小型化毛细管电泳-电化学检测法测定猪尿和猪饲料中的β-兴奋剂

采用小型化毛细管电泳-电化学检测技术测定了猪尿和猪饲料中的克伦特罗(clenbuterol,俗称瘦肉精)及其替代品莱克多巴胺(ractopamine)和沙丁胺醇(salbutamol)。考察了工作电极的氧化电位、运行缓冲液的酸度和浓度、分离电压和进样时间等因素对分离和检测的影响。以直径300 μm的碳圆盘电极为工作电极,检测电极电位为+0.95 V (vs SCE),在100 mmol/L的硼酸盐(pH 9.15)运行缓冲液中,上述3种组分在7 min内实现了较好的分离。被测物浓度与峰电流在3个数量级范围内呈良好的线性关系,检测限为1.20×10-7~2.06×10-7 g/mL。该方法简单可靠,已成功应用于猪尿和猪饲料样品中3种β-兴奋剂的测定,是一种有效的食品安全分析检测方法。     

Simultaneous determination of N,N-dimethylaniline and phenol in wastewater by high-performance liquid chromatography with chemiluminescence detection.

A method using HPLC-CL linkage was developed for simultaneous determination of N,N-dimethylaniline and phenol in wastewater, based on the strong sensitive chemiluminescence of the luminol-K3Fe(CN)6 systems in alkaline medium. The separation was carried out on a Hypersil ODS column with a mobile phase of ethanol-0.01% triethylamine (2:1, v/v). The linear ranges for N,N-dimethylaniline determinations were 2.0 x 10(-7) - 2.5 x 10(-5) g/mL and 4.0 x 10(-5) - 1.5 x 10(-4) g/mL with a detection limit (3sigma) of 1.20 x 10(-8) g/mL; the relative standard deviation (3sigma) for 5.0 x 10(-6) g/mL N,N-dimethylaniline was 1.4% (n = 6). The range for phenol was from 5.1 x 10(-7) to 1.3 x 10(-4) g/mL, and a detection limit (3sigma) of 2.5 x 10(-8) g/mL could be obtained. The method can be useful for the determination of N,N-dimethylaniline and phenol in some environmental samples.     

Determination of clozapine by capillary zone electrophoresis following end-column amperometric detection with simplified capillary/electrode alignment

Capillary zone electrophoresis was employed for the determination of clozapine using an end-column amperometric detection at a carbon fiber array microdisk electrode with simplified capillary/electrode alignment. The optimum conditions of separation and detection are: Britton-Robinson buffer, pH 2.0 (1.3 x 10(-2) mol/L total concentration of acids, 3.2 x 10(-3) mol/L NaOH), 15 kV for separation voltage, 5 kV and 10 s for injection voltage and injection time, respectively. The limit of detection is 4.2 x 10(-7) mol/L or 1.2 fmole (signal to noise, S/N = 2). The relative standard deviation is 1.4% for the migration time and 2.5% for the electrophoretic peak current. The method was applied to the determination of clozapine in human blood. The recovery of the method is between 94-104%.     

Analysis of quinolizidine alkaloids in Sophora flavescens Ait. by capillary electrophoresis with tris(2,2'-bipyridyl) ruthenium (II)-based electrochemiluminescence detection

A capillary electrophoresis method coupled with electrochemiluminescence detection for the analysis of quinolizidine alkaloids was established, especially, oxymatrine (OMT) which could not be measured by previous electrochemiluminescence methods was detected sensitively herein. Complete separation of sophoridine (SR), matrine (MT) and OMT was achieved within 13 min using a background electrolyte of 50mM phosphate buffer at pH 8.4 and a separation voltage of 15 kV. The calibration curves showed a linear range from 2.8 x 10(-8) to 4.4 x 10(-7) M for SR, 2.7 x 10(-8) to 4.4 x 10(-7) M for MT, and 2.5 x 10(-7) to 4.0 x 10(-6)M for OMT, respectively. The relative standard derivations for all analytes were below 3.1%. Good linear relationships were showed with correlation coefficients for all analytes exceeded 0.987. The detection limits were 1.0 nM for SR and MT, and 40 nM for OMT under the optimal conditions, respectively. The developed method was nearly harmless to the human and environment.     

