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.     

Evaluation of the synergistic effect with amino acids for enantioseparation of basic drugs using capillary electrophoresis

The synergistic effect of two acidic amino acids, aspartic and glutamic acid, on the electrophoretic enantioseparation of four basic drugs was evaluated in the BGE containing a CD and at different pHs. Chlorpheniramine, hydroxyzine, propranolol and tramadol were used as the basic model drugs. However, no enantioseparations were achieved with a BGE containing sole amino acid, but the combined use of an acidic amino acid and a CD showed improved enantioseparations (synergistic effect) compared with the single CD system. The results demonstrated that at optimized pH, the electrostatic interactions of the anionic amino acids with the positively charged basic drugs could result in a decrease of the analyte migration velocity and it consequently improved the enantioseparation. The effective parameters such as the amino acid and chiral selector type and concentration, buffer pH, applied voltage, and capillary temperature were optimized. Favorable enantiomeric resolution and migration times of the model drugs were achieved with a 100 mM phosphate buffer solution (pH 3.0) containing 5.0 mM HP‐α‐CD/HP‐β‐CD and 20 mM aspartic acid with an 18 kV applied voltage at 25°C. ¹H NMR experiments were also carried out in a mixture of an analyte and CD in the absence and presence of aspartic acid. The NMR results were consistent with the results obtained by CE which showed the synergistic effect of amino acid.     

Application of capillary electrophoresis technique for the enantioseparation of bioactive ferrocene-based compounds versus DFT calculated data

Herein, a series of bioactive ferrocene-modified N-heterocycles with alkyl linkers was prepared in good to quantitative yields starting from easy accessible ferrocene alcohols and heterocycles under acidic or neutral (for imidazole) conditions in racemic forms. The analytical resolution of a number of bioactive racemic ferrocene azoles 1 – 6 (where azole = imidazole, pyrazole, and benzotriazole derivatives) into enantiomers was first carried out by CE using sulfobuthylether-β-CD (captisol) as a chiral selector. The analytical approaches to highly enantiomeric-enriched ferrocene derivatives are based on the formation of their inclusion complexes. The best chiral separation was achieved using zone CE in a quartz capillary. The ACE was used to evaluate the stability constants of captisol complexes with enantiomeric forms of two ferrocene derivatives 1 , FcCHMe-imidazole, and 6 , FcCHMe-benzotriazole. The optimal conditions for the resolution of the studied (R, S)-ferrocene compounds 1 , 2 , and 6 were predicted on the basis of the performed quantum chemical calculations and then implemented by the electrophoretic method. A high correlation between density functional theory calculation results and experimental electrophoresis data were obtained. Successful enantioseparation of racemic mixtures is of great importance for the characterization and further applications of drug candidates in enantiopure forms and in the development of clinical treatment. The advantages of the CE procedure make it possible to have important practical value and significance for determining the purity and enantiomeric excess of other ferrocene-containing compounds.     

Optimization using central composite design for antihistamines separation by nonaqueous capillary electrophoresis with electrochemical and electrochemiluminescence detections

The first detailed examination of non-aqueous capillary electrophoresis with electrochemical and electrochemiluminescence detections (NACE-EC/ECL), separation parameters and their interactions via central composite design was presented. This concept was demonstrated by examining the optimization separation conditions of seven antihistamines (chlorpheniramine, cyproheptadine, diphenhydramine, doxylamine, methapyrilene, terfenadine, and triprolidine) by NACE-EC/ECL. To evaluate the NACE separation quality, the chromatography resolution statistic function (CRS(-1) function) with regard to the resolution and migration time was established as the response variable. The influences of three experimental variables (buffer apparent pH value (pH*), buffer (TBAP) concentration, and separation voltage) on the response were investigated. A set of optimal conditions was obtained from central composite design: 9.2 mM TBAP in ACN (pH* 4.0) and voltage (12.7 kV), and under these optimum conditions, the seven antihistamines could be well separated in less than 10 min. The obtained electropherograms indicated that the dual EC/ECL detection system was indispensable since the six antihistamines (except for triprolidine) displayed both EC and ECL response, whereas triprolidine only displayed the EC response. This work is instructive for investigators in simplifying the NACE-EC/ECL development procedures for multi-component analysis.     

