Determination of levodopa by capillary electrophoresis with chemiluminescence detection

A rapid and simple method using capillary electrophoresis (CE) with chemiluminescence (CL) detection was developed for the determination of levodopa. This method was based on enhance effect of levodopa on the CL reaction between luminol and potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) in alkaline aqueous solution. CL detection employed a lab-built reaction flow cell and a photon counter. The optimized conditions for the CL detection were 1.0 × 10⁻⁵ M luminol added to the CE running buffer and 5.0 × 10⁻⁵ M K₃[Fe(CN)₆] in 0.6 M NaOH solution introduced postcolumn. Under the optimal conditions, a linear range from 5.0 × 10⁻⁸ to 2.5 × 10⁻⁶ M (r = 9991), and a detection limit of 2.0 × 10⁻⁸ M (signal/noise = 3) for levodopa were achieved. The precision (R.S.D.) on peak area (at 5.0 × 10⁻⁷ M of levodopa, n = 11) was 4.1%. The applicability of the method for the analysis of pharmaceutical and human plasma samples was examined.     

Detection of paracetamol by capillary electrophoresis with chemiluminescence detection

Indirect detection of paracetamol was accomplished using a capillary electrophoresis-chemiluminescence (CE-CL) detection system, which was based on its inhibitory effect on a luminol-potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) CL reaction. Paracetamol migrated in the separation capillary, where it mixed with luminol included in the running buffer. The separation capillary outlet was inserted into the reaction capillary to reach the detection window. A four-way plexiglass joint held the separation capillary and the reaction capillary in place. K₃[Fe(CN)₆] solution was siphoned into a tee and flowed down to the detection window. CL was observed at the tip of the separation capillary outlet. The CL reaction of K₃[Fe(CN)₆] oxidized luminol was employed to provide the high and constant background. Since paracetamol inhibits the CL reaction, an inverted paracetamol peak can be detected, and the degree of CL suppression is proportional to the paracetamol concentration. Maximum CL signal was observed with an electrophoretic buffer of 30 mM sodium borate (pH 9.4) containing 0.5 mM luminol and an oxidizer solution of 0.8 mM K₃[Fe(CN)₆] in 100 mM NaOH solution. Under the optimal conditions, a linear range from 6.6 × 10⁻¹⁰ to 6.6 × 10⁻⁸ M (r = 0.9999), and a detection limit of 5.6 × 10⁻¹⁰ M (signal-to-noise ratio = 3) for paracetamol were achieved. The relative standard deviation (R.S.D.) of the peak area for 5.0 × 10⁻⁹ M of paracetamol (n = 11) was 2.9%. The applicability of the method for the analysis of pharmaceutical and biological samples was examined.     

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.     

Rapid determination of catecholamines in urine samples by nonaqueous microchip electrophoresis with LIF detection

A method was developed for the rapid separation of catecholamines by nonaqueous microchip electrophoresis (NAMCE) with LIF detection, A homemade pump‐free negative pressure sampling device was used for rapid bias‐free sampling in NAMCE, the injection time was 0.5 s and the electrophoresis separation conditions were optimized. Under the optimized conditions, the samples were separated completely in <1 min. The average migration times of the epinephrine (E), dopamine (DA), and norepinephrine (NE) were 34.26, 43.81, and 50.07 s, with an RSD of 1.05, 1.26, and 0.89% (n = 7), respectively. The linearity of the method ranged from 0.0125 to 2.0 mg/L for E and 0.025～4.0 mg/L for DA and NE, with correlation coefficients ranging between 0.9978 and 0.9986. The detection limits of E, DA, and NE were 2.5, 5.0, and 5.0 μg/L, respectively. The recoveries of E, DA, and NE in spiked urine samples were between 86 and 103%, with RSDs of 4.5～6.8% (n = 5). The proposed NAMCE with LIF detection combined with a pump‐free negative pressure sampling device is a simple, inexpensive, energy efficient, miniaturized system that can be successfully applied for the determination of catecholamines in urine samples.     