Resonance Rayleigh scattering for detection of proteins in HPLC

An HPLC-resonance Rayleigh scattering (RRS) (HPLC-RRS) detection system is described for separation and detection of proteins. This system is based on the modification of a commercial HPLC instrument involving the addition of a pump and a T-shaped interface, and a common fluorescence detector was used for detection. The detection principle is based on the change of RRS intensity of the ion-association complex formed from biebrich scarlet (BS) and protein. The RRS signal was detected at lambdaex=lambdaem=376 nm. The utility of the presented method was demonstrated by the separation and determination of four proteins involving cytochrome (Cyt-c), lysozyme (Lys), HSA, and gamma-globulin (gamma-Glo). An LOD of 0.2-1.0 microg/mL was reached and a linear range was found between peak area and concentration in the range of 0.20-3.0 microg/mL for Cyt-c, 0.25-2.5 microg/mL for Lys, 1.5-10 microg/mL for HSA, and 2.0-15 microg/mL for gamma-Glo, with linear regression coefficients all above 0.99. The method presented has been applied to determine HSA and gamma-Glo in human serum samples synchronously.     

Determination of histamine by capillary zone electrophoresis with amperometric detection.

Capillary zone electrophoresis was employed for the determination of histamine using end-column amperometric detection with a carbon fiber microelectrode, at a constant potential. The optimum conditions of separation and detection were 10 mmol/L phosphate buffer, pH 5.6 for the buffer solution, 15 kV for the separation voltage, and 1.35 V (versus SCE) for the detection potential. The linear range was from 6.3 x 10(-7) to 1.5 x 1(-5) mol/L with the regression coefficient of 0.9997, and the detection limit was 4.0 x 10(-7) mol/L (S/N = 3). The proposed method was successfully applied to the direct determination of histamine in the beer samples without any sample clean-up procedures.     

Separation and determination of norepinephrine, epinephrine and isoprinaline enantiomers by capillary electrophoresis in pharmaceutical formulation and human serum

A capillary electrophoresis method with ultraviolet (UV) detection was developed and optimized for the enantiomer separation of norepinephrine (NE), epinephrine (EP) and isoprenaline (IP) using dual cyclodextrins (CDs) of 2-hydroxypropyl-beta-CD (HP-beta-CD) and heptakis (2,6-di-o-methyl)-beta-CD (DM-beta-CD) as chiral selectors. Optimal separation was obtained using a running buffer of 50mM phosphate containing 30mM HP-beta-CD and 5mM DM-beta-CD at pH 2.90 and a field strength of 20kV in 45cmx75mum (40cm effective length) uncoated capillary. The UV absorbance detection was set at 205nm. A 0.1% (w/w) polyethylene glycol or 0.1% (v/v) acetonitrile was used to enhance the detection sensitivity. There was a wide and excellent linear calibration graph for each enantiomer in the range 1.0x10(-3) to 1.0x10(-6)M and the detection limit (S/N=3) was found from 8.5x10(-7) to 9.5x10(-7)M. The method has been applied for the determination of isoprenaline in isoprenaline hydrochloride aerosol and to the analysis of serum samples. The recoveries of NE and EP in serum and IP in drug were ranged from 90 to 110%. The relative standard deviations of all the analyte peaks were less than 2.8% for migration time and less than 4.8% for peak area.     

Study of the separation and determination of monosaccharides in soluble coffee by capillary zone electrophoresis with electrochemical detection

A simple, fast and reliable method, based on capillary electrophoresis with electrochemical detection, for the separation and determination of six monosaccharides, namely glucose, galactose, arabinose, fructose, xylose and ribose, in soluble coffees was developed. A copper disk electrode was used as the working electrode. The optimum conditions for separation and detection were 50 mmol L-1 sodium hydroxide buffer (pH 12.7), separation voltage 5 kV and detection potential 0.65 V (vs. Ag/AgCl). The linear ranges were from 5.0 x 10(-3) to 0.5 mmol L-1 for all six sugars. All regression coefficients were > 0.99. The detection limits for all the sugars were 1.0 x 10(-3) mmol L-1. The RSD of the peak current was < 4.2% (n = 5). The proposed method was applied directly to the separation and determination of the six sugars without prior derivatization, and the assay results were satisfactory.     

