Non‐competitive immunoassay for luteinizing hormone in human serum using capillary electrophoresis with chemiluminescence detection

A non‐competitive immunoassay based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of luteinizing hormone (LH) in human serum. The work involved the development of separation and CL conditions, allowing for routine analysis of serum samples. In this study, horseradish peroxidase (HRP)‐labelled monoclonal anti‐LH can catalyse the luminol–hydrogen peroxide reaction. The determined LH can react with excessive amount of HRP‐labelled anti‐LH. Within 14 min, free enzyme conjugate and immune complex could be separated in alkaline borate buffer by means of a high voltage (15 kV). To improve sensitivity, a series of measures were adopted, including the choice of para‐iodophenol as a CL enhancer, unique design in detect window. Under the optimal conditions, the calibration curve for LH was established in the concentration range 1–200 mIU/mL and the detection limit was 0.08 mIU/mL. Compared with ELISA, this method decreased the detection limit by about 12 times, and it has been successfully employed in the determination of LH in human serum. Copyright © 2007 John Wiley & Sons, Ltd.     

Capillary electrophoresis analysis of isoniazid using luminol-periodate potassium chemiluminescence system

A rapid and simple capillary electrophoresis method coupled with chemiluminescent (CL) detection was proposed for analysis of isoniazid (ISO) based on the enhancement effect of ISO to CL emission of luminol‐periodate potassium reaction. Under the optimal conditions, ISO can be assayed in the range of 7.0 × 10^?7 to 3.0 × 10^?5 g mL^?1 (R² = 0.9990) with a limit of detection of 3.0 × 10^?7 g mL^?1 (signal‐to‐noise ratio of 3). The whole analysis process can be completed within 2.5 min with a theoretical plate number of 6258. The relative standard deviations of the signal intensity and the migration time were 3.1 and 1.4% for a standard sample at 1.0 × 10^?5 g mL^?1 (n = 5), respectively. The presented novel strategy was successfully applied to the determination of ISO in commercial pharmaceutical preparations and spiked human serum samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of a novel capillary electrophoresis chemiluminescence system for amino acid analysis

In the present study, a novel peroxyoxalate CE–CL system was developed to achieve high signal stability and sensitivity based on a design of a new interface including a new mixing mode and a new grounding electrode mode. Amino acids fluorescently tagged with dansyl chloride and naphthalene‐2,3‐dicarboxaldehyde(NDA) were used for the study. Experiment results show this new system is quite effective to separate and detect amine acid with high stability and resolute. The detection limits were 1.1 nmol/L for dansyl‐leucine (Leu) and 2.0 nmol/L for dansyl‐aspartic acid (Asp). The relative standard deviations of peak height and migration time were in the ranges of 2.3–3.8% and 1.2–1.5%, respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K₃[Fe(CN)₆]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10⁻⁴ M luminol added to the CE running buffer and 1.0 × 10⁻⁴ M K₃[Fe(CN)₆] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10⁻⁷ M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10⁻⁷ to 3.0 × 10⁻⁵ M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.     

Chemiluminescence determination of pharmacologically active compounds by capillary electrophoresis.

A simple and rapid capillary electrophoresis with direct chemiluminescence method has been developed for the determination of five natural pharmacologically active compounds including rutin, protocatechuic aldehyde, chlorogenic acid, luteolin and protocatechuic acid. The luminol as a component of the separation electrolyte buffer was introduced at the head of the separation capillary. The separation of five compounds was carried out in a fused-silica capillary with 15.0 mmol/L tetraborate, 1.0 mmol/L SDS and 0.42 mmol/L luminol (pH 8.5). The analytes was determined by enhancing the chemiluminescence of luminol with 0.13 mmol/L K3Fe(CN)6 in 0.05 mol/L NaOH, which was introduced at the post-column stage. The voltage applied was 16 kV. Under the optimum conditions, the analytes were separated within 10 min. The excellent linearity was obtained over two to three orders of magnitude with a detection limit (signal:noise = 3) of 0.012-0.055 micromol/L for all five analytes. The method was successfully used in the analysis of pharmaceutical and biological samples, and the assay results were satisfactory.     