Study on clarithromycin lactobionate based dual selector systems for the enantioseparation of basic drugs in capillary electrophoresis

In this paper, the use of clarithromycin lactobionate, a kind of antibiotic chiral selector, in combination with four neutral cyclodextrin derivatives (glucose‐β‐cyclodextrin, hydroxyethyl‐β‐cyclodextrin, methyl‐β‐cyclodextrin and hydroxypropyl‐β‐cyclodextrin) was reported for the first time. As a result, these dual systems gave much better resolution of nefopam (the Rs increased to 3.58, 2.72, 1.49 and 1.42, respectively) compared to the single systems. The effects of buffer pH and selector concentration on the separation of nefopam were also investigated. Additionally, some other basic drugs including metoprolol, atenolol, propranolol, bisoprolol, esmolol and ritodrine were tested for the investigation and evaluation of the enantiorecognition capability of the four dual systems. As expected, the synergistic effect was observed in four systems. Different results of these dual systems were also summarized.     

Determination of nicotine and its metabolite cotinine in urine and cigarette samples by capillary electrophoresis coupled with electrochemiluminescence

In this paper, CE coupled with electrochemiluminesence (ECL) detection using a 76‐μm Pt disk as working electrode was developed for nicotine (NIC) determination. The major metabolite of NIC is cotinine (COT), which has a similar tertiary amine structure to NIC. However, there is a carbonyl group attached in the structure of COT, which leads to the great decrease in ECL response. In order to improve the ECL response of COT, NaBH₄ was used for carbonyl reduction. After reduction, NIC and COT were separated and detected by CE‐ECL. ECL response plotted with NIC concentration was linear between 5.0×10^?7 and 5.0×10^?5 mol/L (81–8100 μg/L), with LOD of 5.0×10^?8 mol/L (8.1 μg/L). The developed CE‐ECL method was applied for NIC determination in urine and cigarette samples.     

In‐capillary detection of fast antibody‐peptide binding using fluorescence coupled capillary electrophoresis

Herein, we report a technique for detecting the fast binding of antibody‐peptide inside a capillary. Anti‐HA was mixed and interacted with FAM‐labeled HA tag (FAM‐E₄) inside the capillary. Fluorescence coupled capillary electrophoresis (CE‐FL) was employed to measure and record the binding process. The efficiency of the antibody‐peptide binding on in‐capillary assays was found to be affected by the molar ratio. Furthermore, the stability of anti‐HA‐FAM‐E₄ complex was investigated as well. The results indicated that E₄YPYDVPDYA (E₄) or TAMRA‐E₄YPYDVPDYA (TAMRA‐E₄) had the same binding priorities with anti‐HA. The addition of excess E₄ or TAMRA‐E₄ could lead to partial dissociation of the complex and take a two‐step mechanism including dissociation and association. This method can be applied to detect a wide range of biomolecular interactions.     

Evaluation of sulfated maltodextrin as a novel anionic chiral selector for the enantioseparation of basic chiral drugs by capillary electrophoresis

Introducing a new class of chiral selectors is an interesting work and this issue is still one of the hot topics in separation science and chirality. In this study, for the first time, sulfated maltodextrin (MD) was synthesized as a new anionic chiral selector and then it was successfully applied for the enantioseparation of five basic drugs (amlodipine, hydroxyzine, fluoxetine, tolterodine, and tramadol) as model chiral compounds using CE. This chiral selector has two recognition sites: a helical structure and a sulfated group which contribute to three corresponding driving forces; inclusion complexation, electrostatic interaction, and hydrogen binding. Under the optimized condition (buffer solution: 50 mM phosphate (pH 3.0) and 2% w/v sulfated MD; applied voltage: 18 kV; temperature: 20°C), baseline enantioseparation was observed for all mentioned chiral drugs. When instead of sulfated MD neutral MD was used under the same condition, no enantioseparation was observed which means the resolution power of sulfated MD is higher than neutral MD due to the electrostatic interaction between sulfated groups and protonated chiral drugs. Also, the countercurrent mobility of negatively charged MD (sulfated MD) allows more interactions between the chiral selector and chiral drugs and this in turn results in a successful resolution for the enantiomers. Furthermore, a higher concentration of neutral MD (approximately five times) is necessary to achieve the equivalent resolution compared with the negatively charged MD.     

