粒子群算法优化中药化学成分的毛细管电泳分离条件

建立了一种基于粒子群优化算法的毛细管电泳条件辅助优化方法。以丹参为研究对象,将改良的色谱指数方程用于评价酚酸类成分的电泳分离性能,用粒子群优化算法对分离条件进行全局寻优,获得最佳的区带电泳分离条件(5.0 mmol/L硼砂,18.5 mmol/L磷酸二氢钠,6.1%乙腈,运行电压18.2 kV)。为进一步改善分离,在所获优化条件下添加50.0 mmol/L SDS,在胶束电动毛细管色谱分离模式下使酚酸类成分(原儿茶醛、丹参素、丹酚酸B等)得到更好分离。本方法准确可靠,可推广应用于其他复杂化学体系的毛细管电泳分离条件优化。     

Optimization and validation of an enantioselective method for a chiral drug with eight stereo-isomers in capillary electrophoresis

Highly selective capillary electrophoresis (CE) screening methods were applied to find a satisfactory separation of a chiral drug with eight stereoisomeric compounds. The initial separation conditions were further optimized using response surface modelling by applying a Box-Behnken experimental design. This approach resulted in a rapid and efficient optimization of the buffer concentration, the concentration of two cyclodextrins, and the run voltage, in order to obtain final separation conditions of the method. Further optimization and validation of the system in terms of sensitivity and robustness resulted in a method that is suitable for quality control release purposes.     

A screening strategy for the development of enantiomeric separation methods in capillary electrophoresis

Method development of enantiomeric separations in capillary electrophoresis (CE) is a time-consuming task, since finding the appropriate chiral selector is usually a "trial and error" process. It is impossible to predict the selectivity of a selector towards a certain enantiomer. Therefore, the affinity of all selectors has to be examined one at a time. In order to speed up this process, a strategy is proposed based on simple experimental design methodology. The approach includes first a screening in function of the pH to determine the optimal migration conditions followed by a selection of the right chiral selector by means of Taguchi designs. In the approach several variables, such as the type and concentration of cyclodextrin, the concentration of buffer electrolyte, and the percentage of organic modifier, are varied simultaneously to find initial separation conditions rapidly. The resulting initial separation conditions can be optimized in further steps to be more reproducible. We discuss the results of the approach when applied on a number of selected compounds that are recently in development at Johnson & Johnson--Pharmaceutical Research and Development. Parameters, such as quality of the separation and analysis time, are evaluated to determine initial separation conditions for each compound.     

Application of Artificial Neural Networks in Multifactor Optimization of Selectivity in Capillary Electrophoresis

ABSTRACT

A methodology based on the coupling of experimental design and artificial neural networks (ANNs) was proposed in the optimization of selectivity in capillary electrophoresis. The effect of the buffer composition, concentration, SDS concentration, ethanol percentage and the applied voltage on the separation of six choice solutes was examined by using orthogonal design. Feedforward-type neural networks with faster back propagation (BP) algorithm were applied to model the separation process, and then optimization of the experimental conditions was carried out in the modeled neural network with 5-7-1 structure, which had been confirmed to be able to provide the maximum performance. It was demonstrated that by combining ANN modeling with experimental design, the number of experiments necessary to search and find optimal separation conditions can be reduced significantly. Because of its general validity, the new proposed approach can also be applied in other separation conditions.     

Identifying the orientation of DNA fragment in recombinant plasmid by capillary electrophoresis with a non-gel sieving solution.

It was demonstrated that a capillary electrophoresis (CE) method with a non-gel sieving solution has been developed to identify the orientation of DNA fragments in recombinant plasmids in molecular biology. The influences of the concentration of sieving polymer HEC, the applied electric field strength and sampling on CE separation were analyzed concerning the optimization of separation. YO-PRO-1 was used as a DNA intercalating reagent to facilitate fluorescence detection. Under the chosen conditions (buffer, 1 x TBE containing 1 microM YO-PRO-1 and 1.2% HEC; applied electric field strength, 200 V/cm; electrokinetic sampling: time, 5 s; voltage, -6 kV), three DNA markers (phi 174/HaeIII, pBR322/HaeIII and lambda DNA/HindIII) were tested for further evaluating the relationship between the DNA size and the mobility. The established CE method conjugated with the enzymatic approach was successfully applied to identifying the DNA orientation of recombinant plasmid in transgene operations of a newly cloned gene from Arabidopsis Thaliana.     

Method development and validation for the chiral separation of peptides in the presence of cyclodextrins using capillary electrophoresis and experimental design.

