Stacking and quantitative analysis of lovastatin in urine samples by the transient moving chemical reaction boundary method in capillary electrophoresis

A simple, sensitive, and useful concentration method for lovastatin (Lvt) in urine has been developed based on the transient moving chemical reaction boundary method (tMCRBM) in capillary electrophoresis. The MCRB is formed with acidic sample buffer (Gly-HCl) and alkaline running buffer (Gly-NaOH). The following optimal conditions were determined for stacking and separation: electrophoretic buffer of 100 mM Gly- NaOH (pH 11.52), sample buffer of 20 mM Gly-HCl (pH 4.93), fused-silica capillary of 76 cm × 75-μm i.d (67 cm from detector), sample injection at 14 mbar for 3 min. A 21- to 26-fold increase in peak height was achieved for detection of Lvt in urine under the optimal conditions compared with normal capillary zone electrophoresis. By combining the sample pretreatment procedure with the stacking method, the sensitivity of Lvt in urine was increased by 105- to 130-fold. The limits of detection (LOD) and quantification (LOQ) for Lvt in urine were decreased to 8.8 ng/mL and 29.2 ng/mL, respectively. The intra-day and inter-day precision values (expressed as RSD) were 2.23–3.61% and 4.03–5.05%, respectively. The recoveries of the analyte at three concentration levels changed from 82.65 to 100.49%.     

Determination of carbohydrates as their 3-aminophthalhydrazide derivatives by capillary zone electrophoresis with on-line chemiluminescence detection

A method based on pre-capillary derivatization with luminol (3-aminophthalhydrazide) for carbohydrate analysis using capillary electrophoresis with on-line chemiluminescence (CL) detection was developed. The derivatives of seven monosaccharides were separated and detected by using 200 mM borate buffer containing 100 mM hydrogen peroxide at pH 10.0 as separation electrolyte and 25 mM hexacyanoferrate in 3 M sodium hydroxide solution as post-capillary chemiluminescence reagent with separation efficiencies ranging from 160,000 to 231,000 plates per metre. The minimum amount of carbohydrate derivatized was 2 pmol (corresponding to the concentration of 2 microM). The method also provided a linear response for glucose in the concentration range of 0.1-250 microM with a mass detection limit of 420 amol or a concentration detection limit of 0.1 microM. Preliminary work using the CE-CL format to determine glucose in a rat brain microdialysis sample is presented as a typical case.     

Simultaneous determination of nitrite, nitrate, thiocyanate and uric acid in human saliva by capillary zone electrophoresis and its application to the study of daily variations

Simultaneous determination of nitrite (NO2-), nitrate (NO3-), thiocyanate (SCN-) and uric acid in human saliva was performed by capillary zone electrophoresis using a coated capillary with reversed electroosmotic flow (EOF), using a 100 mM sodium phosphate buffer at pH 6.5 as a running buffer. Saliva samples were deproteinized with acetonitrile and filtered through a membrane filter. The important advantages of the reported method are: simple operation, short analysis time, minimal sample pre-treatment and sample dilution. In order to evaluate the daily variations of the anionic components, the concentrations were determined in the human saliva of four healthy volunteers upon waking and at 2qh intervals during a day.     

Improved method for the simultaneous determination of whey proteins, caseins and para-kappa-casein in milk and dairy products by capillary electrophoresis

A capillary electrophoresis method for the simultaneous determination of whey proteins, caseins and their degradation products, such as para-kappa-casein, was proposed. The effect of several parameters (pH, ionic strength and concentration of urea in the electrophoresis buffer and applied voltage) on the analysis time and on the separation efficiency of the major milk proteins was studied. Using a hydrophilically coated capillary, in combination with electrophoresis buffer 0.48 M citric acid-13.6 mM citrate-4.8 M urea at pH 2.3, and a separation voltage of 25 kV, a complete separation of beta-lactoglobulin and para-kappa-casein was achieved, permitting the quantification of both components.     