毛细管电泳-安培检测法测定3种拟肾上腺素药物

建立了毛细管电泳-安培检测法测定盐酸去氧肾上腺素(phenylephrine hydrochloride, PHE)、重酒石酸间羟胺(metaraminol bitartrate, MR)和盐酸异丙肾上腺素(isoprenaline hydrochloride, IP)3种拟肾上腺素药物的方法。检测电位为0.950 V(Ag/AgCl为参比电极),硼酸盐浓度为50 mmol/L(pH 10.00),分离电压为18 kV,进样时间为10 s。在最佳实验条件下,3种物质在18 min内达到基线分离,在2～100 μmol/L浓度范围内峰面积与浓度呈良好的线性关系,线性相关系数不小于0.9991。盐酸去氧肾上腺素、重酒石酸间羟胺和盐酸异丙肾上腺素的检出限分别为0.8、0.8和1.0 μmol/L。将所建立的方法应用于针剂样品的分析,结果令人满意。     

Mechanism of change in enantiomer migration order of enantioseparation of tartaric acid by ligand exchange capillary electrophoresis with Cu(II) and Ni(II)-D-quinic acid systems

The mechanism of change in the enantiomer migration order (EMO) of tartarate on ligand exchange CE with Cu(II)- and Ni(II)-D-quinic acid systems was investigated thoroughly by circular dichroism (CD) spectropolarimetry. The (13) C NMR spectra of solutions containing D-quinate (pH 5.0) with Cu(II) or Ni(II) revealed the coordination of carboxylate and hydroxyl groups on D-quinate. The D-quinic acid concentration dependence of the CD spectra at a fixed Cu(II) concentration at pH 5.0 indicates that the 1:1, 1:2 and 1:3 Cu(II)-D-quinate complexes were formed with an increase in the concentration of D-quinic acid. The CD spectral behavior revealed that D-tartarate is selectively coordinated to the 1:1 complex to give the 1:1:1 Cu(II)-D-quinate-D-tartarate ternary complex while L-tartarate is selectively bound to the 1:2 and 1:3 complexes to form the 1:2:1 ternary complex. In the Ni(II)-D-quinic acid system, it became apparent that the 1:2 Ni(II)-D-quinate complex is mainly formed in the wide range of D-quinic acid concentration at pH 5.0 and D-tartarate is selectively coordinated to the 1:2 complex to form the 1:2:1 ternary complex. The change in EMO of tartarate on ligand exchange CE was explainable by the change in coordination selectivity for D- and L-tartarates in the Cu(II)- and Ni(II)-D-quinic acid systems depending on the compositions of the complexes formed in BGE.     

Pressurized fluid extraction of essential oil from Lavandula hybrida using a modified supercritical fluid extractor and a central composite design for optimization

Essential oil components were extracted from lavandin (Lavandula hybrida) flowers using pressurized fluid extraction. A central composite design was used to optimize the effective extraction variables. The chemical composition of extracted samples was analyzed by a gas chromatograph-flame ionization detector column. For achieving 100% extraction yield, the temperature, pressure, extraction time, and the solvent flow rate were adjusted at 90.6°C, 63 bar, 30.4 min, and 0.2 mL/min, respectively. The results showed that pressurized fluid extraction is a practical technique for separation of constituents such as 1,8-cineole (8.1%), linalool (34.1%), linalyl acetate (30.5%), and camphor (7.3%) from lavandin to be applied in the food, fragrance, pharmaceutical, and natural biocides industries.     

Tris(2,2'-bipyridyl)ruthenium(II)-zirconia-Nafion composite films applied as solid-state electrochemiluminescence detector for capillary electrophoresis

The major goal of this work was to develop a new solid-state electrochemiluminescence (ECL) detector suitable for capillary electrophoresis (CE). The detector was fabricated by coating a sol-gel derived zirconia (ZrO(2))-Nafion composite film on a graphite electrode, then the zirconia-Nafion modified electrode was immersed in tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3) (2+)) solution to immobilize this active chemiluminescence reagent. The voltammetric and ECL behaviors of the detector were investigated and optimized in tripropylamine solution. The ratio of 53% for zirconia in the zirconia-Nafion composite provided the highest luminescence intensity of immobilized Ru(bpy)(3) (2+). The ECL can maintain its stability very well in the phosphate solution in the period of 5-90 h when the solid-state ECL detector was immersed in the solution all the time. The optimum distance of capillary outlet to the solid-state ECL detector has been found to be ca. 50-80 microm for a 75 microm capillary. The effects of ionic strength and pH of ECL solution on peak height were investigated. The CE with solid-state ECL detector system was successfully used to detect tripropylamine, lidocaine, and proline. The detection limits (S/N = 3) were 5 x 10(-9) mol.L(-1) for tripropylamine, 1 x 10(-8) mol.L(-1) for lidocaine and 5 x 10(-6) mol.L(-1) for proline, and the linear ranges were from 1.0 x 10(-8) to 1.0 x 10(-5) mol.L(-1) for tripropylamine, 5.0 x 10(-7) mol.L(-1) to 1.0 x 10(-5) mol.L(-1) for lidocaine and 1.0 x 10(-5) to 1.0 x 10(-3) mol.L(-1) for proline, respectively.     