The present study describes the application of statistical experimental design to the optimization of enantioselective separations of peptides in capillary electrophoresis in order to obtain optimal operating conditions for routine work. Hydroxypropyl-beta-cyclodextrin was used as chiral selector and Ala-PheOMe as model peptide. The experiments were performed according to a face centered cube response surface experimental design for obtaining information how the factors such as concentration of the chiral selector, pH, buffer concentration and voltage affected the two response goals, resolution and analysis time. In order to achieve the simultaneous optimization of these two major electrophoretic performance goals for efficient and fast separation, the Derringer desirability functions were tested. While in the predefined experiments the analysis time for baseline separation was 25 min the desirability functions proposed a CE method, which diminished the analysis time and permitted the complete separation of the peptide enantiomers within 9 min.     

Analysis of Salvia officinalis plant extracts by capillary electrophoresis

Salvia officinalis (commonly called Sage) and similar plants contain many compounds of pharmaceutical interest and are used as a tea or in various pharmaceutical products. In this work, the use of CE for analysis of aqueous or ethanolic extracts from various Salvia plants has been studied. Especially, several buffers like borate, phosphate, acetate, etc., were examined under different concentrations, pH, separation voltage, injection time, and other parameters to find the optimal separation conditions. The optimization was also performed using experimental design and artificial neural networks. The optimal conditions were: separation voltage +20 kV, 40 mM buffer borate, pH 9.2, injection time 5 s, and UV detection at 280 nm. A new CE method has been developed, validated, and applied to analyze samples of S. officinalis from various countries.     

Separation of natural and synthetic heparin fragments by high-performance capillary electrophoresis.

The application of capillary electrophoresis (CE) for the analysis of natural and synthetic low-molecular-mass heparin fragments at low pH is described. It is demonstrated that under the applied conditions the separation is based on charge, charge distribution and molecular mass of the heparin molecules, yielding a high resolution. It is shown that the presence of sodium chloride in the sample solution has hardly any effect on the CE performance. However, the pH of the electrophoresis buffer is a critical parameter. The resolutions obtained with CE and high-performance anion-exchange chromatography (HPAEC) are compared for various heparin fragments and it is concluded that, at least for this type of molecule, CE forms an attractive alternative to HPAEC.     

Recent advances in the analysis of antibiotics by CE and CEC

This article reviews the most recent developments concerning the determination of antibiotics by CE and CEC. The most employed CE separation modes were CZE and MEKC although microemulsion electrokinetic capillary chromatoghraphy was also employed. For the first time, CE was coupled to MS that was applied as a specific and confirmatory detection technique for the analysis of antibiotics. The analytical characteristics of the developed methodologies as well as the different applications reported in the literature on this subject from June 2005 until May 2007 are included in this article. To give the most relevant information on this topic, the experimental conditions employed to achieve the analysis of antibiotics by CE and CEC are provided together with the main applications performed in the pharmaceutical, agrochemical, biological, food, and environmental fields, emphacizing sample preparation requirements needed in each case.     

A capillary electrophoresis and off-line capillary electrophoresis/electrospray ionization-quadrupole time of flight-tandem mass spectrometry approach for ganglioside analysis

A systematic study for the optimization and implementation of high-performance capillary electrophoresis (HPCE) in conjunction with negative ion electrospray ionization-quadrupole time of flight-tandem mass spectrometry (ESI-QTOF-MS/MS) for the analysis of complex glycolipids is described. The performance of the capillary electrophoresis (CE) and off-line CE/ESI-QTOF-MS approach has been explored for screening a complex ganglioside mixture from bovine brain. All instrumental and solution parameters demonstrated to require special adjustment and to have the most substantial effect on the CE separation, abundance of product ions produced in a low-energy collision-induced dissociation (CID) process and their detection by MS/MS, when attempting to identify and sequence single ganglioside molecular species from CE eluted fractions. Upon optimization of the experimental parameters, an efficient methodology emerged providing the general basic requirements for combined CE/ESI-MS analysis of this type of complex glycoconjugate.     

