Simultaneous analysis of six cardiovascular drugs by capillary electrophoresis coupled with electrochemical and electrochemiluminescence detection,using a chemometrical optimization approach

CE coupled with dual electrochemical (EC) and electrochemiluminescence (ECL) detection was optimized for simultaneous analysis of six cardiovascular drugs (alprenolol, propafenone, acebutolol, verapamil, atenolol and metoprolol) via central composite design. Following this study, three critical electrophoretic factors governing the CE separation were investigated: Tris‐H₃PO₄ buffer concentration, buffer pH value and separation voltage. A modified chromatographic response was adopted for evaluating CE separation quality. Optimum conditions were achieved using Tris‐H₃PO₄ buffer 35.6 mM (pH 2.3) separated at 13.9 kV, which was employed experimentally and led to the successful simultaneous separation of the above six drugs. The good agreement of the chromatographic response was observed between predicted data and actual experimental results using these optimized conditions (RSD=3.75%). The proposed method was validated for linearity, repeatability and sensitivity, and subsequently successfully applied to determine six basic drugs in urine samples.     

Analysis of amphetamines in urine with liquid-liquid extraction by capillary electrophoresis with simultaneous electrochemical and electrochemiluminescence detection

Amphetamines including methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine were separated and detected by CE using simultaneous electrochemical (EC) and electrochemiluminescence (ECL) detection (CE-EC/ECL). Factors that influenced the separation and detection performance, such as the detection potential, the pH value and concentration of the running buffer, the separation voltage and the pH of the detection buffer, were investigated. LODs of 3.3x10(-8) mol/L (0.16 fmol), 1.6x10(-7) mol/L (0.78 fmol) and 3.3x10(-8) mol/L (0.16 fmol) were obtained for methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine, respectively. For practical application, a liquid-liquid extraction with ethyl acetate procedure was developed for urine sample pretreatment and extraction efficiencies higher than 90% were obtained. The established simultaneous CE-EC/ECL was successfully applied for urine sample analysis.     

毛细管电泳-电致化学发光法测定兔血浆中的羟考酮

基于稀土Eu(Ⅲ)掺杂的类普鲁士蓝膜修饰的铂电极为工作电极,建立了测定羟考酮的毛细管电泳-电致化学发光分析方法。考察了检测电位、运行缓冲溶液的酸度及浓度、分离电压、进样条件等对电泳分离效果及检测灵敏度的影响。在最佳的实验条件下,羟考酮可在4 min内得到分离,其ECL强度值与羟考酮的质量浓度在7.0×10-2～7.0μg/mL和7.0～70.0μg/mL范围内呈良好的线性关系,检出限为4.2×10-2μg/mL(3σ),峰高和迁移时间的相对偏差分别为3.6%和0.48%(n=6)。方法用于兔血浆中羟考酮含量的检测,加标回收率在99.7%～101.0%之间。     

Application of response surface methodologies in capillary electrophoresis

A fast method of determining ascorbic acid and isoascorbic acid by capillary zone electrophoresis with a photodiode array detector was developed. Response surface methodologies based on three-level, three-variable designs, such as the Box-Behnken design, central composite face-centered and full fractional design, were used comparatively for optimization of buffer pH, buffer concentration and operation voltage. Statistical interpretation of the variables concerning different responses, such as resolution and migration time of the last migrated analyte, were performed. The optimum conditions of these variables were predicted using a second-order polynomial model fitted to the results obtained by applying three designs. The response surface plots using three experimental designs revealed a separation optimum with Tris–HCl buffer of pH 8.5, a concentration of 50 mM, and an operation voltage of 30 kV. The significance of the statistical designs were confirmed by the generally good agreement obtained between predicted responses and actual experimental data. We concluded that experimental designs offer a rapid means of optimizing several variables and provide an efficient test for the robustness of the analytical method.     