Capillary electrophoresis of some tetracycline antibiotics coupled with reductive fast cyclic voltammetric detection.

The separation and quantitative performance parameters for tetracycline, chlortetracycline and oxytetracycline antibiotics were investigated by capillary zone electrophoresis coupled with fast cyclic voltammetric detection. Optimization of pH and complexation with a boric acid-sodium tetraborate buffer provided good resolution of all compounds. Detection by electrochemical reduction using fast on-line cyclic voltammetric detection with a Hg-film-microm electrode gave detection limits (2 x peak-to-peak baseline noise) of 7 x 10(-7) mol/l for tetracycline and chlortetracycline, and 1.5 x 10(-6) mol/l for oxytetracycline. The influence of electrode material, potential range and scan rate was examined and discussed. Optimal electrochemical detection was obtained at a Hg-film electrode with a waveform that consisted of an initial constant potential of -0.6 V for 200 ms followed by a cyclic voltammetry (CV) scan at 300 V/s from - 0.6 V to a vertex potential of 1.7 V. The analytical signal was obtained by plotting the integrated values of the CV current from each applied waveform as a function of time. The calibration plot (peak areas) for each separated peak was found to be linear over three-orders of magnitude.     

Post-column fluorescence derivatization of proteins and peptides in capillary electrophoresis with a sheath flow reactor and 488 nm argon ion laser excitation

We report the use of a sheath flow reactor for post-column fluorescence derivatization of proteins. The derivatization reaction employed naphthalene-2,3-dicarboxaldehyde (NDA) and beta-mercaptoethanol, which were added in the sheath buffer. The labeled proteins were detected by laser-induced fluorescence with an argon-ion laser beam at 488 nm. The performance of this detection scheme was evaluated by separation of some protein standards. A column efficiency of 450,000 plates/m was obtained without stacking. The limits of detection for those standard proteins were determined to be from 8 to 32 nM. Excellent linear relationship was obtained with correlation coefficient of 0.9998 for alpha-lactalbumin concentration ranging from 3.91 x 10(-7) to 1.25 x 10(-5) M. Separation of protein standards at low pH was also demonstrated by reversing the electroosmotic flow (EOF) with addition of cetyltrimethylammonium bromide (CTAB) to the running buffer. Different separation selectivity was achieved, but the sensitivity is poorer than that at high pH. This post-column derivatization detection system was applied successfully to analyze the protein extract from HT29 human colon cancer cells as well as tryptic peptides.     

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.     

End-column amperometric detection of melatonin by high performance capillary electrophoresis

A rapid and sensitive method is described for the detection of melatonin in rat pineal gland and melatonin tablets. Capillary electrophoresis (CE) with electrochemical detection (EC) was used. This method had high sensitivity and good reproducibility. The detection limit of melatonin was as low as 1.3x10(-9) mol/l (0.30 fg). There was a linear range of concentration from 3.9x10(-7) to 3.25x10(-5) mol/l with a correlation coefficient of 0.999. The linear equation was Y=8.27+0.0042X with a slope of 0.0042 pA/nM. The relative standard deviations of the peak current response and the migration time for 16 continuous injections of 6.5 muM melatonin were 4.5 and 0.48%, respectively. The proposed method was applied to detect melatonin in the rat pineal gland and the melatonin tablets. Good results were obtained compared with previous reports.     