Separation strategy for acidic chiral pharmaceuticals with capillary electrochromatography on polysaccharide stationary phases

The effect of five factors on the capillary electrochromatographic enantioseparation of acidic compounds was studied using an experimental design. The studied factors were pH, acetonitrile content in the mobile phase, temperature, buffer concentration, and applied voltage. These experiments allowed defining a generic separation strategy applicable on acidic compounds with chemical and structural diversity. The starting screening conditions consist of a 45 mM ammonium formate electrolyte at pH 2.9 mixed with 65% acetonitrile, an applied voltage of 15 kV, and a temperature of 25 degrees C. The screening phase occasionally can be followed by an optimization procedure. Evaluation of the proposed strategy pointed out that it allows achieving baseline resolution within a relatively short time when a beginning of separation is obtained at the starting conditions. This strategy revealed enantioselectivity for 11 compounds out of 15, of which 10 could be baseline-separated after the proposed optimization steps.     

Optimization of a nano‐enzymatic reactor for on‐line tryptic digestion of polypeptide conjugates by capillary electrophoresis

This work aims at studying the optimization of an on‐line capillary electrophoresis (CE)‐based tryptic digestion methodology for the analysis of therapeutic polypeptides (PP). With this methodology, a mixture of surrogate peptide fragments and amino acid were produced on‐line by trypsin cleavage (enzymatic digestion) and subsequently analyzed using the same capillary. The resulting automation of all steps such as injection, mixing, incubation, separation and detection minimizes the possible errors and saves experimental time. In this paper, we first study the differents parameters influencing PP cleavage inside the capillary (plug length, reactant concentration, incubation time, diffusion and electrophoretic plugs mixing). In a second part, the optimization of the electrophoretic separation conditions of generated hydrolysis products (nature, pH and ionic strength (I) of the background electrolyte (BGE)) is described. Using the optimized conditions, excellent repeatability was obtained in terms of separation (migration times) and proteolysis (number of products from enzymatic hydrolysis and corresponding amounts) demonstrating the robustness of the proposed methodology.     

On-line preparation of microsamples prior to CE

An experimental setup is presented here for the automated analysis of microsamples, based on the on-line coupling of a capillary SPE module and a CE unit using a two-position six-port valve, an open-closed valve to isolate electrically the sample preparation from the CE unit and a "T" interface. A C18 trapping microcolumn (dimensions 2.5 cm x 100 microm id x 360 microm od) was used for the SPE step. The utility of the proposed experimental setup was demonstrated by applying it to the determination of quinolone antibiotics in serum microsamples, which was efficiently carried out in less than 20 min (4 min for protein denaturation and 15 min for analytes preconcentration and CE-UV separation-determination). A complete optimization study was performed for preconcentration and cleanup of quinolones, the coupling of sample preparation module to the CE unit and electrophoretic separation of quinolones. A preconcentration factor of 10.4 was achieved. The volume injected with the proposed method was 125 nL versus 160 nL introduced by hydrodynamic injection. The volume required for the analysis was 2 microL, which makes the proposed experimental setup very useful for the analysis of microsamples in fields of current interest such as metabolomics or proteomics.     

Response surface examination of the relationship between experimental conditions and product distribution in electrophoretically mediated microanalysis

This work presents the first known use of response surface methodology (RSM) in electrophoretically mediated microanalysis. This concept is demonstrated by examining the optimization of reaction conditions for the conversion of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced form by glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in the conversion of glucose-6-phosphate to 6-phosphogluconate. Experimental factors including voltage, enzyme concentration, and mixing time of reaction at the applied voltage were selected at three levels and tested in a Box-Behnken response surface design. Upon migration in a capillary under CE conditions, plugs of substrate and enzyme are injected separately in buffer and allowed to react at variable conditions. Extent of reaction and product ratios were subsequently determined by CE. The model predicted results are shown to be in good agreement (7.1% discrepancy difference) with experimental data. The use of chemometric RSM provides a direct relationship between electrophoretic conditions and product distribution of microscale reactions using CE, thereby offering a new and versatile approach to optimizing enzymatic experimental conditions.     

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.     

An Integrated Approach Based on Experimental Designs for Fingerprint Development of the Complex Herbal Prescription Sheng-Mai-San by MEKC

An integrated strategy based on experimental designs for the development, optimization and validation of the fingerprint method of Sheng-Mai-San by MEKC has been described. Orthogonal and sequential uniform designs were employed to select important experimental parameters and optimize CE conditions. Method validation was performed in terms of injection precision, sample stability test and robustness testing. Additionally, conventional modeling method was used to predict the optimum separation conditions for comparative purpose. The strategy described can also be utilized for fingerprint development in the quality control of other herbal medicines.     