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 cmx25 micro m (ID) filled with acidic phosphate buffer (200 mmol/L phosphate, pH 2.0) - 1mol/L phosphoric acid (9:1 v/v) and a separation voltage of 5 kV (25 micro A), 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 1.9x10(-7) mol/L and 7.6x10(-9) mol/L for Spd and Spm, respectively. Intraday and interday relative standard deviations of ECL peak intensities are less than 8%. The main advantages of this CE-ECL detection technique for polyamines analysis presented herein are the omission of chemical derivatization of the analytes and the high selectivity.     

Application of Central Composite Design for the Determination of Exfoliating Agents in Cosmetics by Capillary Electrophoresis with Electroosmotic Flow Modulation

Multivariate analysis within central composite design is applied to simplify an optimization procedure and explore the interactions among experimental parameters in analytical chemistry. In this study, central composite design was used to identify the optimal capillary electrophoresis conditions with electroosmotic flow modulation to determine seven exfoliating agents in cosmetics. The influence of phosphate concentration, cetyltrimethylammonium bromide concentration, and methanol percentage on the response was evaluated by the use of the chromatographic exponential function to simultaneously investigate the resolution and separation under sixteen sets of capillary electrophoresis conditions. The optimized conditions were 150 mM phosphate solution (pH = 7) containing 0.5 mM cetyltrimethylammonium bromide, 3 mM γ-cyclodextrin, and 25% methanol as the running buffer. To shorten the analysis time, an electroosmotic flow modulating agent (cetyltrimethylammonium bromide) was added to the separation buffer. Calibration plots were linear (r ≧ 0.998) with high precision and accuracy in the homemade cosmetic matrix. The exfoliating agents in two commercial cosmetic products were determined using the optimized conditions, and the results correlated well with results obtained by high-performance liquid chromatography–mass spectrometry.     

Determination of hydroxy acids in cosmetics by chemometric experimental design and cyclodextrin‐modified capillary electrophoresis

A CD‐modified CE method was established for quantitative determination of seven hydroxy acids in cosmetic products. This method involved chemometric experimental design aspects, including fractional factorial design and central composite design. Chemometric experimental design was used to enhance the method's separation capability and to explore the interactions between parameters. Compared to the traditional investigation that uses multiple parameters, the method that used chemometric experimental design was less time‐consuming and lower in cost. In this study, the influences of three experimental variables (phosphate concentration, surfactant concentration, and methanol percentage) on the experimental response were investigated by applying a chromatographic resolution statistic function. The optimized conditions were as follows: a running buffer of 150 mM phosphate solution (pH 7) containing 0.5 mM CTAB, 3 mM γ‐CD, and 25% methanol; 20 s sample injection at 0.5 psi; a separation voltage of ?15 kV; temperature was set at 25°C; and UV detection at 200 nm. The seven hydroxy acids were well separated in less than 10 min. The LOD (S/N = 3) was 625 nM for both salicylic acid and mandelic acid. The correlation coefficient of the regression curve was greater than 0.998. The RSD and relative error values were all less than 9.21%. After optimization and validation, this simple and rapid analysis method was considered to be established and was successfully applied to several commercial cosmetic products.     

Multivariate approach for the enantioselective analysis in MEKC‐MS: II. Optimization of 1,1′‐binaphthyl‐2,2′‐diamine in positive ion mode

Enantiomeric separation and detection of 1,1′‐binaphthyl‐2,2′‐diamine (BNA) has been successfully optimized by MEKC‐ESI‐MS using a polymeric surfactant polysodium N‐undecenoxycarbonyl‐L‐leucinate (poly‐L‐SUCL) as a pseudostationary phase. In the first step, MEKC conditions were optimized by a five‐factor three‐level central composite design (CCD) of experiment. All five MEKC factors (buffer pH, percentage of ACN in the running buffer, concentration of surfactant, concentration of ammonium acetate (NH₄OAc), and voltage) were found significant to the responses (measured as the chiral resolution and analysis time). The interactions between MEKC factors were further evaluated using a quadratic model equation which allowed the generation of 3‐D response surface image to reach the optimum conditions. To obtain the best S/N, sheath liquid composition and spray chamber parameters were successfully optimized using the same strategy. Baseline enantiomeric resolution in less than 20 min and optimum MS signal of BNA enantiomers (S/N = 45 at 0.4 mg/mL) were ultimately achieved at the optimized conditions. The adequacy of the model was validated by experimental runs at the optimal predicted conditions. The predicted results were found to be in good agreement with the experimental data.     

