Capillary electrophoresis coupled with electrochemiluminescence detection using porous etched joint

A new setup to couple capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection is described in which the electrical connection of CE is achieved through a porous section at a distance of 7 mm from the CE capillary outlet. Because the porous capillary wall allowed the CE current to pass through and there was no electric field gradient beyond that section, the influence of CE high-voltage field on the ECL procedure was eliminated. The porous section formed by etching the capillary with hydrofluoric acid after only one side of the circumference of 2-3 mm of polyimide coating of the capillary was removed, while keeping the polyimide coating on the other part to protect the capillary from HF etching makes the capillary joint much more robust since only a part of the circumference of it is etched. A standard three-electrode configuration was used in experiments with Pt wire as a counter electrode, Ag/AgCl as a reference electrode, and a 300-mum diameter Pt disk as a working electrode. Compared with CE-ECL conventional decoupler designs, the present setup with a porous joint has no added dead volume created.

Drug-human serum albumin binding studied by capillary electrophoresis with electrochemiluminescence detection

A new technique for investigating drug-protein binding was developed employing capillary electrophoresis (CE) coupled with tris(2,2'-bipyridyl) ruthenium(II) [Ru(bPY)(3)(2+)] electrochemiluminescence (ECL) (CE-ECL) detection after equilibrium dialysis. Three basic drugs, namely pridinol, procyclidine and its analogue trihexyphenidyl, were successfully separated by capillary zone electrophoresis with end-column Ru(bPY)(3)(2+) ECL detection. The relative drug binding to human serum albumin (HSA) for each single drug as well'as for the three drugs binding simultaneously was calculated. It was found that the three antiparkinsonian drugs compete for the same binding site on HSA. This work demonstrated that Ru(bPY)(3)(2+) CE-ECL can be a suitable technique for studying drug-protein binding.

Characterization of prolidase activity using capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection and application to evaluate collagen degradation in diabetes mellitus

A new method for prolidase (PLD, EC 3.4.13.9) activity assay was developed based on the determination of proline produced from enzymatic reaction through capillary electrophoresis (CE) with tris(2,2'-bipyridyl)ruthenium(11) [Ru(bpy)(3)(2+)] electrochemiluminescence detection (ECL). A detection limit of 12.2 fmol (S/N = 3) for proline, corresponding to 1.22 x 10(-8) units of prolidase catalyzing for 1 min was achieved. PLD activity determined by CE-ECL method was in agreement with that obtained from the classical Chinard's one. CE-ECL showed its powerful resolving ability and selectivity as no sample pretreatmentwas needed and no interference existed. The clinical utility of this method was successfully demonstrated by its application to assay PLD activity in the serum of diabetic patients in order to evaluate collagen degradation in diabetes mellitus (DM). The results indicated that enhanced collagen degradation occurred in DM.

Capillary electrophoresis with electrochemiluminescence detection for measurement of aspartate aminotransferase and alanine aminotransferase activities in biofluids

A new sensitive assay for aspartate aminotransterase (AST) and alanine aminotransferase (ALT) activities in biofluids was developed, based on the separation and detection of alanine, glutamate, and aspartate using capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection. The three amino acids were separated in 5 mM phosphate of pH 2.1 as background electrolyte, and detected on a 500 mu m platinum disk electrode at 1.2 V (versus Ag/AgCl) in the presence of 10 mM tris(2,2'-bipyridyl)ruthenium(II) dissolved in 80 mM phosphate of pH 10.5. A mass detection limit of 37.3 fmol (or 81.5 fmol) for glutamate, corresponding to the product in the enzyme reaction catalyzed by 1.24 x 10(-9) U AST (or 2.72 x 10(-9) U ALT) in a 30 min reaction period, was achieved. This assay was applied to investigate the cytotoxicity effect of ethanol on HepG2 cells and differentiating nonalcoholic steatohepatitis (NASH) from alcoholic liver disease, indicating that the technique is promising for the application in the cell biological and clinical fields.