Flow injection chemiluminescence determination of isoniazid using the lucigenin-periodate system.

A weak chemiluminescence (CL) signal was observed during the mixing of isoniazid with lucigenin in alkaline aqueous solution. The CL signal was enhanced more than 100 times in the presence of potassium periodate. This CL system was developed for the determination of isoniazid using a flow injection mode. The CL intensity is proportional to the concentration of isoniazid in the range 0.005-1.0 mg/L. The limit of detection is 0.0034 mg/L and the relative standard deviation is 2.0% for 0.2 mg/L isoniazid solution in 11 repeated measurements. The method was applied to the determination of isoniazid in pharmaceutical preparations and satisfactory results were obtained.     

Electrophoresis–chemiluminescence detection of phenols catalyzed by hemin

Based on the catalytic activity of hemin, an efficient biocatalyst, an indirect capillary electrophoresis–chemiluminescence (CE‐CL) detection method for phenols using a hemin–luminol–hydrogen peroxide system was developed. Through a series of static injection experiments, hemin was found to perform best in a neutral solution rather than an acidic or alkaline medium. Although halide ions such as Br^? and F^? could further enhance the CL signal catalyzed by hemin, it is difficult to apply these conditions to this CE‐CL detection system because of the self‐polymerization of hemin, as it hinders the CE process. The addition of concentrated ammonium hydroxide to an aqueous/dimethyl sulfoxide solution of hemin–luminol afforded a stable CE‐CL baseline. The indirect CE‐CL detection of five phenols using this method gave the following limits of detections: 4.8 × 10^?8 mol/L (o‐sec‐butylphenol), 4.9 × 10^?8 mol/L (o‐cresol), 5.4 × 10^?8 mol/L (m‐cresol), 5.3 × 10^?8 mol/L (2,4‐dichlorophenol) and 7.1 × 10^?8 mol/L (phenol). Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of clopidogrel and its carboxylic acid metabolite by capillary electrophoresis

A capillary electrophoresis method was developed and validated for the first time for the analysis of clopidogrel and its carboxylic acid metabolite. Prior to method optimization, the pH dependence of effective mobility of both compounds was determined in order to define the initial pH of the running buffer. The optimized method demonstrated to be selective, and linear in the concentration range of 2–100 μM for both compounds. The method limits of detection and quantification were, respectively, 1.2 and 3.7 μM for clopidogrel and 1.1 and 3.2 μM for the carboxylic acid metabolite. Moreover, method validation demonstrated acceptable results for method repeatability (RSD < 7%), intermediate precision (RSD < 7%) and accuracy (85–96%) and is suitable for the quantitative analysis of clopidogrel and its metabolite in serum samples. The validated method was also applied to the determination of the kinetic parameters of the enzymatic hydrolysis of clopidogrel. An apparent K_m of 145 ± 30 μM and V_max of 0.4, 1.5 and 3.4 μM/min, respectively for the enzyme concentrations 1.0, 2.0 and 4.0 U/ml, were obtained.     

Simultaneous determination of the main metabolites in rice leaves using capillary electrophoresis mass spectrometry and capillary electrophoresis diode array detection

The study of the metabolomics of primary metabolites using conventional chemical analyses requires a high-throughput method. Chemical derivatizations are a prerequisite for gas-chromatographic separation, and a large sample quantity is needed for liquid-chromatographic separation and nuclear magnetic resonance detection systems. Recently, we have developed a capillary electrophoresis-mass spectrometry (CE-MS) technology that can simultaneously quantify a large number of primary metabolites, using only a small quantity of samples, and without any chemical derivatizations. Parallel use of a capillary electrophoresis-diode array detector (CE-DAD) system further enables almost all water-soluble intracellular metabolites to be analyzed. We demonstrate, with rice leaves, a simple and rapid method of sample preparation for CE analysis; using this method, we have successfully measured the levels of 88 main metabolites involved in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, photorespiration, and amino acid biosynthesis.     