Determination of chlorpheniramine and its binding with human serum albumin by capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection.

Tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence (ECL) detection in a capillary electrophoresis separation system was used for the determination of chlorpheniramine (CPM). The experimental conditions, such as the applied potential, separation voltage, injection voltage, injection time and the pH of the separation buffer were considered in detail. The ECL intensity showed two linear responses to CPM, i.e., from 15 microM to 1 mM and from 0.8 microM to 15 microM with a detection limit of 0.5 microM. The binding of CPM with human serum albumin was also monitored using this method and the binding constant was estimated to be 4.1 x 103 M(-1).     

卤代乙酸及其结构相近化合物的高效毛细管电泳分离

氟、氯、溴等卤代乙酸是结构非常相近的离子型化合物,对它们的分离测定比较困难。用高效毛细管电泳法在碱性或酸性缓冲液条件下可将它们分离。在酸性缓冲液条件下,可提高有机酸分离的选择性。较低的操作电压有利于提高阴离子的分离度,而改变温度对分离度的影响不大。     

毛细管电泳电致化学发光检测血浆中布比卡因

布比卡因是一种外科局部麻醉剂,使用过量会导致中枢神经系统和心脏血管系统中毒^[1],可引起心脏停博.高效液相色谱和毛细管电泳(CE)^[2]是该药常用的检测方法.     

Enantioseparation of atropine by capillary electrophoresis using sulfated beta-cyclodextrin: application to a plant extract

A capillary zone electrophoresis (CZE) method, with sulfated beta-CD as chiral selector, was optimized by means of an experimental design for the enantioseparation of atropine. In this study, a central composite design was used and the following factors were varied simultaneously: buffer concentration, buffer pH and sulfated beta-CD concentration. The resolutions between littorine and its positional isomer ((-)-hyoscyamine) and between atropine enantiomers, as well as the separation time and generated current were established as responses. A model was obtained for each response by linear multiple regression of a second-degree mathematical expression. The most favorable conditions were determined by maximizing the resolution between atropine enantiomers and by setting the other responses at threshold values. Successful results were obtained with a 55 mM phosphate buffer at pH 7 in the presence of 2.9 mM sulfated-beta-CD at 20 degrees C and 20 kV. Under these optimized conditions, a baseline separation of littorine and atropine enantiomers was achieved in less than 5 min. Finally, the method allowed the enantiomeric separation of atropine in a pharmaceutical formulation and was also found to be suitable for the enantiomeric purity evaluation of (-)-hyoscyamine in plant extracts, in relation with the extraction procedure. It was demonstrated that supercritical fluid extraction induced less racemization than classical liquid-solid extraction procedures.     

Experimental design optimization of the capillary electrophoresis separation of leucine enkephalin and its immune complex

To optimize the capillary electrophoretic separation conditions for leucine enkephalin (LE) and the immune complex of the LE and anti-LE reaction, an analysis using a three-level, three-factorial Box-Behnken design was performed. Three separation parameters, buffer pH (X(1)), buffer concentration (X(2)), and applied voltage (X(3)), were chosen to observe the effect on separation responses. The responses were theoretical plate number, migration time of the LE peak, and resolution between the peaks. The optimum conditions and process validation were determined using statistical regression analysis and surface plot diagrams. The capillary electrophoresis optimum separation conditions were established to be 75 mM phosphate buffer at pH 7.00 with an applied separation voltage of 15 kV. By using the analysis technique, the prediction of responses was satisfactory and process verification yielded values within the +/-5% range of the predicted efficiency.     