Determination of catecholamines by ion chromatography coupled to acidic potassium permanganate chemiluminescence detection

A simple,fast,sensitive,highly selective and eco-friendly analytical method for the determination of catecholamines in human urine by ion chromatography（IC） with chemiluminescence（CL） detection was described in this paper.Using 12 mmol/L H₂SO₄ without any organic additive as eluent,three catecholamines including epinephrine（EP）,norepinephrine（NE） and dopamine（DA） were well separated on a cation-exchange column.The CL detection was based on the reaction of analytes with acidic potassium permanganate in the presence of formaldehyde as an enhancer.The absence of methanol and acetonitrile in eluent made the proposed method more sensitive and eco-friendly.Under the optimal conditions,the linear range of the proposed method was in the range of 0.02-0.5μg/mL.The limit of detection（LOD） was in the range of 0.6 and 5.1μg/L.The relative standard deviations （RSD） for 0.1μg/mL mixed standard solution were in the range of 0.8-1.9%（n = 11）.The method has been applied to the determination of catecholamines in human urine successfully.Excellent spiked recoveries were achieved for catecholamines ranged from 91.2%to 112.7%.     

Determination of clenbuterol by capillary electrophoresis immunoassay with chemiluminescence detection

A competitive immunoassay for clenbuterol (CLB) based on capillary electrophoresis with chemiluminescence (CL) detection was established. The method was based on the competitive reaction of horseradish peroxidase (HRP)-labeled CLB (CLB-HRP) and free CLB with anti-CLB antiserum. The factors affecting the electrophoresis and CL detection were systematically investigated with HRP as a model sample. Under the optimal conditions, the tracer CLB-HRP and the immunoassay complex were separated, and the linear range and the detection limit (S/N = 3) for CLB were 5.0-40 nmol l⁻¹ and 1.2 nmol l⁻¹, respectively. The proposed method has been applied satisfactorily in the analysis of urine sample.     

Aptamer‐based detection and quantitative analysis of human immunoglobulin E in capillary electrophoresis with chemiluminescence detection

A novel aptamer‐based CE with chemiluminescence (CL) assay was developed for highly sensitive detection of human immunoglobulin E (IgE). The IgE aptamer was conjugated with gold nanoparticles (AuNPs) to form AuNPs‐aptamer that could specifically recognize the IgE to produce an AuNPs‐aptamer‐IgE complex. The mixture of the AuNPs‐aptamer‐IgE complex and the unbounded AuNPs‐aptamer could be effectively separated by CE and sensitively detected with luminol‐H₂O₂ CL system. By taking the advantage of the excellent catalytic behavior of AuNPs on luminol‐H₂O₂ CL system, the ultrasensitive detection of IgE was achieved. The detection limit of IgE is 7.6 fM (S/N = 3) with a linear range from 0.025 to 250 pM. Successful detection of IgE in human serum samples was demonstrated and the recoveries of 94.9–103.2% were obtained. The excellent assay features of the developed approach are its specificity, sensitivity, adaptability, and very small sample consumption. Our design provides a methodology model for determination of rare proteins in biological samples.     

A simple and sensitive CE method for the simultaneous determination of catecholamines in urine with in-column optical fiber light-emitting diode-induced fluorescence detection

A simple and sensitive method has been developed for simultaneous analysis of three catecholamines: dopamine (DA), epinephrine (EP) and norepinephrine (NE) in urine by capillary electrophoresis (CE) coupled with in-column fiber-optic light-emitting diode-induced fluorescence detection (ICFO-LED-IFD). Fluorescein isothiocyanate was used as the fluorescence tagged reagent for derivatization of DA, EP and NE. The CE conditions for separation of these catecholamines were systematically investigated. It was found that catecholamines could be more effectively separated by adding β-cyclodextin (β-CD) and acetonitrile (ACN) to a background electrolyte (BGE) of sodium borate. The migration times are 10.61, 10.83 and 11.14 min for DA, EP and NE, respectively and the catecholamines are completely separated within 11.5 min under the optimal condition of a BGE containing 10% v/v ACN, 20 mM β-CD and 20 mM sodium borate (pH 9.5), and an applied voltage of 13 kV. The relative standard deviations of migration time and peak area for these catecholamines are less than 0.16 and 2.0%, respectively. The limit of quantifications (LOQs) for DA, EP and NE are 3.5, 1.0 and 3.1 nM whereas the limit of detections (LODs) for DA, EP and NE are 1.0, 0.3 and 0.9 nM, respectively. Our proposed CE method provides low LOQ and LOD values. This CE-ICFO-LED-IFD methodology has been successfully applied to analyze catecholamines in human urine samples with good accuracy and satisfactory recovery.     

