Capillary electrophoresis and microchip capillary electrophoresis with electrochemical and electrochemiluminescence detection

Recent advances and key strategies in capillary electrophoresis and microchip CE with electrochemical detection (ECD) and electrochemiluminescence (ECL) detection are reviewed. This article consists of four main parts: CE-ECD; microchip CE-ECD; CE-ECL; and microchip CE-ECL. It is expected that ECD and ECL will become powerful tools for CE microchip systems and will lead to the creation of truly disposable devices. The focus is on papers published in the last two years (from 2005 to 2006).     

毛细管电泳-电致化学发光法测定淮山药中胆碱和尿囊素

基于胆碱和尿囊素均能增强联吡啶钌的电致化学发光信号,建立了毛细管电泳-电化学发光法(CE-ECL)分离检测淮山药中胆碱和尿囊素的方法。在优化实验条件下,胆碱和尿囊素均在0.5～1000μg/L范围内与发光强度呈良好线性,检出限分别为0.02μg/L和0.04μg/L。对0.5 mg/L胆碱和0.5 mg/L尿囊素混合溶液连续6次测定,胆碱和尿囊素峰高的RSD分别为1.8%和2.0%,迁移时间的RSD分别为0.98%和1.1%。该方法已用于样品中胆碱和尿囊素测定,加标回收率在95.6%～101.1%之间,RSD≤2.5%。     

Capillary electrophoresis with gold nanoparticles enhanced electrochemiluminescence for the detection of roxithromycin

Tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)(3)(2+))-roxithromycin based electrochemiluminescence (ECL) was enhanced greatly by gold nanoparticles 10 nm in diameter. Capillary electrophoresis (CE) was coupled with the resultant ECL system as a detector for roxithromycin. This ECL emission is explained by the coreactant mechanism where roxithromycin behaves as a coreactant to generate strong reducing species and gold nanoparticles act as "floating nanoelectrodes". The reaction of Ru(bpy)(3)(3+) with the generated strong reducing species on the Pt working electrode as well as on "floating nanoelectrodes" releases Ru(bpy)(3)(2+*), resulting in enhancement of ECL emission. The selectivity of this detection system towards roxithromycin was examined by CE. Under the optimized conditions, the intensity of ECL emission varies linearly with the concentration of roxithromycin from 24 nM to 0.24 mM. The detection limit is 8.4 nM, while without adding gold nanoparticles it is only 84 nM. The detection of roxithromycin in pharmaceutical and urine samples was also performed by the proposed CE-ECL method.     

Capillary electrophoresis with solid-state electrochemiluminescence detector

We report capillary electrophoresis coupling to a solid-state electrochemiluminescence (ECL) detector for the first time. The solid-state ECL detector was fabricated by immobilizing the ECL reagent tris(2,2'-bipyridyl)ruthenium (TBR) in poly-(p-styrenesulfonate)-silica-poly(vinyl alcohol) grafting 4-vinylpyridine copolymer films. The excellent stability of the solid-state ECL detector in the phosphate solution satisfied application in CE. The CE with solid-state ECL detector system was characterized using tripropylamine (TPA) and proline. The influences of detection potential, the concentration of TBR in the film, and pH value of ECL buffer were investigated. The linear range for TPA and proline was 0.005-10 microM and 5-10 mM with correlation coefficients of 0.997 and 0.998, respectively. The detection limit (signal-to-noise ratio S/N = 3) was estimated to be 0.002 and 2.0 microM for TPA and proline, respectively. The relative standard deviations for 1.0 microM TPA and 1.0 mM proline were 8.7% and 7.5% with theoretical plate numbers of 70 000 and 16 000, respectively. Compared with the CE-ECL of TBR in aqueous solution, the CE coupling with solid-state ECL detector system gave the same sensitivity of analysis.     