Experimental design optimization of a capillary zone electrophoresis method for the screening of several diuretics and ACE inhibitors

Experimental design methodologies are applied to the development of a capillary zone electrophoretic method for the separation of the angiotensin-converting enzyme inhibitor enalapril and its derivative enalaprilat and the diuretics xipamide and hydrochlorothiazide. The effects of pH, buffer concentration, proportion of boric acid in the mixed boric acid-potassium dihydrogen phosphate background electrolyte, temperature, applied voltage, and percentage of organic modifier are studied. Critical factors are identified in a screening design (a 2(6-2) fractional factorial design), and afterwards, optimal conditions for the separation are reached by means of an optimization design (a 2(2) + 2 x 2 + k central composite design). The studied response is the resolution between peaks. The four studied compounds can be separated in less than 3.5 min using an electrolyte of 20mM boric acid-potassium dihydrogen phosphate (75:25, v/v) with 5% MeOH adjusted to pH 8.0 with KOH, at a potential of 30 kV. The detection wavelength and temperature are 206 nm and 35 degrees C, respectively.     

Enhanced separation of seven quinolones by capillary electrophoresis with silica nanoparticles as additive

This paper describes the enhanced separation of lomefloxacin, sparfloxacin, fleroxacin, norfloxacin, ofloxacin, gatifloxacin and pazufloxacin by capillary zone electrophoresis (CZE) using silica nanoparticles (SiNPs) as running buffer additive. The impact of SiNPs concentration on the resolution and selectivity of separation was investigated and a given value of SiNPs was finally chosen under the optimum conditions. The addition of the SiNPs to the running buffer enabled electroosmotic flow (EOF) decrease and permitted full interaction between SiNPs and analytes. The influence of separation voltage, pH and buffer concentration on the separation in the presence of SiNPs was examined. Interactions between drugs and nanoparticles during the separation are discussed; the determination of interaction constants is also achieved. A good resolution of seven quinolones was obtained within 15 min in a 50 cm effective length fused-silica capillary at a separation voltage of +10 kV in a 12 mM disodium tetraborate-phosphate buffer (pH 9.08) containing 5.2 μg mL⁻¹ SiNPs.     

Determination of galanthamine in Bulbus Lycoridis Radiatae by coupling capillary electrophoresis with end‐column electrochemiluminescence detection

A novel method for the determination of galanthamine (GAL) in Bulbus Lycoridis Radiatae has been developed based on coupling CE with an end‐column tris(2,2′‐bipyridyl)ruthenium(II) electrochemiluminescence (ECL). Parameters affecting CE separation and ECL detection were investigated and optimized. Baseline separation of GAL from other components in the Bulbus Lycoridis Radiatae sample was achieved with an 18 mmol/L phosphate running buffer at pH 9.0. Under the optimized conditions: 12 kV CE‐separation voltage, ECL detection potential at 1.25 V with 5 mmol/L and 50 mmol/L phosphate buffer at pH 7.5 in the detection reservoir, the linear range of GAL concentration was from 0.8 ng/mL to 2 μg/mL, whereas the detection limit was 0.25 ng/mL (S/N=3). The proposed method was successfully demonstrated for the determination of GAL in Bulbus Lycoridis Radiatae.     

