Use of cyclodextrins for the enantioselective separation of ergot alkaloids by capillary zone electrophoresis.

Ergot alkaloid enantiomer derivatives were resolved using capillary zone electrophoresis. The effect of cyclodextrins, added to the background electrolyte, on the migration time and the resolution was studied. Good separation for epimeric ergot alkaloid derivatives was also obtained using phosphate buffer at pH 2.5. Separation was improved by supplementing the background electrolyte with 30 mM of gamma-cyclodextrin. Good resolution of racemic ergot alkaloid derivatives in their enantiomers was achieved in a background electrolyte containing either beta-cyclodextrin or its derivative, or gamma-cyclodextrin.     

Enantiomeric separations by use of polymeric surfactant electrokinetic chromatography

This review surveys the enantiomeric separation of drugs by electrokinetic chromatography using polymeric chiral surfactant pseudostationary phases. These phases have recently been shown to provide better mass transfer and increased rigidity and stability than regular micelles in micellar capillary electrophoresis. Characterization of the polymeric chiral surfactants is presented. Solution interactions of the pseudostationary phases via thermodynamics and fluorescence probe studies are evaluated. Also, case studies of enantiomeric separation of drugs using a single amino acid surfactant and the synergistic effect of the addition of gamma-cyclodextrin to the buffer is discussed. The use of dipeptide surfactants for chiral drug separations is described as well.     

Assay of protein drug substances present in solution mixtures by fluorescamine derivatization and capillary electrophoresis.

A method is described to enhance the resolution and detection sensitivity of proteins, peptides, and amino acids in capillary electrophoretic analysis of solution mixtures. The method consists of derivatizing the analytes with fluorescamine, which is normally used as a fluorogenic reagent for compounds containing a reactive primary amine functional group, and then using the derivative as an ultraviolet chromophore to enhance detection sensitivity (measured at 280 nm) in capillary electrophoresis. The results demonstrated a significant improvement in the separation and detection sensitivity of the derivatized analytes as compared to their underivatized counterparts. The use of chromophores, such as fluorescamine, in capillary electrophoresis facilitates the analysis of components of solution mixtures, such as pharmaceutical formulations, that could not be resolved and/or detected by conventional capillary electrophoresis procedures.     

Separation of quaternary ammonium diastereomeric oligomers by capillary electrophoresis

The separation of novel diastereomeric trimers (3M) and pentamers (5M), derived from quaternary ammonium salts, was studied in conventional, uncoated and coated capillaries using capillary zone electrophoresis (CZE) with a variety of buffers and additives. Resolution of 5M diastereomers was best achieved using gamma-cyclodextrin (gamma-CD) as a chiral selector, while no diastereomeric resolution was realized for the 3M material.     

Enhancing separation in short‐capillary electrophoresis via pressure‐driven backflow

We present a novel easy‐to‐operate and efficient method to improve the separation efficiency in short‐capillary electrophoresis by introducing steady backflow to counterbalance electro‐osmotic flow without the use of any external pressure. The backflow was easily generated by tapering the capillary end, which was achieved by heating a straight capillary and stretching it with a constant force. We investigated the net fluidic transport rate under different tip lengths and separation voltages. Good run‐to‐run repeatability and capillary‐to‐capillary reproducibility of the present method were obtained with RSD less than 1.5%, indicating the stability of the fluid transport rate in the tapered capillary, which ensures the quantification and repeatability of capillary zone electrophoresis (CZE) analysis. Enhanced separation of the tapered short capillary electrophoresis was demonstrated by CZE analyzing amino acids and positional isomers. Baseline separations were achieved in less than 60 s using a tapered capillary with the effective length of 5 cm, while no separation was achieved using a normal capillary without a tapered tip. The present study provides a promising method to use pressure‐driven backflow to enhance separation efficiency in short‐capillary electrophoresis, which would be of potential value in a wide application for fast analysis of complex samples.     