Analysis of intact heparin by capillary electrophoresis using short end injection configuration

A capillary electrophoresis method for the analysis of intact heparin was developed using a phosphate buffer and a fused silica capillary. Operational parameters such as pH and concentration of the running buffer were investigated. The short end injection configuration permitted a gain on peak efficiency, on the analysis time and on the repeatability of both migration times and peak areas, through a reduction of the migration distance. Moreover, the beneficial effect of the presence of sodium chloride in the heparin sample on the peak efficiency was demonstrated and the influence of the salts on the conformation of the heparin was discussed. The optimized method (short end injection configuration, 50mM phosphate buffer pH 3, heparin sample prepared in 10 g/L NaCl solution) was validated in terms of linearity, reproducibility and specificity according to ICH requirements.     

Determination of histamine and histidine by capillary zone electrophoresis with pre-column naphthalene-2,3-dicarboxaldehyde derivatization and fluorescence detection

A rapid and sensitive method was developed for the simultaneous determination of histamine and histidine by capillary zone electrophoresis with lamp-induced fluorescence detection. A fluoregenic derivatization reagent, naphthalene-2,3-dicarboxaldehyde (NDA) was successfully applied to label the histamine and histidine respectively. The derivatization conditions and separation parameters including pH and concentration of electrolyte and sample injection were optimized in detail. The optimal derivatization reaction was performed with 1.0 mM NDA, 20 mM NaCN, and 20 mM borate buffer, pH 9.1 for 15 min. The separation of NDA-tagged histamine and histidine could be achieved in less than 200 s with 40 mM phosphate buffer (pH 5.8) as the running buffer. The detection limits for histamine and histidine were 5.5 x 10(-9) and 3.8 x 10(-9) M, respectively (S/N = 3). The relative standard derivations for migration time and peak height of derivatives were less than 1.5 and 5.0%, respectively. The method was successfully applied to the analysis of histamine and histidine in the P815 mastocytoma cells and the beer samples.     

Discontinuous electrolyte systems for improved detection of biologically active amines and acids by capillary electrophoresis with laser-induced native fluorescence detection

On-line concentration and separation of biologically active amines and acids by capillary electrophoresis (CE) in conjunction with laser-induced fluorescence using an Nd:YAG laser at 266 nm under discontinuous conditions is presented. The suitable conditions for simultaneous analysis of amines and acids were: samples were prepared in a solution (pH* 3.1) consisting of 10 mM citric acid, 89% acetonitrile (ACN), and water; a capillary was filled with 1.5 M Tris-borate (TB) buffer (pH 10.0); and the anodic vial contained PTG10 buffer (pH* 9.0) that consists of 50 mM propanoic acid, Tris, 10% glycerol, and water. After injecting a large-volume sample, amines and acids were separately stacked at the front (cathodic side) and back (anodic side) of the acidic sample zone, mainly because of changes in their electrophoretic mobilities as a result of changes in pH, viscosity, and electric field when high voltage was applied. When the sample was injected at 15 kV for 360 s, the concentration limits of detection (LODs) for 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) were 0.27 and 0.31 nM, respectively, which are about 400- and 800-fold sensitivity improvements when compared to those injected at 1 kV for 10 s. For the analysis of amines, samples were prepared in 100 mM citric acid (pH* 1.8) containing 89% ACN and both the capillary and anodic vial were filled with 400 mM PTG20 (propanoic acid, Tris, 20% glycerol, and water) at pH* 4.5. Using a large injection volume (15 kV for 360 s), we achieved concentration LODs of 17 pM and 0.3 nM for tryptamine and epinephrine, which are about 5200- and 14,000-fold sensitivity improvements, respectively, in comparison with those injected at 1 kV for 10 s. The features of simplicity (no sample pretreatment), rapidity (12 min), and sensitivity for identification of amines and acids of interest in urine samples show diagnostic potential of the two approaches developed in this study.     

Analysis of phenolic acids by non-aqueous capillary electrophoresis after electrokinetic supercharging

Electrokinetic supercharging (EKS), a new and powerful on-line preconcentration method for capillary electrophoresis, was utilized in non-aqueous capillary electrophoresis (NACE) to enhance the sensitivity of phenolic acids. The buffer acidity and concentration, leader and terminator length and electrokinetic injection time were optimised, with the optimum conditions being: a background electrolyte of 40 mM Tris-acetic acid (pH 7.9), hydrodynamic injection of 50 mM ammonium chloride (22 s, 0.5 psi) as leader, electrokinetic injection of the sample (180 s, -10 kV), hydrodynamic injection of 20 mM CHES (32 s, 0.5 psi) as terminator, before application of the separation voltage (-25 kV). Under these conditions the sensitivity was enhanced between 1333 and 3440 times when compared to a normal hydrodynamic injection with the sample volume <3% of the capillary volume. Detection limits for the seven phenolic acids were in the range of 0.22-0.51 ng/mL and EKS was found to be 3.6-7.9 times more sensitive than large-volume sample stacking and anion selective exhaustive injection for the same seven phenolic acids.     