Enantiomeric separation of Novel Psychoactive Substances by capillary electrophoresis using (+)‐18‐crown‐6‐tetracarboxylic acid as chiral selector

In the recent years, hundreds of Novel Psychoactive Substances (NPS) have entered both the European and the global drug market. These drugs, which are mainly used for recreational matters, have caused serious social problems. Every year, the spectrum of these misused drugs is enlarged by new derivatives, which are produced by modifications of basic structures of already well‐known substances. Additionally, a lot of them possess a stereogenic center which leads to 2 enantiomeric forms. The fact that the pharmacological effects and potencies of the enantiomers of these chiral NPS may differ can be assumed from a broad spectrum of active pharmaceutical ingredients. For this reason, analytical method development regarding enantiomeric separation for these classes of substances is of great pharmaceutical and medical interest. The aim of this work was to create an easy‐to‐prepare chiral capillary electrophoresis method for the enantioseparation of NPS which contains a primary amino group by means of (+)‐18‐crown‐6‐tetracarboxylic acid as chiral selector. Novel Psychoactive Substances were purchased at various Internet stores or represent samples seized by Austrian police. The effects of selector concentration, the electrolyte composition, and the addition of organic modifiers to the background electrolyte on enantioseparation were investigated. Under optimized conditions, the use of 20‐mM (+)‐18‐crown‐6‐tetracarboxylic acid, 10‐mM Tris, and 30‐mM citric acid buffer at pH 2.10 turned out to be effective. Fifteen of 24 tested NPS were resolved in their enantiomers within 15 minutes. It was found that all NPS were traded as racemic mixtures.     

On-line chemiluminescence determination of mitoxantrone by capillary electrophoresis

A novel capillary electrophoresis (CE) with chemiluminescence (CL) detection method for the determination of mitoxantrone (MTX) has been developed, which based on the CL reaction of potassium ferricyanide with luminol in sodium hydroxide medium sensitized by MTX. Under optimum analytical conditions, MTX is determined over the range of 7.0 × 10⁻⁸–1.0 × 10⁻⁶ M with a detection limit of 1.0 × 10⁻⁸ M. The relative standard deviation (RSD) was 3.7%, 2.6% and 3.0% for 7.0 × 10⁻⁸, 5.0 × 10⁻⁷ and 1.0 × 10⁻⁶ M MTX (n = 11), respectively. In laboratory-built CE–CL apparatus, the proposed method has been applied to determination of MTX in commercial drug and spiked in human urine and plasma with satisfactory results.     

Determination of trimethylamine in fish by capillary electrophoresis with electrogenerated tris(2,2'-bipyridyl)ruthenium(II) chemiluminescence detection.

A capillary electrophoresis with electrogenerated chemiluminescence (CE-ECL) method for the determination of trimethylamine (TMA) in fish was studied. In the presence of TMA, ECL from the reaction of analyte and in situ generated tris(2,2'-bipyridyl)ruthenium(III) [Ru(bpy)(3) (3+)] at electrode surface could be produced. The ECL detection was performed using a Pt working electrode biased at 1.23 V (vs. Ag/AgCl) potential in a 10 mmol/L sodium borate buffer solution, pH 9.2, containing 3 mmol/L Ru(bpy)(3) (2+). A linear calibration curve (correlation coefficient = 0.9996) was obtained in the range 8 x 10(-5)-4 x 10(-8) mol/L for TMA concentration. Recoveries obtained were in the range 98.78-101.46%. The method was successfully applied for the assay of TMA in fish, in combination with solid phase extraction (SPE) disks for sample clean-up and enrichment.     

