as intermediate running buffer, the voltages to be applied for plug driving and product analysis, and the duration of voltage application, all of which are important for effective in-capillary derivatization. An example of the analysis of maltooligosaccharides by this technique is presented. It was shown that maltooligosaccharides were quantitatively derivatized with PMP in 35 min at 57 degrees C, and the derivatives could be analyzed in ca. 15 min by CE immediately after derivatization. Separation was satisfactory in 200 mM borate buffer, pH 8.2 containing sodium dodecyl sulfate to a concentration of 200 mM. Although the theoretical plate number, and accordingly the resolution, were significantly lower than the corresponding values in pre-capillary derivatization, reasonable reproducibility was ensured for both migration time (RSD 3.5% on average) and peak area (RSD less than 3%) under the optimized conditions. It is notable that sample amount could be lowered to the 10 fmol level, in contrast to the 10 pmol level in pre-capillary derivatization. In addition, since the technique employed here (the modified at-inlet technique of in-capillary derivatization) is easily automated, the established system will be highly beneficial for routine analysis of carbohydrates. Analysis by this technique was also shown to be useful for kinetic study of the derivatization reaction.