In‐capillary self‐assembly study of quantum dots and protein using fluorescence coupled capillary electrophoresis

As a vast number of novel materials in particular inorganic nanoparticles have been invented and introduced to all aspects of life, public concerns about how they might affect our ecosystem and human life continue to arise. Such incertitude roots at a fundamental question of how inorganic nanoparticles self‐assemble with biomolecules in solution. Various techniques have been developed to probe the interaction between particles and biomolecules, but very few if any can provide advantages of both rapid and convenient. Herein, we report a systematic investigation on quantum dots (QDs) and protein self‐assembly inside a capillary. QDs and protein were injected to a capillary one after another. They were mixed inside the capillary when a high voltage was applied. Online separation and detection were then achieved. This new method can also be used to study the self‐assembly kinetics of QDs and protein using the Hill equation, the K_D value for the self‐assembly of QDs and protein was calculated to be 8.8 μM. The obtained results were compared with the previous out of‐capillary method and confirmed the effectiveness of the present method.     

Determination of phenol in landfill leachate by using microchip capillary electrophoresis with end-channel amperometric detection

Phenol, 2,4-dichlorophenol (2,4-DCP), and 2,4,6-trichlorophenol (2,4,6-TCP) were baseline separated by using a homemade microchip CE with an end-channel amperometric detector where a 50 microm Pt microdisk working electrode (WE) and a Pt cathode were integrated onto the microchip itself. Separation parameters such as injection time and voltage, pH of the buffer, online pretreatment condition for WE, reproducibility, and detection potential were investigated. Under the selected separation conditions, the linear ranges for phenol, 2,4-DCP, and 2,4,6-TCP were 2-200, 4-400, and 4-400 microM, respectively. The LODs were 0.4, 0.5, and 0.7 microM for phenol, 2,4-DCP, and 2,4,6-TCP, respectively (S/N = 3). The standard addition method was successfully applied to the analysis of landfill leachate samples and the concentration of phenol in the landfill leachate samples was measured to be 0.32 and 0.21 mM, respectively. The recoveries were in the range of 85-103% and corresponding RSDs were less than 5.5%.     

Separation and identification of different therapeutic pharmaceutical drugs on humic acid-based HPLC column: Method optimization using central composite design

Multimode chromatographic separations are highly desirable in pharmaceutical and environmental sciences. Current study deals with the application of newly developed mixed-mode end capped-immobilized humic acid onto an aminopropyl silica based chromatographic column for separation and identification of six drugs belonging to different therapeutic groups for its applicability in pharmaceutical industries. For this, central composite design was used to evaluate the separation and resolution by optimization of three most effective parameters (acetonitrile%, flow rate, and pH of mobile phase). Second-order quadratic model was used to evaluate their effect on resolution of peaks; the probability value (<0.05) obtained from analysis of variance suggested the best applicability of the model. Desirability function was applied to calculate optimum conditions (44.8% acetonitrile, 1.75 mL/min of flow rate, and 7.5 pH) required to achieve maximum separation with good resolution within 11 min. The method was validated for linearity, precision accuracy, selectivity, and sensitivity. The results revealed a highly precise (coefficient of variance > 1%), linear (R² = 0.99), and highly selective method. Moreover, the limit of detection/quantification values revealed acceptable sensitivity of the method. The developed column was compared for its efficiency with a commercially available column and found to be highly applicable for industrial applications.     

Dispersive liquid–liquid microextraction coupled with capillary electrophoresis for simultaneous determination of sulfonamides with the aid of experimental design

A novel method for the simultaneous determination of sulfonamides (SAs) in water samples has been developed by using dispersive liquid–liquid microextraction (DLLME) coupled with CE. Orthogonal and Box–Behnken designs were employed together to assist the optimization of DLLME parameters, including volumes of extraction and disperser solvents, ionic strength, extraction time, and centrifugation time and speed as variable factors. Under the optimum extraction and detection conditions, successful separation of the five SAs was achieved within 5 min, and excellent analytical performances were attained, such as good linear relationships (R>0.980) between peak area and concentration for each SA from 0.5 to 50 μg/mL, low limits of detection for the five SAs between 0.020 and 0.570 μg/mL and the intra‐day precisions of migration time below 0.80%. The method recoveries obtained at fortified 10 μg/mL for three water samples ranged from 53.6 to 94.0% with precisions of 1.23–5.60%. The proposed method proved highly sensitive and selective, rapid, convenient and cost‐effective, showing great potential for the simultaneous determination of SAs in water samples.     