Determination of puerarin, daidzein and rutin in Pueraria lobata (Wild.) Ohwi by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection was developed for the determination of puerarin, daidzein and rutin. Effects of several important factors such as the acidity and concentration of running buffer, separation voltage, injection time, and detection potential were investigated to acquire the optimum conditions. The working electrode was a 300-microm diameter carbon disc electrode positioned opposite the outlet of capillary. The three analytes could be well separated within 12 min in a 40 cm length capillary at a separation voltage of 9 kV in a 50 mmol/l borate buffer (pH 9.0). The relationship between peak currents and analyte concentrations was linear over about three orders of magnitude with detection limits (SIN=3) ranging from 0.241 x 10(-6) to 0.511 x 10(-6) mol/l for all compounds. This proposed method demonstrated long-term stability and reproducibility with relative standard deviations of less than 5% for both migration time and peak current (n=7). It has been successfully applied for the determination of puerarin, daidzein and rutin in Chinese traditional drugs, the vines of Pueraria lobata (Wild.) Ohwi and Puerariae Radix.     

A new method of quantification of pipemidic-acid by capillary zone electrophoresis with end-column amperometric detection

Capillary zone electrophoresis was employed for the determination of pipemidic acid using an end-column amperometric detection with a carbon fiber microdisk array electrode, at a constant potential of -1.10 V vs. saturated calomel electrode. The optimum conditions of separation and detection were 1.2 x 10(-4) mol/LNaOAc - 8.8 x 10(-4) mol/ LHOAc for the buffer solution, 20 kV for the separation voltage, 5 kV and 10 s for the injection voltage and the injection time. The limit of detection was 1.05 x 10(-7) mol/L or 189 amol (S/N=3). The relative standard deviation was 0.31% for the migration time and 2.0% for the electrophoretic peak current. The method was applied to determining pipemidic acid in human serum.     

Facile separation and determination of Aconitine alkaloids in traditional Chinese medicines by CE with tris(2,2'-bipyridyl) ruthenium(II)-based electrochemiluminescence detection

A facile CE method coupled with tris(2,2'-bipyridyl) ruthenium(II)-based electrochemiluminescence [Ru(bpy)(3) (2+)] detection was developed for simultaneous determination of Aconitum alkaloids, i.e., hypaconitine (HA), aconitine (AC), and mesaconitine (MA) in baseline separation. The optimal separation of these Aconitum alkaloids was achieved in a fused-silica capillary column (50 cm x 25 microm id) with 30 mM phosphate solution (pH 8.40) as running buffer at 12 kV applied voltage. The three alkaloids can be determined within 10 min by a single run. The calibration curves showed a linear range from 2.0 x 10(-7) to 2.0 x 10(-5) M for HA, 3.4 x 10(-7) to 1.7 x 10(-5) M for AC, and 3.8 x 10(-7) to 1.9 x 10(-5) M for MA. The RSDs for all analytes were below 3.01%. Good linear relationships were found with correlation coefficients for all analytes exceeding 0.993. The detection limits were 2.0 x 10(-8) M for HA, 1.7 x 10(-7) M for AC, and 1.9 x 10(-7) M for MA under optimal conditions. This method was successfully applied to determine the three alkaloids in Aconitum plants.     

Rapid and ultrasensitive determination of ephedrine and pseudoephedrine derivatizated with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein by micellar electrokinetic chromatography with laser-induced fluorescence detection

This paper presents a micellar electrokinetic chromatography method with laser-induced fluorescence detection to analyze ephedrine (E) and pseudoephedrine (PE) after derivatizated with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein. The optimum derivatization conditions were: 0.05 M Na2CO(3/NaHCO3 (pH 9.5), reaction time 30 min at 45 degrees C, molar ratio of DTAF to E and PE mixture 20:1. The baseline separation was achieved within 8 min with running buffer composed of 20 mM borate+20 mM SDS+15% acetonitrile (v/v) (adjusted pH 9.8), and applied voltage of 20 kV. Good linearity relationships (correlation coefficients: 0.9906 for E and 0.9941 for PE) between the peak heights and concentration of the analytes were obtained (2.5-50 ngmL(-1)). The detection limits for E and PE were 3.85 x 10(-4) and 1.41 x 10(-4)ngmL(-1), respectively, which indicated that the proposed method surpassed other chromatographic alternatives in terms of limit of detection by at least 10(3) folds. The method was applied to the analysis of the two alkaloids in ephedra herb plants and its preparations with recoveries in the range of 89.6-107.0%.     