癌胚抗原毛细管电泳-化学发光均相免疫分析

设计了一种测定人血清中癌胚抗原的毛细管电泳-化学发光检测均相免疫分析新方法。采用四苯硼酸钠增强luminol-H2O2-HRP体系化学发光的原理,化学发光检测经毛细管电泳分离的,用辣根过氧化物酶(HRP)标记的癌胚抗体及免疫复合物。测定癌胚抗原的线性范围2.0~80.0μg/L(R=0.9921),检出限为0.1μg/L(绝对检出限为0.75 fg)。     

Capillary electrophoresis with contactless conductivity detection for uric acid determination in biological fluids

The suitability of capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) for the direct determination of uric acid in human plasma and urine was investigated. It was found that a careful optimization of the buffer composition and pH was necessary to achieve selective determination in the complex sample matrices. An electrolyte solution consisting of 10 mM 2-morpholinoethanesulfonic acid (MES), 10 mM histidine and 0.1 mM hexadecyltrimethylammonium bromide (CTAB), pH 6.0, was finally found suitable for use as running buffer for both sample matrices. The limit of detection (3 S/N) was determined as 3.3 μM. The linearity of the response was tested for the range between 10 and 500 μM and a correlation coefficient of 0.9996 was obtained. Intra- and inter-day variabilities were <10%. Quantitative analysis of urine and plasma samples showed a good correlation with the routine enzymatic method currently used at the University Hospital of Basel.     

Capillary electrophoresis microchip coupled with on-line chemiluminescence detection

In the present work, chemiluminescence detection was integrated with capillary electrophoresis microchip. The microchip was designed on the principle of flow-injection chemiluminescence system and capillary electrophoresis. It has three main channels, five reservoirs and a detection cell. As model samples, dopamine and catechol were separated and detected using a permanganate chemiluminescent system on the prepared microchip. The samples were electrokinetically injected into the double-T cross section, separated in the separation channel, and then oxidized by chemiluminescent reagent delivered by a home-made micropump to produce light in the detection cell. The electroosmotic flow could be smoothly coupled with the micropump flow. The detection limits for dopamine and catechol were 20.0 and 10.0 μM, respectively. Successful separation and detection of dopamine and catechol demonstrated the distinct advantages of integration of chemiluminescent detection on a microchip for rapid and sensitive analysis.     

癌胚抗原毛细管电泳-化学发光均相免疫分析

建立了一种测定人血清中癌胚抗原的毛细管电泳-化学发光检测的均相免疫分析新方法.采用四苯硼钠增强luminol-H2 O2-HRP体系化学发光的原理,化学发光检测经毛细管电泳分离的,用辣根过氧化物酶(HRP)标记的癌胚抗体及免疫复合物.测定癌胚抗原的线性范围2.0～80.0 μg/L(R=0.9921),检出限为0.1 μg/L(绝对检出限为0.75 fg).     

Capillary electrophoresis chemiluminescence assay of naphthol isomers in human urine and river water using Ni(IV) complex‐luminol system

A capillary electrophoresis method involving online indirect chemiluminescence (CL) detection was used to determine naphthol (NAP) isomers. The method was based on the quenching effect of 1‐ and 2‐NAP on a new CL reaction of luminol with Ni(IV) complex in an alkaline medium. Separation was conducted with a 25.0 mM sodium borate buffer containing 0.8 mmol/L luminol. Under optimized conditions, 1‐ and 2‐NAP were baseline separated and detected in less than 8 min. The limits of detection of 1‐ and 2‐NAP were 3.1 and 2.7 μg/L, respectively (S/N = 3), with a linear range of 4.0–80.0 μg/L (r > 0.995). Analysis of real samples demonstrated that the spiked recoveries were in the range of 89.2–107.5% (n = 3). The proposed method was successfully used to determine 1‐ and 2‐NAP contents in three environmental water samples and 14 human urine samples. No derivatization or tedious pretreatment was required in the analysis. The proposed method is a potential approach for routine tests of naphthol isomers in a facile CE–CL system.     

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%.     

Analysis of antioxidants using a capillary electrophoresis with chemiluminescence detection system

We developed a capillary electrophoresis with chemiluminescence detection system using 2-methyl-6-p-methoxyphenylethynylimidazopyrazinone as a chemiluminescence reagent for determination of antioxidants of superoxide anions. 2-Methyl-6-p-methoxyphenylethynylimidazopyrazinone reacted with superoxide anions generated through the reaction of hypoxanthine and xanthine oxidase, and then emitted chemiluminescence. Suppression of the chemiluminescence in the presence of antioxidants for superoxide anions was introduced as a detection principle for antioxidants into the capillary electrophoresis with chemiluminescence detection system. After optimizing the analytical conditions, various antioxidants, such as superoxide dismutase, nitroblue tetrazolium, ascorbic acid, and catechin, were subjected to the present system. They gave negative peaks due to the quenching effect; the detection limits of superoxide dismutase, nitroblue tetrazolium, ascorbic acid, and catechin were 1, 100, 100, and 10 μM, respectively (S/N = 2). A model sample consisting of superoxide dismutase and nitroblue tetrazolium was satisfactorily separated and detected within ca. 10 min. We also applied the present system to analysis of catechin in green tea as a real sample.     