Field-amplified sample stacking capillary electrophoresis with electrochemiluminescence applied to the determination of illicit drugs on banknotes

Capillary electrophoresis (CE) with Ru(bpy)(3)(2+) electrochemiluminescence. (ECL) detection system was established to the determination of contamination of banknotes with controlled drugs and a high efficiency on-column field-amplified sample stacking (FASS) technique was also optimized to increase the ECL intensity. The method was illustrated using heroin and cocaine, which are two typical and popular illicit drugs. Highest sample stacking was obtained when 0.01 mM acetic acid was chosen for sample dissolution with electrokinetical injection for 6 s at 17 kV. Under the optimized conditions: ECL detection at 1.2 V, separation voltage 10.0 kV, 20 mM phosphate-acetate (pH 7.2) as running buffer, 5 mM Ru(bpy)(3)(2+) with 50 mM phosphate-acetate (pH 7.2) in the detection cell, the standard curves were linear in the range of 7.50 x 10(-8) to 1.00 x 10(-5) M for heroin and 2.50 x 10(-7) to 1.00 x 10(-4) M for cocaine and detection limits of 50 nM for heroin and 60 nM for cocaine were achieved (S/N = 3), respectively. Relative standard derivations of the ECL intensity and the migration time were 3.50 and 0.51% for heroin and 4.44 and 0.12% for cocaine, respectively.The developed method was successfully applied to the determination of heroin and cocaine on illicit drug contaminated banknotes without any damage of the paper currency.

Analysis of nephroloxic and carcinogenic aristolochic acids in Aristolochia plants by capillary electrophoresis with electrochemical detection at a carbon fiber microdisk electrode

Aristolochic acids (AAs) are the main bioactive ingredients in the most of Aristolochia plants, which are used to make dietary supplements, slimming pills and Traditional Chinese Medicines (TCMs). Excessive ingestion of AAs can lead to serious nephropathy. Therefore, quantitative analysis and quality control for the plants containing AAs is of great importance. In this paper, capillary electrophoresis (CE) with electrochemical detection (ED) at a 33 mu m carbon fiber microdisk electrode (CFE) has been applied to detect AA-I and AA-II in Aristolochia plants. Under the optimum conditions: detection potential at 1.20 V, 2.0 x 10(-2) mol L-1 phosphate buffer solution (PBS) (pH 10.0), injection time 25 s at a height of 17 cm and separation voltage at 12.5 kV, the AA-I and AA-II were baseline separated within 5 min. Low detection limits for AA-I and AA-II were 4.0 x 10(-8) mol L-1 and 1.0 x 10(-7) mol L-1, respectively. Wide linear ranges were from 4.0 x 10(-8) mol L-1 to 1.9 x 10(-5) mol L-1 and 1.0 X 10(-7) mol L-1 to 5.0 x 10(-5) mol L-1 for AA-I and AA-II, respectively. The proposed method has been successfully applied to analyze AAs contents in plant extracts. The results indicated that the contents of AAs in each part of Aristolochia debilis Sieb. Et Zucc.

Chinese herb medicines analyses by capillary electrophoresis with amperometric detection

Capillary electrophoresis (CE) with amperometric detection (AD) has been widely used in various fields of analytical science, especially in the pharmaceutical industry recently due to its high separation efficiency and low detection limit. The determination of active ingredients in Chinese herb medicines by CE-AD is of great importance in developing the researches on pharmacology of herbs, quantitative analysis and quality control. Analyses of the effective components in Chinese herb medicines and compound Chinese herb medicine by CE-AD are reviewed in this paper. In contrast with other analysis methods, the advantage of CE-AD is discussed. The development in analyses of traditional Chinese medicine (TCM) by CE-AD in future is mentioned.

Miniaturized tris(2,2 '-bipyridyl)ruthenium(II) electrochemiluminescence detection cell for capillary electrophoresis and flow injection analysis

The design and performance of a miniaturized chip-type tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)(3)(2+)] electrochemiluminescence (ECL) detection cell suitable for both capillary electrophoresis (CE) and flow injection (FI) analysis are described. The cell was fabricated from two pieces of glass (20 x 15 x 1.7 mm), and the 0.5-mm-diameter platinum disk was used as working electrode held at +1.15 V (vs silver wire quasi-reference), the stainless steel guide tubing as counter electrode, and the silver wire as quasi-reference electrode. The performance traits of the cell in both CE and FI modes were evaluated using tripropylamine, proline, and oxalate and compared favorably to those reported for CE and FI detection cells. The advantages of versatility, sensitivity, and accuracy make the device attractive for the routine analysis of amine-containing species or oxalate by CE and FI with Ru(bPY)(3)(2divided by) ECL detection.