Simultaneous determination of aminomethylbenzoic acid,cefminox sodium and etamsylate in human urine by capillary electrophoresis

A sensitive and selective capillary electrophoresis method is developed, for the first time, for effective separation and simultaneous determination of aminomethylbezoic acid (PAMBA), cefminox sodium (CMNX) and etamsylate (ETM). The electrophoresis conditions were investigated and optimized. A 25 mM phosphate solution (pH 8.5) was used as a buffer and the peak area was determined with UV detection at 216 nm wavelength under 18 kV separation voltage. Under optimal conditions, the three drugs can be separated effectively. Good linearity was achieved in 3.13–150 μg/mL for PAMBA, 6.25–150 μg/mL for CMNX and 3.13–150 μg/mL for ETM, with the correlation coefficients of >0.999. The limit of detection (LOD) for PAMBA, CMNX and ETM was 1.04, 2.08 and 1.04 μg/mL, respectively. Their recoveries in human urine were in the range from 90.2% to 101% with the RSD (n = 5) of 0.7–3.1%. The proposed method is simple, rapid and accurate, and provides the sensitivity and linearity necessary for analysis of the test drugs in human urine at clinically relevant concentrations.     

Separation and determination of anesthetics by capillary electrophoresis with mixed micelles of sodium dodecyl sulfate and Tween 20 using electrochemiluminescence detection

A simple and new method for the simultaneous determination of procaine (Pro), lidocaine (Lid), ropivacaine (Rop) and bupivacaine (Bup) was developed using capillary electrophoresis separation with mixed micelles and electrochemiluminescence detection. The use of mixed micelles of 2.0 × 10^–3 mol/L sodium dodecyl sulfate (SDS) and 8.0 × 10^–3 mol/L Tween 20 greatly improved separation selectivity. The detection sensitivities of four drugs with a Pt working electrode were increased by modification of the Pt electrode with europium(III)–doped Prussian Blue analog (Eu–PB). Under optimal conditions, the four local anesthetics were well separated and detected. The limits of detection (LOD, S/N = 3) of Pro, Lid, Rop and Bup in standard solution are 2.5 × 10^–8, 1.3 × 10^–8, 3.0 × 10^–8 and 4.1 × 10^–8 mol/L, respectively. The limits of quantitation (LOQ, S/N = 10) of Pro, Lid, Rop and Bup are 2.3 × 10^–7, 1.2 × 10^–7, 3.7 × 10^–7 and 5.6 × 10^–7 mol/L in a human urine sample, and 8.5 × 10^–7, 6.9 × 10^–7, 2.8 × 10^–6 and 1.1 × 10^–6 mol/L in a human serum sample, respectively. The recoveries of four drugs at different spiked concentrations in human urine and serum samples were between 86.5 and 107.6%. The proposed method has been successfully applied to determine local anesthetics in biofluids. Copyright © 2012 John Wiley & Sons, Ltd.     

Selective determination of human immunoglobulin G by flow‐injection chemiluminescence

A novel flow‐injection chemiluminescence method was developed for the selective determination of human immunoglobulin G (IgG) in the presence of thiomersal by changing the flow rates of peristaltic pump. The study was based on the independence and additivity of the CL signals of human IgG and thiomersal in the galangin–potassium permanganate–polyphosphoric acid system. In meantime, two equations relating to the concentrations of mixing solutions of human IgG and thiomersal vs the CL intensity were established and solved, on the basis of which the content of thiomersal included in samples was simultaneously determined too. The enhanced CL intensity was in proportion to concerntrations in the range 8.0 × 10^?7 to 8.0 × 10^?5 g/mL for human IgG and 1.0 × 10^?7 to 2.0 × 10^?6 g/mL for thiomersal with the detection limits of 5.0 × 10^?7 g/mL for human IgG and 6.0 × 10^?8 g/mL for thiomersal, respectively. The relative standard deviation for 1.0 × 10^?5 g/mL human IgG was 0.8% and for 2.0 × 10^?7 g/mL thiomersal it was 2.0% (n = 10). The proposed method was applied to determine three synthetic samples with recoveries of 91.5–109.5%. In addition, the possible chemiluminescence mechanisms are discussed as well. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis and interaction of bovine serum albumin with p‐hydroxybenzoic acid derivatives