Development of a capillary zone electrophoresis method for the separation of a furan combinatorial library

Nine component mixtures of a furan library were simultaneously separated by capillary zone electrophoresis (CZE) using a phosphate buffer as a background electrolyte at low pH. The effects of buffer concentration, buffer pH, type and concentration of organic solvents on the electrophoretic mobility, resolution, and analysis time were systematically investigated. Resolution and efficiency of furan library components were further improved using cyclodextrin (CD)-modified CZE. Under optimum conditions, eight of the nine furans were baseline-resolved in less than 10 min at 30 kV using 50 mM phosphate buffer, 10% v/v acetonitrile (ACN), pH 2.0, with 5 mM gamma-CD.     

Direct chiral resolution of lactic acid in food products by capillary electrophoresis

Chiral resolution of native DL-lactic acid was performed by capillary electrophoresis using 2-hydroxypropyl-beta-cyclodextrin as a chiral selector. Various factors affecting chiral resolution, migration time, and peak area of lactic acid were studied. The running conditions for optimum separation of lactic acid were found to be 90 mM phosphate buffer (pH 6.0) containing 240 mM 2-hydroxypropyl-beta-cyclodextrin with an effective voltage of -30 kV at 16 degrees C, using direct detection at 200 nm. In order to enhance the sensitivity, sample injection was done under a pressure of 50 mbar for 200 s. On-line sample concentration was accomplished by sample stacking. With this system, D- and L-lactic acids in food products were analyzed successfully.     

Analysis of polymyxin B sulfate by capillary zone electrophoresis with cyclodextrin as additive. Method development and validation

A capillary zone electrophoresis method for analysis of polymyxin B sulfate is described. In this method, triethanolamine (TEA)-phosphate buffer at pH 2.5 was employed to reduce the adsorption of analyte onto the capillary wall. Methyl-beta-cyclodextrin (M-beta-CD) and 2-propanol (IPA) were found to be necessary for selectivity enhancement. In order to optimize the method and to control its robustness, a central composite design was performed with four parameters, i.e. concentration of M-beta-CD, TEA, IPA and buffer pH. The optimal separation conditions were as follows: capillary, 55 cm (50 microm I.D., 47 cm effective length); 130 mM TEA-phosphate buffer (pH 2.5) containing 5 mM M-beta-CD and 5% IPA; 24 kV (51 microA) applied voltage; column temperature, 20 degrees C. Further, linearity and limits of detection quantification were examined. Three commercial samples were analyzed quantitatively.     

Experimental design optimization of the capillary electrophoresis separation of leucine enkephalin and its immune complex

To optimize the capillary electrophoretic separation conditions for leucine enkephalin (LE) and the immune complex of the LE and anti-LE reaction, an analysis using a three-level, three-factorial Box-Behnken design was performed. Three separation parameters, buffer pH (X(1)), buffer concentration (X(2)), and applied voltage (X(3)), were chosen to observe the effect on separation responses. The responses were theoretical plate number, migration time of the LE peak, and resolution between the peaks. The optimum conditions and process validation were determined using statistical regression analysis and surface plot diagrams. The capillary electrophoresis optimum separation conditions were established to be 75 mM phosphate buffer at pH 7.00 with an applied separation voltage of 15 kV. By using the analysis technique, the prediction of responses was satisfactory and process verification yielded values within the +/-5% range of the predicted efficiency.     