Use of neutral capillaries for the enantioseparation of N‐benzoylated amino acids by capillary electrophoresis with bromobalhimycin as chiral selector

In this study, the partial filling technique on both polycationic polymer hexadimethrine bromide (HDB) modified capillary and eCAP neutral capillary were systematically compared in order to enhance the enantioseparation ability of bromobalhimycin as CE additive. The separation conditions, such as pH, the plug length, and the concentration of bromobalhimycin, etc., were optimized in order to obtain satisfactory separations. As expected, for all tested 28 N‐benzoylated amino acids, up to five times higher enantioresolutions were obtained on the eCAP neutral capillary compared to that on the polycationic polymer hexadimethrine bromide modified capillary. Moreover, 26 of 28 tested racemic compounds were almost baseline‐ resolved without observing any interference from the front of the plug of bromobalhimycin. Although the limitation of longer running time on the neutral capillary, it allows the use of higher content of bromobalhimycin in the running buffer without any interference on the detection of analytes when enantioseparations are more difficult to obtain.     

A fluorescent derivatization method of proteins for the detection of low‐level impurities by microchip capillary gel electrophoresis

A novel pre‐chip fluorescent derivatization method is presented for protein sizing and quantification by microchip CGE. The derivatization reaction employed a water‐soluble and stable fluorescent dye and was performed under conditions that favored the formation of homogeneous reaction products. The method delivered in terms of protein sizing similar results as microchip CGE with on‐chip staining but showed an extended linear dynamic range for protein quantification encompassing four orders of magnitude. The sensitivity of the method was similar to standard silver‐stained planar gels. The characterization of derivatization reaction products by MS and preparative isoelectric focusing indicated that a constant degree of dye molecule tagging was obtained over a broad range of protein/dye ratios. The method allowed detecting and quantifying an impurity spiked into an antibody preparation down to a level of 0.05%. Advantages of this method compared with CGE approaches with pre‐column derivatization include a shorter analysis time and an increased robustness and ease of use.     

Affinity capillary electrophoresis coupling with partial filling technique and field‐amplified sample injection for enantioseparation and determination of DL‐tetrahydropalmatine

A novel, simple and sensitive method for the enantioseparation and determination of DL ‐tetrahydropalmatine (DL ‐THP) was developed using ACE in combination with partial filling technique and field‐amplified sample injection. A chiral selector, i.e. BSA, was used for the enantioseparation of DL ‐THP in ACE. Effects of BSA concentration, pH and separation voltage on the effectiveness of the enantiomer separation were evaluated. In an optimal condition, D ‐ and L ‐THP were completely enantio‐separated in less than 9 min by partially filling an electrophoretic capillary with 50 μmol/L BSA (50 mbar, 100 s) and carrying out an electrophoresis with 20 mmol/L phosphate buffer (pH 7.4) at 15 kV. The sensitivity was further improved by making use of field‐amplified sample injection to lower the LOD (defined as S/N=3) down to 6 ng/mL. Real samples were also tested and promising results for the determination of DL ‐THP enantiomers were obtained.     

Optimization for quantitative determination of four flavonoids in Epimedium by capillary zone electrophoresis coupled with diode array detection using central composite design

Herba Epimedii (family Berberidaceae), Ying-Yang-Huo in Chinese, is a famous Chinese herbal medicine. Flavonoids are thought to be the major active components in it. A capillary zone electrophoresis (CZE) separation were developed for simultaneous determination of four flavonoids including icariin, epimedin A, epimedin B and epimedin C in Epimedium. The effects of the experimental variables on CZE had been optimized by using central composite design (CCD). The best separation of four flavonoids could be obtained using 50 mM borate buffer (pH 10.0) containing 22% acetontrile as modifier, while separation voltage was 15 kV and temperature was at 25 degrees C. The method developed is accurate, simple and reproducible, which could be used for quality control of Epimedium and its medical preparations.