Three novel p‐hydroxybenzoic acid derivatives (HSOP, HSOX, HSCP) were synthesized from p‐hydroxybenzoic acid and sulfonamides (sulfamonomethoxine sodium, sulfamethoxazole and sulfachloropyridazine sodium) and characterized by elemental analysis, HNMR and MS. Interactions between derivatives and bovine serum albumin (BSA) were studied by fluorescence quenching spectra, UV–vis absorption spectra and time‐resolved fluorescence spectra. Based on fluorescence quenching calculation and Förster's non‐radioactive energy transfer theory, the values of the binding constants, basic thermodynamic parameters and binding distances were obtained. Experimental results indicated that the three derivatives had a strong ability to quench fluorescence from BSA and that the binding reactions of the derivatives with BSA were a static quenching process. Thermodynamic parameters showed that binding reactions were spontaneous and exothermic and hydrogen bond and van der Waals force were predominant intermolecular forces between the derivatives and BSA. Synchronous fluorescence spectra suggested that HSOX and HSCP had little effect on the microenvironment and conformation of BSA in the binding reactions but the microenvironments around tyrosine residues were disturbed and polarity around tyrosine residues increased in the presence of HSOP. Copyright © 2012 John Wiley & Sons, Ltd.     

Chiral separation of 2,3-allenoic acid by capillary zone electrophoresis using cyclodextrin derivatives

In this paper, five of six samples of 2,3-allenoic acid enantiomers were separated by capillary zone electrophoresis (CZE) using hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) as chiral selectors. Using HP-beta-CD for chiral separation, three of the six enantiomers were separated. Five experimental conditions including HP-beta-CD concentration, pH, buffer concentration, temperature, and running voltage were investigated for their influence on separation and migration using enantiomers of 2-methyl-4-phenyl-2,3-butadienoic acid (A) and 2-(n-propyl)-4-phenyl-2,3-butadienoic acid (B) as samples. Good separation results were observed when [HP-beta-CD] = 3-12 mmol/L and pH = 7-9 for samples A and B. The temperature range of 15-25 degrees C can be selected for convenience. According to the chiral separation results, HP-beta-CD and HP-gamma-CD should be valuable selectors to separate 2,3-allenoic acids and HP-gamma-CD was suggested to separate the 2,3-allenoic acid samples with a group at 4-position bulkier than phenyl.     

Sensitive determination of gentiopicroside in medicine and bio‐fluids using luminol‐myoglobin chemiluminescence combined with flow injection technique

A novel chemiluminescence method for the determination of gentiopicroside is presented, which was based on the inhibitory effect of gentiopicroside on the chemiluminescence reaction between luminol and myoglobin in a flow‐injection system. The decrement of chemiluminescence intensity was linear with the logarithm of gentiopicroside concentration over the range from 10.0 pg mL^?1 to 500.0 ng mL^?1 (r² = 0.9992), with a detection limit of 3.0 pg mL^?1 (3σ). At a flow rate of 2.0 mL min^?1, a complete analytical process could be performed within 0.5 min, including sampling and washing, with a relative standard deviation of less than 3.0% (n = 5). The proposed procedure was applied successfully in the determination of gentiopicroside in pharmaceutical preparations, human urine and serum without any pretreatment procedure. The possible mechanism of the reaction was also discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

响应曲面法优化毛细管区带电泳分析rhEPO的方法

为了在采用国内常用的60cm毛细管柱检测的条件下,能够完全分离检测重组人促红细胞生成素(rhEPO)8个组分,运用响应面分析法(RSM)对《欧洲药典》中规定的rhEPO的毛细管区带电泳检测法所用缓冲液组成作细微调整,以达到pH值操作允许范围略加宽松,易于控制,又保证所有组分清晰分离的目的。数学分析得到的缓冲液组分为:12 mmol/L氯化钠,12 mmol/L麦黄酮,12 mmol/L醋酸钠,3.0 mmol/L腐胺;用此缓冲液,pH值可拓宽至5.2~5.8范围内。实例检测验证结果表明,采用60 cm毛细管柱,在此缓冲液条件下检测样品,pH值在5.2~5.8范围内,rhEPO的8个糖基化形式在38 min内实现了基线分离,各相邻峰间的分离度达1.63~3.29,柱效达每米1.63×105~3.23×105理论塔板数,峰形良好。本研究提供了一个可行、可靠、高效的rhEPO分析方法。