Determination of levofloxacin in human urine by adsorptive square-wave anodic stripping voltammetry on a glassy carbon electrode

The adsorption behavior of levofloxacin on a glassy carbon electrode was explored by cyclic and square-wave voltammetry. The drug was accumulated on a glassy carbon electrode and a well-defined oxidation peak was obtained in acetate buffer pH 5.0. Using square-wave anodic stripping voltammetry and accumulation at +0.4 V versus Ag/AgCl (saturated KCl) for 300 s, linear calibration graph was obtained from 6.0x10(-9) to 5.0x10(-7) M levofloxacin. The detection limit was calculated to be 5.0x10(-9) M. The R.S.D. determined from ten determinations at the 1.0x10(-7) M level was 1.7%. The method was applied for the direct determination of levofloxacin in diluted urine samples. It was validated using high-performance liquid chromatography (HPLC) as a reference method.     

Attomole sensitivity for unlabeled proteins and polypeptides with on-chip capillary electrophoresis and universal detection by interferometric backscatter

A universal detector based on backscatter interferometry has been developed to perform nanoliter volume refractive index measurements for on-chip sodium dodecyl sulfate (SDS) gel based (polyethylene oxide gel) separations and quantification label-free proteins. The on-chip interferometric backscatter detector (OCIBD) system consists of a simple, folded optical train based on the interaction of a laser beam with an etched channel in the shape of half cylinder in a fused-silica plate. The backscattered light from the channel takes on the form of a high-contrast interference pattern that contains information related to the bulk properties of the fluid located within the probe or detection volume of 2.32 x 10(-9) L. Depending on capillary electrophoresis (CE) injection method, the positional changes of the interference pattern extrema (fringes) allow for the quantification of unlabeled proteins at levels ranging from 11 to 310 amol (2.7 x 10(-8)mol/L) with a linear dynamic range of 2.5 decades (egg albumin). Using OCIBD microchannel-based SDS capillary gel electrophoresis (SDS/CGE), separation and detection of five label-free proteins was achieved in less than 100 seconds with detection limits ranging from 0.95 pg (1.1 x 10(-16)mol or 2.5 x 10(-7)mol/L) of calmodulin to 7.0 pg (1.0 x 10(-16)mol or 2.4 x 10(-7)mol/L) for bovine serum albumin (BSA) without signal filtering or active thermal control. This development shows that a universal detector based on backscatter interferometry can be used effectively for on-chip label-free solute analysis.     

Determination of diclofenac sodium by capillary zone electrophoresis with electrochemical detection

Capillary zone electrophoresis was employed for the determination of diclofenac sodium using an end-column amperometric detection with a carbon fiber microelectrode, at a constant potential of 0.83 V vs. saturated calomel electrode. The optimum conditions of separation and detection are 4.90 x 10(-3) mol/l Na2HPO4-3.10 x 10(-3) mol/l NaH2PO4 (pH 7.0) for the buffer solution, 10 kV for the separation voltage, 5 kV and 10 s for the injection voltage and the injection time, respectively. The limit of detection is 2.5 x 10(-6) mol/l or 5.2 fmol (S/N=2). The relative standard deviation is 0.8% for the migration time and 4.7% for the electrophoretic peak current. The method was applied to the determination of diclofenac sodium in human urine.     

Capillary electrophoresis with end-column electrochemiluminescence for the analysis of chloroquine phosphate and the study on its interaction with human serum albumin

This paper describes a novel and sensitive method for the estimation of chloroquine phosphate (CQ) using capillary electrophoresis with end-column electrochemiluminescence (ECL) detection. Under the optimized condition, linear calibration curve was obtained for the system over two orders of magnitude with a detection limit of 3x10(-7) M (S/N=3). The relative standard deviations of the ECL intensity and the migration time were 1.4% and 0.05%, respectively (n=6, 5x10(-5) M CQ). Successful separation of chloroquine phosphate, difenidol hydrochloride and clomifene citrate was obtained at pH 7.0. Using the proposed electrochemiluminescence system, a simple method was proposed to study the interaction between chloroquine phosphate and human serum albumin (HSA), and the number of binding sites and binding constant were estimated to be 32.6 and 7.7x10(3) M(-1), respectively.