Microchip capillary electrophoresis with an integrated indium tin oxide electrode-based electrochemiluminescence detector

This paper describes an indium tin oxide (ITO) electrode-based Ru(bPY)(3)(2+) electrochemiluminecence (ECL) detector for a microchip capillary electrophoresis (CE). The microchip CE-ECL system described in this article consists of a poly(dimethylsiloxane) (PDMS) layer containing separation and injection channels and an electrode plate with an ITO electrode fabricated by a photolithographic method. The PDMS layer was reversibly bound to the ITO electrode plate, which greatly simplified the alignment of the separation channel with the working electrode and enhanced the photon-capturing efficiency. In our study, the high separation electric field had no significant influence on the ECL detector, and decouplers for isolating the separation electric field were not needed in the microchip CE-ECL system. The ITO electrodes employed in the experiments displayed good durability and stability in the analytical procedures. Proline was selected to perform the microchip device with a limit of detection of 1.2 muM (S/N = 3) and a linear range from 5 to 600 muM.

Direct tris(2,2 '-bipyridyl)ruthenium(II) electrochemiluminescence detection of polyamines separated by capillary electrophoresis

Capillary electrophoresis (CE) with tris(2,2'-bipyridyl) ruthenium (II) (Ru(bpy)(3)(2+)) electrochemiluminescence (ECL) detection technique was developed for the analysis of four polyamines (putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)) analysis. The four polyamines contain different amine groups, which have different ECL activity. There are several parameters which influence the resolution and ECL peak intensities, including the buffer pH and concentrations, separation voltage, sample injection, electrode materials, and Ru(bpy)(3)(2+) concentrations. Polyamines are separated by capillary zone electrophoresis in an uncoated fused-silica capillary (50 cm x 25 mum (ID) filled with acidic phosphate buffer (200 mmol/L phosphate, pH 2.0) - 1 mol/L phosphoric acid (9:1 v/v) and a separation voltage of 5 kV (25 muA), with end-column Ru(bpy)(3)(2+) ECL detection. A 5 mmol/L Ru(bpy)(3)(2+) solution plus 200 mmol/L phosphate buffer (pH 11.0) is added into the reagent reservoir. The calibration curve is linear over a concentration range of two or three orders of magnitude for the polyamines. The analysis time is less than 25 min. Detection limits for Put and Cad are 1.9 x 10(-7) mol/L and 7.6 x 10(-9) mol/L for Spd and Spm, respectively.

New technique for capillary electrophoresis directly coupled with end-column electrochemiluminescence detection

capillary electrophoresis (CE) is characterized. A 300 mum diameter Pt working electrode was used to directly couple with a 75 mum inner diameter separation capillary without an electric field decoupler. The hydrodynamic cyclic voltammogram (CV) of Ru(bpy)(3)(2+) showed that electrophoretic current did not affect the ECL reaction. The presence of high-voltage (HV) field only resulted in the shift of the ECL detection potential. The distance of capillary to electrode was an important parameter for optimizing detection performance as it determined the characteristics of mass transport toward the electrode and the actual concentration of Ru(bpy)(3)(2+) in the detection region. The optimum distance of capillary to electrode was decided by the inner diameter of the capillary, too. For a 75 mum capillary, the working electrode should be placed away from the capillary outlet at a distance within the range of 20-260 mum. The effects of pH value of ECL solution and molecular structure of analytes on peak height and theoretical plate numbers were discussed. Using the 75 mum capillary, under the optimum conditions, the method provided a linear range for tripropylamine (TPA) between 1 x 10(-10) and 1 X 10(-5) mol/L with correlation coefficient of 0.998. The detection limit (signal-to-noise ratio S/N = 3) was 5.0 x 10(-11) mol/L. The relative standard deviation in peak height for eight consecutive injections was 5.6%. By this new technique lidocaine spiked in a urine sample was determined. The method exhibited the linear range for lidocaine from 5.0 x 10(-8) to 1.0 X 10(-5) mol/L with correlation efficient of 0.998. The limit of detection (S/N = 3) was 2.0 x 10(-1) mol/L.