On the surface modification of microchannels for microcapillary electrophoresis chips

This paper presents systematic investigation of the microchannel surface properties in microCE chips. Three popular materials for microCE chips, polydimethylsiloxane (PDMS), quartz, and glass, are used. The zeta potentials of these microchannels are calculated by measuring the EOF velocity to evaluate the surface properties after surface modification. The hydrophobic PDMS is usually plasma-treated for microCE applications. In this study, a new method using a high-throughput atmospheric plasma generator is adopted to treat the PDMS surface under atmospheric conditions. In this approach, the cost and time for surface treatment can be significantly reduced compared with the conventional vacuum plasma generator method. Experimental results indicate that new functional groups could be formed on the PDMS surface after treatment, resulting in a change in the surface property. The time-dependent surface property of the plasma-treated PDMS is then measured in terms of the zeta potential. Results show that the surface property will reach a stable condition after 1 h of plasma treatment. For glass CE chips, two new methods for changing the microchannel surface properties are developed. Instead of using complicated and time-consuming chemical silanization procedures for CE channel surface modification, two simple and reliable methods utilizing organic-based spin-on-glass and water-soluble acrylic resin are reported. The proposed method provides a fast batch process for controlling the surface properties of glass-based CE channels. The proposed methods are evaluated using PhiX-174 DNA maker separation. The experimental data show that the surface property is modified and separation efficiency greatly improved. In addition, the long-term stability of both coatings is verified in this study. The methods proposed in this study show potential as an excellent solution for glass-based microCE chip surface modification.     

Design of experiments for capillary electrophoretic enantioresolution of salbutamol using dermatan sulfate

Statistical experimental design was used for the optimization and for robustness evaluation of a capillary electrophoretic method developed for the enantioresolution of salbutamol. Dermatan sulfate was used as chiral selector. The goal of the study was to obtain an efficient and fast separation. An eight-run Plackett-Burman matrix was used during the optimization process for the screening of the factors and to adjust the experimental domain under study. Response surface methodology was adopted after the screening phase to obtain information about how the factors percentage of chiral selector, pH and voltage affected the considered responses resolution and analysis time. The Derringer desirability function, which makes it possible to combine results obtained for properties measured on different scales, was used to simultaneously optimize the two responses. Robustness testing was carried out using a Plackett-Burman matrix. The method was found robust as regards the response resolution while voltage and chiral selector were found to be critical factors for the robustness of analysis time response. The proposed CE method permitted the complete enantioseparation of racemic salbutamol and was applied to its chiral resolution in spiked urine samples.     

Development of capillary electrophoresis for the determination of metal ions using mixed partial and complete complexation techniques

A new capillary electrophoretic (CE) method was developed for the selective and sensitive determination of common metal ions. The proposed method is based on conventional CE separation of metal cations followed by complete complexation of separated analytes with 1,10-phenanthroline using the zone-passing technique. This approach combines both partial and complete complexation modes and, thus, enables rapid, selective, efficient separation together with sensitive direct UV detection of metal species. The optimal conditions for the separation and derivatization reaction were established by varying type of electrolyte, electrolyte pH, introduction time and concentration of 1,10-phenanthroline. The optimized separations were carried out in 50 mmol l(-1) glycolic acid electrolyte (pH 6.0 with imidazole) using direct UV detection at 254 nm. Five common metal cations (Fe2+, Co2+, Ni2+, Cu2+ and Zn2+) were separated in less than 4 min. The proposed system was applied to the determination of Fe(II) and Zn(II) in snow samples. The recovery tests established for snow samples were within the range 100+/-12%.     

Modelling and optimization of the separation of anions in ion chromatography--capillary electrophoresis

The influence of varying experimental conditions on the mobilities of inorganic and organic anions in ion chromatography-capillary electrophoresis (IC-CE) was studied. A theoretical model derived from both IC and CE was used to explain the influence on analyte mobility caused by varying the concentration of polymer and increasing the salt concentration in the background electrolyte. The influence of the type of competing ion was also accounted for by including the analyte selectivity coefficient in the model equation. The validity of the model was shown using electrolyte systems containing four different competing anions, with correlation between experimental and mobilities predicted being excellent (r2 > 0.98) for all systems. Selectivity coefficients determined via nonlinear regression enabled quantitative comparisons of different competing ion strength, with the eluting strength increasing in the order of fluoride, acetate, chloride, and sulfate. Optimization of the polymer and eluent concentration was performed for all electrolyte systems using the normalized resolution product optimization criterion, requiring only seven experiments to obtain the optimum conditions for complete separation. The minimum resolution criterion was used to optimize the fluoride system which gave a different separation selectivity from both CE and IC.