Capillary electrophoresis with electrochemiluminescence detection for the analysis of quinolone drugs and pharmacokinetics study

A novel method for the determination of two quinolone drugs norfloxacin （NOR） and levofloxacin （LVX） was described by capillary electrophoresis with electrochemiluminescence detection. The good relationship （r ≥ 0.9991） between peak area and concentration of analytes was established over two orders of magnitude. The limits of detection （LOD, S/N = 3） in standard solution are 4.8 × 10^-7 mol/L for NOR and 6.4 × 10^-7 mol/L for LVX, respectively. The limits of quantitation （LOQ, S/N = 10） in real human urine samples are 1.2 × 10^-6 mol/L for NOR and 1.4 × 10^-6 mol/L for LVX, respectively. The present method was successfully applied to the determination of NOR and LVX in human urine and the studv of oharmacokinetics of NOR.     

Amperometric enzyme electrode for the determination of aspartate aminotransferase and alanine aminotransferase in serum

Glutamate oxidase (E.C. 1.4.3.7) was immobilized at a platinized activated carbon electrode and the enzyme electrodes were used for the amperometric determination of L-glutamate in a stirred aqueous solution by the electrochemical detection of enzymically produced hydrogen peroxide at + 320 mV vs. Ag/AgCl. A linear calibration graph was obtained between 2 μM and 2 mM with a steady-state response time of 1 min. The glutamate oxidase electrode was subsequently applied to the measurement of aspartate aminotransferase (AST) (E.C. 2.6.1.1) and alanine aminotransferase (ALT) (E.C. 2.6.1.2) in serum. The performance of the electrode was compared with that of techniques used in the hospital diagnostic laboratory. The responses of the enzyme electrode to AST and ALT activities were linear over the clinically relevant range (5-500 U l ^?1), and correlated well (r=0.99) with the methods used for routine clinical analysis.     

Capillary electrophoresis with electrochemiluminescence detection: fundamental theory,apparatus, and applications

This review presents a comprehensive survey of recent progress on electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE). The fundamental theories involved in CE-ECL, e.g., the mechanism involving both coreactant-based and inhibitor-based ECL, as well as the possible analytes to be detected by CE-ECL are summarized. Different schemes for the construction of CE-ECL apparatus, including methods for preparing the working electrode, approaches for addition of ECL reagents, ways to fabricate electrical decouplers, and factors affecting ECL efficiency are reviewed. Discussion of the literature related to the application of CE-ECL from January 2005 to September 2010 is sorted by the corresponding analyte matrixes, namely, the standard solution, urine, serum and plasma, and other matrixes. Finally, possible trends for CE-ECL in the near future are discussed.     

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

This paper describe a Ru(bpy)₃²⁺ 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)₃²⁺. Parameters affecting separation and detection were optimized, leading to a detection limit of 1×10⁻⁹ 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%.     

Capillary electrophoresis coupled with electrochemiluminescence detection for the separation and determination of thyreostatic drugs in animal feed

A rapid, simple, and practical method for the determination of four of the most used thyreostatic drugs (methimazole, 2‐thiouracil, 6‐methyl‐2‐thiouracil, and 6‐propyl‐2‐thiouracil) using CE coupled to electrochemiluminescence detection has been established, based on the electrochemiluminescence enhancement of tris(2,2‐bipyridyl)ruthenium(II) with these analytes. Parameters that affect separation and detection were optimized. Under the optimum experimental conditions, the four analytes could be well separated within 11 min at the separation voltage of 16 kV in a running solution containing 20 mM phosphate buffer (pH 9.0) and 1.0 × 10^?4 M Ru(bpy)₃²⁺, with a solution of 20 mM phosphate buffer (pH 12.0) containing 1.0 × 10^?4 M Ru(bpy)₃²⁺ in the electrochemiluminescence detection cell. The detection limits for methimazole, 6‐methyl‐2‐thiouracil, 6‐propyl‐2‐thiouracil, and 2‐thiouracil were 0.1, 0.05, 0.05, and 0.01 μM, respectively. The proposed method was applied to analyze these drugs in spiked animal feed samples. The recoveries were 88.2～99.0 and 86.4～98.7% for the intraday and interday analyses, respectively. The RSDs were 2.7～4.8 and 1.8～5.0% for the intraday and interday analyses, respectively. The results demonstrate that the proposed method has promising applications in the detection of thyreostatic drugs in animal feeds.     