Capillary electrophoresis with electrochemiluminescence detection of procyclidine in human urine pretreated by ion-exchange cartridge

This paper describe a Ru(bpy)(3)(2+) based electrochemiluminescence (ECL) method to detect procyclidine in human urine following separation by capillary electrophoresis (CE). An ECL detection cell was designed for post-column addition of Ru(bpy)(3)(2+). Parameters affecting separation and detection were optimized, leading to a detection limit of 1 x 10(-9) mol/l in an on-capillary stacking mode. For application in urine, a cartridge packed with slightly acidic cation-exchange resin was used to eliminate the matrix effects of urine and improve the detection sensitivity. Extraction recovery was nearly 90%.

Progress in amperometric detection for capillary electrophoresis

As a high efficiency separation technique, capillary electrophoresis(CE) has been widely used in various fields of analytical science. Amperometry is one of the most sensitive electrochemical detection methods in CE. The capillary/electrode decoupling mechanism, applications,of new electrode systems in CE, detection cell technique are discussed in detail. Amperometric detection is compatible with microfabricated CE chips and will make the concept of lab-on-a-chip become a reality. Because of these progresses, amperometry is becoming a widely acceptable detection method,for more chemical and biological analytes.

Determination of sulpiride by capillary electrophoresis with end-column electrogenerated chemiluminescence detection

Background: Capillary electrophoresis (CE) with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)(3)(2+)]-electro-generated chemiluminescence (ECL) detection is a promising method for clinical analysis. In this study, a method combining CE with Ru(bpy)(3)(2+) ECL (CE-ECL) detection that can be applied to amine-containing clinical species was developed, and the performance of CE-ECL as a quantitative method for determination of sulpiride in human plasma or urine was evaluated. Methods: Sulpiride was separated by capillary zone electrophoresis in uncoated fused-silica capillaries [510 cm x 25 mum (i.d.)] filled with phosphate buffer (pH 8.0 and a driving voltage of +15 kV, with end-column Ru(bpy)(3)(2+) ECL detection. A platinum disc electrode was used as working electrode. Sulpiride in human plasma or urine samples (100 muL) was extracted by a double-step liquid-liquid extraction procedure, dried under nitrogen at 35 degreesC in a water bath, and reconstituted with 100 muL of filtered water. The extraction solvent was ethyl acetate-dichloromethane (5:1 by volume). Results: Under optimum conditions (pH 8.0 phosphate buffer, injection for 6 s at 10 kV, and +1.2 V as detection potential), separation of sulpiride was accomplished within 4 min. The calibration curve was linear over a concentration range of 0.05-25.0 mumol/L, and the limit of detection was 2.9 x 10(-8) mol/L for sulpiride. Intra- and interday CVs for ECL intensities were <6%. Extraction recoveries of sulpiride were 95.6-101% with CVs of 2.9-6.0%. The method was,clinically validated for patient plasma and urine samples. Conclusions: CE combined with Ru(bpy)(3)(2+) ECL is reproducible, precise, selective, and enables the analysis of sulpiride in human plasma and urine. It thus is of value for rapid and efficient analysis of amine-containing analytes of clinical interest.

Determination of three beta-blockers by capillary electrophoresis with end-column electrochemical detection

Three beta -blockers (propranolol, timolol, acebutolol) were separated by capillary electrophoresis (CE) and detected by end-column electrochemical detection (EC). In the present work, a carbon fiber (33 mum) electrode was used as the working electrode. The effect of the buffer concentration, buffer pH, detection potential and separation voltage on the separation of analytes and behavior of electrochemical detection was systematically investigated. The optimum conditions determined were as following: 40 cm length, 25 mum i.d. capillary; 17.5 kV separation voltage; 2 s injection at 15 kV; 70mM phosphate buffer, pH 3.5; detection potential + 1.2V (vs. Ag/AgCl). Under these conditions, the linear ranges of beta -blockers were over three orders of magnititude and the low detection limit of 10(-8)M was obtained. This method was also applied to detect the simulated urine sample.