A sensitive and rapid immunoassay for quantification of CA125 in human sera by capillary electrophoresis with enhanced chemiluminescence detection

In this paper we have presented a sensitive and rapid immunoassay (IA) method by capillary electrophoresis with an enhanced chemiluminescence detection system (CE-CL) based on the catalytic effects of horseradish peroxidase (HRP) on the luminol-hydrogen peroxide reaction. The conditions for the CL reaction and electrophoresis were systematically investigated using HRP as a model sample. The linear range from 2.5 x 10(-11) to 1.0 x 10(-9) mol/L (R = 0.999), and the detection limit of 1.0 x 10(-12) mol/L (signal-to-noise ratio = 3) for HRP were achieved using para-iodophenol as CL enhancer. The relative standard deviations of the migration time and peak area for 5.0 x 10(-10) mol/L HRP (n = 7) were 0.26 and 4.8%, respectively, using a CE system with a home-built CL detector. Under the optimal condition, the HRP-labeled CA125 antibody (Ab) and the Ab-antigen complex were well separated within 4 min by CE using a high-pH buffer (pH 10.20). The assay was successfully used for quantification of CA125 in human sera from health controls and patients associated with ovarian cancer, and the recoveries of the standard addition experiments were 93-109%. Our primary results demonstrated that IA based on CE-CL detection is a powerful tool for clinical diagnosis combined with these commercial IA kits.     

毛细管电泳化学发光在线检测

评论了毛细管区带电泳化学发光检测联用技术这一新兴的研究领域。化学发光检测具有背景低、热力学范围宽、灵敏度高的优点，适于毛细管电泳柱后微量样品的在线检测。论述了该检测器与毛细管电泳联用的接口和应用状况。     

On-line stacking and sweeping capillary electrophoresis for detecting heroin metabolites in human urine

Heroin metabolites including morphine, codeine, and 6-acetylmorphine were determined by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI–sweep-MEKC). Liquid–liquid extraction was used for urine pretreatment. An uncoated fused silica capillary (Ld = 30 cm, 50 μm ID) was filled with phosphate buffer (50 mM, pH 2.5) containing 30% methanol, then high conductivity buffer (100 mM phosphate, 41.3 kPa for 18 s) was followed. Samples were injected electrokinetically (20 kV, 300 s). The sweeping and separation were performed at −25 kV using phosphate buffer (20 mM, pH 2.5) and 80 mM sodium dodecyl sulfate. The baseline separation was done within 10 min. During method validation, the calibration curves were linear over a range of 50–500 ng/mL (r ≧ 0.994). The RSD and RE values in intra-day and inter-day assays were all below 20%, which showed good precision and accuracy. Their detection limits were 10 ng/mL (S/N = 3). The optimized method was applied to determine real urine samples from addicts. These samples were confirmed by liquid chromatography/mass spectrometry.     

Capillary electrophoresis chemiluminescent detection system equipped with a two-step postcolumn flow interface for detection of some enkephalin-related peptides labeled with acridinium ester

Despite its low equipment cost and simple design, as one of the sensitive detectors for CE, the chemiluminescence (CL) detector was less developed compared to the detectors of MS and LIF. The main reasons were the limitation of CL reagents, the repeatability problems and the relatively low sensitivity compared to LIF. In this paper, a highly sensitive CE-CL detection system was developed for detection of some enkephalin-related peptides labeled with acridinium ester. A new detection interface was designed for CE with CL detection of acridinium ester and its labeled analytes. The interface included two sections: one was used to acidify the capillary outflow so that the corresponding acridinium pseudo-base form can be changed into acridinium ester form by adding excess acid to the system; the other was designed to provide a suitable solution to produce the CL from acridinium ester. The effect factors, such as pH, the concentration of reaction reagents and the flow rates of the reagents, were investigated. The results showed that acridinium ester had similar CL properties in this interface when pH values of CE BGE were changed from 2.0 to 10.8. The interface was used to detect acridinium ester and three acridinium ester-labeled enkephalin-related peptides, the corresponding LODs were found to be in the attomole range. This CL detection system proved to be of high sensitivity, good repeatability, and relatively low cost.