Determination of free and protein-bound glutathione in HepG2 cells using capillary electrophoresis with laser-induced fluorescence detection

A rapid method using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was developed to determine free and protein-bound glutathione (GSH) in human HepG2 hepatocarcinoma cells. The samples were derivatized with 5-iodoacetamidofluorescein (5-IAF), and analyzed at 22 kV using sodium phosphate buffer (10mM, pH 11.4) and an uncoated 58 cm x 75 microm I.D. fused silica capillary. The analysis time was less than 10 min and N-acetylcysteine was used as internal standard. The derivatization conditions, such as reaction time, 5-IAF concentration, running buffer and cartridge temperature were optimized. Argon gas was used in the study to prevent the oxidization of GSH during sample preparation. The optimized method required only 30-40 nl sample per analysis and was fast and sensitive. The method was applied to the analyses of HepG2 cells treated with the small metal chelating agent, pyrrolidine dithiocarbamate (PDTC). The results demonstrate that the amount of protein-bound GSH, which reflects the amount of protein S-glutathionylation, increased in a time-dependent manner upon cell treatment with PDTC, reaching a maximum of over 50% increase 2h post-PDTC.     

Field-amplified sample stacking in capillary electrophoresis for on-column concentration of alkaloids in Sophora flavescens Ait

A simple and rapid capillary zone electrophoresis method was developed for the separation of the main alkaloids from Sophora flavescens Ait. with the optimum buffer solution containing 110 mM NaH(2)PO(4) and 15% 2-propanol (pH 3.0). The field-amplified sample stacking (FASS) technique was applied to the on-line concentration of the alkaloids. The data presented in this work demonstrate that the use of a short water plug at the column inlet is essential for improving the reproducibility of FASS with electro-injection, and that the water plug injection time affected the sensitivity significantly. The sample concentration was further increased by about 2-3-fold by the introduction of a relatively longer water plug. With this stacking measure, the concentration sensitivity was about 3-4 orders of magnitude higher than in hydrodynamic injection.     

Analysis of destruxins produced from Metarhizium anisopliae by capillary electrophoresis

Destruxins are insecticidal metabolites of a fungus, Metarhizium anisopliae. These metabolites are usually secreted into the culture medium during growth. The structure of destruxins is classified as being a cyclic hexadepsipeptide. More than 35 different destruxins have been characterized with a wide range of insecticidal activities. In this report, the destruxins are extracted by acetonitrile and crystallization by lyophilization. The final crystal is subjected for capillary electrophoresis analysis. Because destruxins are relatively hydrophobic compounds, micellar electrokinetic capillary chromatography is used in this series of experiments. The borate-based running buffer is optimized according to (1) boric acid concentration, (2) sodium dodecyl sulfate (SDS) concentration, (3) acetonitrile concentration, and (4) the pH of the running buffer. Optimization is based on resolution and running speed. The results indicate that 20 mM boric acid with 40 mM SDS plus 10% acetonitrile with pH 9.24 is the best set of conditions for both resolution and running speed.     

Performance of throughout in-capillary derivatization capillary electrophoresis employing an on-line sample and run buffer loading device

We report on the effect on performance of varying the length of the capillary during throughout in-capillary derivatization (TICD) capillary electrophoresis (CE). Performance was evaluated by on-line coupling with a sample and CE runbuffer loading device that was newly introduced for this study. The device was assembled with a low cost using two 5 mm inner diameter (ID) disposable polyethylene syringes. First, a sequence was manually formed consisting of a 200 microL run buffer solution plug, a 100 microL sample plug and another 200 microL run buffer solution plug. Each plug was separated from its neighbor by a 100 microL air plug. When each plug reached the injection point where both a platinum-wire anode and the end of the separation capillary tube were located, 340 V/cm separation voltage (electrophoresis voltage) and 34 V/cm injection voltage were applied to the capillary for 3 s. Then the analytes were derivatized during migration in 50 microm ID capillaries filled with 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in a 20 mM phosphate-borate buffer (pH 10), followed by separating and detecting of OPA derivatives by absorbance of 340 nm. Derivatization, separation, and detection were performed systematically using capillaries which varied in length from 5 to 80 cm. In the case of TICD-CE of a mixture containing 1 mM aspartic acid (Asp) and 20 mM m-nitorophenol (MNP) as a test solution, it was determined that peak area and peak width ratios of Asp to MNP did not depend on capillary length. Enantiomeric separations of DL-alanine (Ala) and Asp were examined using a run buffer consisting of a 45 microM beta-cyclodextrin (CD)-2 mM OPA/NAC-20 mM phosphate-borate buffer (pH 10). Even though the resolution of these enantiomeric pairs decreased with decreasing capillary length, as expected, the peaks corresponding to both enantiomeric amino acids were identified even when a 5 cm capillary was used. An 8-component amino acid mixture was also tested with 5 cm and 10 cm capillaries.     