Determination of enoxacin and ofloxacin by capillary electrophoresis with electrochemiluminescence detection in biofluids and drugs and its application to pharmacokinetics

A novel and sensitive method for the simultaneous determination of enoxacin and ofloxacin has been established using capillary electrophoresis (CE) coupled with electrochemiluminescence (ECL) detection based on the ECL enhancement of tri(2,2‐bipyridyl)ruthenium(II). The conditions for sample solvent type, CE separation and ECL detection were investigated systematically. The analytes were well separated and detected within 7 min. The limits of detection (S/N = 3) of enoxacin and ofloxacin are 9.0 × 10^?9 and 1.6 × 10^?8 mol/L, respectively. The precisions (RSD%) of intraday and interday are less than 2.1 and 4.0%, respectively. The limits of quantitation (S/N = 10) of enoxacin and ofloxacin are 3.2 × 10^?7 and 5.4 × 10^?7 mol/L in human urine samples and 4.1 × 10^?7 and 6.9 × 10^?7 mol/L in human serum samples, respectively. The recoveries of enoxacin and ofloxacin at different concentration levels in human urine, serum and eye drop samples are between 94.0 and 106.7%. The proposed method was successfully applied to the determination of the enoxacin and ofloxacin in human urine, serum and eye drop samples and the monitoring of pharmacokinetics of ofloxacin in human body. Copyright © 2009 John Wiley & Sons, Ltd.     

Capillary electrophoresis with electrochemiluminescence detection for the simultaneous determination of cisatracurium besylate and its degradation products in pharmaceutical preparations

Capillary electrophoresis with electrochemiluminescence detection for the simultaneous analysis of cisatracurium besylate and its degradation products (laudanosine, quaternary monoacrylate) in pharmaceutical preparation was developed and fully validated. The significant parameters that influence capillary electrophoresis separation and electrochemiluminescence detection were optimized. The total analysis time of the analytes was 15 min. The linearities of the method were 0.1～40.0 μg/mL for cisatracurium besylate and 0.04～8.00 μg/mL for laudanosine, with correlation coefficients (r) of 0.999 and 0.998, respectively. The detection limits (S/N = 3) were 83.0 ng/mL for cisatracurium besylate and 32.0 ng/mL for laudanosine. The intraday relative standard deviations of the analytes were <3.0%, and the interday relative standard deviations were <8.0%. The developed method was cost‐effective, sensitive, fast, and resource‐saving, which was suitable for the ingredient analysis in pharmaceutical preparation.     

Determination of mefenacet by capillary electrophoresis with electrochemiluminescence detection

Electrochemiluminescence (ECL) detection with capillary electrophoresis (CE) separation system was used to the rapid analysis of mefenacet within 7 min. The linear response range of mefenacet was from 1.07 × 10⁻⁸ to 5.0 × 10⁻⁷ M with a detection limit of 4.0 × 10⁻⁹ M. This technique was also applied to analyze residues of mefenacet in seedling and soil.     

Capillary electrophoresis with microwave-enhanced electrochemical detection

A new experimental development concerning microwave-assisted electrochemical detection in conjunction with capillary electrophoresis is presented. Focused microwaves are readily incorporated into end-column detection systems for capillary electrophoresis, they induce strong localised thermal activation at microelectrodes, and they affect and modulate, in particular, signals for chemically irreversible redox processes.     

Discrete wavelets transform for signal denoising in capillary electrophoresis with electrochemiluminescence detection

Discrete wavelets transform (DWT) was applied to noise on removal capillary electrophoresis-electrochemiluminescence (CE-ECL) electropherograms. Several typical wavelet transforms, including Haar, Daublets, Coiflets, and Symmlets, were evaluated. Four types of determining threshold methods, fixed form threshold, rigorous Stein's unbiased estimate of risk (rigorous SURE), heuristic SURE and minimax, combined with hard and soft thresholding methods were compared. The denoising study on synthetic signals showed that wave Symmlet 4 with a level decomposition of 5 and the thresholding method of heuristic SURE-hard provide the optimum denoising strategy. Using this strategy, the noise on CE-ECL electropherograms could be removed adequately. Compared with the Savitzky-Golay and Fourier transform denoising methods, DWT is an efficient method for noise removal with a better preservation of the shape of peaks.     