毛细管电泳-电化学发光法检测泛昔洛韦

建立了毛细管电泳-电化学发光(CE-ECL)法检测泛昔洛韦的新方法。考察了检测电位、运行高压、进样电压与时间、检测池中磷酸盐的pH值、运行缓冲溶液的pH值及浓度等测试条件对电化学发光强度的影响。在最优化的实验条件下,泛昔洛韦在5.0×10-6～2.5×10-4mol/L浓度范围内与电化学发光强度有良好的线性关系,相关系数为0.9973,检出限为3.5×10-6mol/L。该法灵敏度高,选择性好,可应用于泛昔洛韦原料药及制剂的质量控制。初步探讨了CE-ELC检测泛昔洛韦的机理。     

Application of capillary zone electrophoresis to the screening of some angiotensin II receptor antagonists

A capillary zone electrophoretic (CZE) method was optimized for the separation of five angiotensin II receptor antagonists (Losartan, Irbesartan, Valsartan, Telmisartan and Eprosartan) and two of their metabolites (EXP 3174 and Candesartan M1) by means of experimental design methodologies. The aim of this study was to define rapidly experimental conditions under which the analytes can be resolved for quantitation. The effects of the buffer (pH, concentration and composition), the organic modifier and voltage were studied. Critical factors were identified in a screening design (fractional factorial design) and sequentially an optimization design (central composite design) was used to choose optimal conditions for separation. The most favorable electrophoretic conditions were found by setting the resolution at a threshold value (Rs < or = 1.5) and minimizing, if possible, analysis time. Successful results were obtained with a 50 mM potassium dihydrogen phosphate:boric acid (25:75 v/v) buffer at pH 5.5 in the presence of 5% methanol and application of a 25 kV voltage. Analysis time was 8 min in a conventional fused-silica capillary (50 cm effective length) in a normal cationic mode (anode at the inlet and cathode at the outlet) after hydrostatical sample injection for 30 s.     

Quality by design-based capillary electrophoresis method development for the quantitative analysis of four iridoid compounds in Gentiana macrophylla Radix

In this study, the capillary electrophoresis-photodiode array detector was employed for the analysis of four iridoid compounds in Gentiana macrophylla Radix (RGM), and the method was optimized based on the concept of analytical quality by design (AQbD). The peak areas relative standard deviation (n = 3) and resolution of the four analytes were selected as critical method attributes. Fractional factorial design test combined with Pareto analysis were employed to screen critical method parameters (buffer concentration, pH, sodium dodecyl sulfate [SDS] micelle concentration, temperature, and voltage). Subsequently, three main factors (buffer concentration, buffer pH, and SDS concentration) were selected by central composite design test for constructing the design space. The optimal separation conditions as follows: capillary column (50.2 cm × 50 µm, detection length 40 cm). Working background electrolyte consisted of 51 mmol/L borax solution (pH = 9.47) and 40 mmol/L SDS. The samples were injected by pressure (5 s at 0.5 psi) and the detection was performed at 254 nm. Applied voltage was 20 kV and column temperature was 23°C. The developed method is rapid and reliable for the quantitative analysis of four iridoid compounds in RGM, providing a reference for the application of AQbD concept in the analysis of natural products.     

Investigation of chondroitin sulfate D and chondroitin sulfate E as novel chiral selectors in capillary electrophoresis

Various chiral selectors have been utilized successfully in capillary electrophoresis (CE); however, the number of polysaccharides used as chiral selectors is still small and the mechanism of enantiorecognition has not been fully elucidated. Chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE), belonging to the group of glycosaminoglycans, are linear, sulfated polysaccharides with large mass. In this paper, they were investigated for the first time for their potential as chiral selectors by CE. The effect of buffer composition and pH, chiral selector concentration, and applied voltage were systematically examined and optimized. A variety of drug enantiomers were resolved in the buffer pH range of 2.8–3.4 using 20 mM Tris/H₃PO₄ buffer with 5.0 % CSD or CSE and 20 kV applied voltage. A central composite design was used to validate the optimized separation parameters and satisfactory uniformity was obtained. As observed, CSE allowed satisfactory separation of the enantiomers of amlodipine, laudanosine, nefopam, sulconazole, and tryptophan methyl ester, as well as partial resolution of citalopram, duloxetine, and propranolol under the optimized conditions. CSD allowed partial or nearly baseline separation of amlodipine, laudanosine, nefopam, and sulconazole. The results indicated that CSE has a better enantiorecognition capability than CSD toward the tested drugs.