Use of capillary electrophoresis and laser-induced fluorescence for attomole detection of amino acids

A capillary electrophoresis and laser-induced fluorescence (CE-LIF) method was developed to identify and quantitate at amol (10(-18)) concentration. Amino acids were derivatized with 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde prior to CE-LIF analysis. The assay was developed by varying the sodium borate concentration, buffer pH, operating voltage, and operating temperature. A run buffer system containing 6.25 mM borate, 150 mM sodium dodecyl sulfate, and 10 mM tetrahydrofuran (pH 9.66) at 25 degrees C, and 24 kV provided analysis conditions for a high-resolution, sensitive, and repeatable assay of amino acids. The rate of derivatization, stability of the labeled amino acids, and amino acid quantitation varied for each amino acid. Amino acids were detected with greater efficiency by this method than automated HPLC amino acid analysis. The repeatability of the assay ranged from 0.3 to 0.9% within a day and 0.7 to 1.5% between analysis days. Bacterial amino acid utilization in a chemically defined medium was successfully monitored using this method. This work defines a sensitive and repeatable method for the detection of amino acids during bacterial metabolism.     

Determination of Phosphoamino Acids by 6-Oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl)fluorescein and MEKC with LIF Detection

The fluorescent reagent 6-oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl)fluorescein (SAMF), has been newly synthesized for use as a label for characterization of phosphoamino acids by micellar electrokinetic capillary electrophoresis (MEKC) with laser-induced fluorescence (LIF) detection. The conditions for derivatization and separation of the phosphoamino acids, including pH, concentration of electrolyte, and Brij-35 concentration were optimized in detail. Derivatization was performed at 35 °C for 10min in borate buffer (pH 8.0). The derivatives were separated to baseline by use of running buffer containing 50 mM borate and 20 mM Brij-35 at pH 9.3. Detection limits ranged from 5 × 10^?11 to 1 × 10^?9 mol L^?1 (signal to noise ratio = 3). The method was used for characterization of the phosphoamino acids in a sample from hydrolysis of a novel protein kinase from tobacco cells.     

Single-strand conformation polymorphism analysis by capillary zone electrophoresis in neutral pH buffer

Sensitivity of single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products was reported to be lower in capillary zone electrophoresis (CZE) compared to conventional slab gel electrophoresis. We examined the effects of buffer ion type, pH, and temperature in an attempt to improve the mutation detectability in the SSCP-CZE mode. It was noted that, by utilizing short-chain polyacrylamide as sieving media while simultaneously lowering the temperature, there was no improvement of conformer detectability. On the contrary, there was a large increment in conformers' resolution by running samples in a lower-pH buffer system. The effects of different buffering ions and pH values were investigated. By using a new buffer system, consisting of 35 mM 2-(N-morpholino)propanesulfonic acid (MES), 30 mM tris(hydroxymethyl)aminomethane (Tris), 1 mM ethylene diaminetetraacetic acid (EDTA), pH 6.8, and keeping constant all the other conditions, such as temperature, sieving, applied voltage, capillary length, and inner diameter (ID), there was a remarkable improvement in resolution and the sensitivity became comparable to that of slab gel systems.     

Determination of antibacterial flomoxef in serum by capillary electrophoresis

A determination method of flomoxef (FMOX) concentration in serum by capillary electrophoresis is developed. Serum samples are extracted with acetonitrile. After pretreatment, they are separated in a fused-silica capillary tube with a 25 mM borate buffer (pH 10.0) as a running buffer that contains 50mM sodium dodecyl sulfate. The FMOX and acetaminophen (internal standard) are detected by UV absorbance at 200 nm. Linearity (0-200 mg/L) is good, and the minimum limit of detection is 1.0 mg/L (S/N = 3). The relative standard deviations of intra- and interassay variability are 1.60-4.78% and 2.10-3.31%, respectively, and the recovery rate is 84-98%. This method can be used for determination of FMOX concentration in serum.