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

A new end-column electrochemiluminescence (ECL) detection technique coupling to capillary electrophoresis (CE) is characterized. A 300 microm diameter Pt working electrode was used to directly couple with a 75 microm 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 microm capillary, the working electrode should be placed away from the capillary outlet at a distance within the range of 220-260 microm. 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 microm 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(-8) mol/L.     

Microchip capillary electrophoresis coupled with an end-column electrochemiluminescence detection

An easy and universal wall-jet configuration for microchip CE-ECL detection system was constructed and investigated in this work. Two detection modes of pre-column and post-column were applied to the above system. TPA, tramadol and lidocaine were chosen as model analytes to estimate the system in both modes. The important operational parameters such as the concentration of luminescent reagent and the distance between the separation outlet and the working electrode were optimally obtained and compared for the first time.     

毛细管电泳电致化学发光法测定洛贝林的研究

基于稀土掺杂类普鲁士蓝化学修饰电极对Ru(bpy)32 的电催化氧化可增敏电致发光信号,建立了一种毛细管电泳-电致化学发光测定洛贝林的新方法。研究了工作电极电位、缓冲液的酸度及其浓度、分离电压和进样时间等实验参数对洛贝林测定的影响。在优化的实验条件下,其线性范围为1.5×10-7mol/L~1.5×10-4mol/L,检出限(S/N=3)为5.0×10-8mol/L。本法可直接用于注射液和空白人尿中洛贝林的测定,回收率为98.3%~101.2%。     

Determination of dioxopromethazine hydrochloride by capillary electrophoresis with electrochemiluminescence detection

The paper presents a rapid method for the determination of dioxopromethazine hydrochloride (DPZ), an antihistamine drug, by the capillary electrophoresis with electrochemiluminescene detection (CE–ECL) using tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺) reagent. This CE–ECL detection method has high sensitivity, good selectivity and reproducibility for DPZ analysis. Under the optimized conditions: separation capillary, 38 cm length (25 μm i.d.); sample injection, 10 s at 8 kV; separation voltage, 12.5 kV; running buffer, 20 mmol L⁻¹ sodium phosphate of pH 6.0; detection potential, 1.15 V; 50 mmol L⁻¹ of phosphate buffer (pH 7.14) containing 5 mmol L⁻¹ of Ru(bpy)₃²⁺ in ECL detection cell, the detection limit of DPZ was 0.05 μmol L⁻¹ (S/N = 3). The linear range extended from 5 to 100 μmol L⁻¹. The linear curve obtained was Y = 181.62 + 9.28X with a correlation coefficient of 0.9970. The relative standard deviations of the ECL intensity and the migration time for six continuous injections of 5 μmol L⁻¹ DPZ were 3.7% and 0.92%, respectively. The CE–ECL method was applied to analyze DPZ in real samples including tablets, rat serum and human urine, and satisfactory results were obtained without interference from samples matrix. The CE–ECL technique was proved to be a potential method for the detection of DPZ in clinic analysis.     

Analysis of ethambutol and methoxyphenamine by capillary electrophoresis with electrochemiluminescence detection

A capillary electrophoresis (CE) coupled with electrochemiluminescence (ECL) detection method for the analysis of ethambutol (EB) and methoxyphenamine (MP) has been investigated. Complete separation of EB and MP was achieved in 8 min using a background electrolyte of 20 mM sodium phosphate at pH 10.0 and a separation voltage of 9 kV. ECL detection was performed with an indium/tin oxide (ITO) working electrode biased at 1.4 V (versus a Pt wire reference) in a 200 mM sodium phosphate buffer (pH 8.0) containing 3.5 mM Ru(bpy)3(2+) (where bpy = 2,2'-bipyridyl). Linear correlation (r > or = 0.993) between ECL intensity and drug concentration was obtained in the range 2-50 ng/ml. The limits of detection (LODs) for EB and MP in water were 1.0 and 0.9 ng/ml, respectively. The relative standard deviation values on peak size (10 ng/ml level) and migration time for the two drugs were in the ranges 5-8 and 0.2-0.7% (n = 7), respectively. Applicability of the CE-ECL method to the analysis of human plasma spiked with EB and MP was examined. The LODs for EB and MP in plasma were 0.4 and 0.3 microg/ml, respectively.