Chiral separation of homocysteine by derivatization with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole followed by capillary electrophoresis using gamma-cyclodextrin

Homocysteine was derivatized with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) to form an inclusion complex with cyclodextrin and to facilitate UV detection. ABD-homocysteine showed interaction with beta- and gamma-cyclodextrin in capillary electrophoresis at pH 2.25 as indicated by the decreased migration time. However, chiral separation of D,L-ABD-homocysteine was observed using gamma-CD only. Optimal separation was obtained at pH 2.25, 50 mM gamma-CD concentration, and 20 kV applied voltage. L-ABD-Homocysteine migrated faster than the D-isomer as demonstrated by a spiking experiment using dithiothreitol-reduced L-homocystine.     

Comparison of capillary electrophoresis and reversed-phase liquid chromatography for determination of the enantiomeric purity of an M3 antagonist

The chiral separation of an M3 antagonist was investigated using capillary electrophoresis (CE) with various sulfated cyclodextrins and by reversed-phase liquid chromatography with derivatized cellulose, derivatized amylose, and two protein stationary phases. Operational parameters for each technique, such as the concentration of the chiral selectors, background electrolyte (or mobile phase) pH and type, organic modifiers, injection mode and temperature were varied in order to achieve a desired elution order and to meet a 0.1% limit of quantitation (LOQ) criteria. Based on the advantages and disadvantages of each technique, a practical CE method using sulfated gamma-cyclodextrin was selected. The method was validated in terms of linearity, LOQ, accuracy, ruggedness and precision.     

Some factors affecting enantiomeric impurity determination by capillary electrophoresis using ultraviolet and laser-induced fluorescence detection.

The key factors influencing enantiomer trace determination were investigated; these include resolution capillary diameter, limit of detection, linear range and type of detection. Chiral reagents, (+)- and (-)-1-(9-fluorenyl)ethyl chloroformate (FLEC), were employed as probes to demonstrate the influence of the variables. In order to find the best resolution, separation variables were optimized in both capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) modes by the application of factorial design experiments. A highly efficient chiral separation of the (+/-)-FLEC, derivatized with nonchiral amino acids, was achieved when using gamma-cyclodextrin as the chiral selector. The benefits of using a small diameter capillary for direct determination of both (+) and (-)-FLEC impurity (0.05-0.1% area/area) were demonstrated using UV detection and applying a sample stacking condition. A frequency-doubled argon ion laser (244 nm) was used as light source for laser-induced fluorescence (LIF) detection. Excitation light was provided by means of an optical fiber directed into the Hewlett Packard 3D capillary cartridge. The signals from UV and LIF were monitored simultaneously. The application of LIF detection greatly improved sensitivity and linear range. Further, as a consequence of the increased sensitivity, sample loading could be decreased, which led to an improvement of separation efficiency. Direct determination of 0.005% impurity could be achieved within the linear range.     

Absolute determination method for trace quantities of enantiomer of glufosinate by gamma-cyclodextrin modified capillary zone electrophoresis combined with solid-phase extraction and on-capillary concentration.

A highly sensitive and accurate determination method for trace quantities of enantiomers of glufosinate (D,L-GLUF), a phosphorus-containing amino acid-type herbicide, has been studied. The present method is based on the change in the mole ratio of the enantiomeric isomers after spiking of a known amount of an isomer (L-GLUF). The chiral separation and detection were made by gamma-cyclodextrin modified capillary zone electrophoresis (gamma-CD-CZE) with fluorescence detection. Solid-phase extraction of D,L-GLUF with titania was investigated as the pre-separation method to eliminate coexisting materials such as inorganic salts and organic compounds in river water. A separated D,L-GLUF was labeled with dansyl chloride and subjected to the on-capillary concentration using large-volume sample stacking (LVSS) before gamma-CD-CZE. The detection limit of the present method was as low as 2.0 x 10(-9) M. The present method was successfully applied to a model sample containing 2.0 x 10(-7) M D,L-GLUF in river water. It was confirmed that trace quantities of D-and L-GLUF in environmental samples can be accurately determined without any calibration curves and comparison standards.     

氨基酸衍生物在毛细管区带电泳下的分离研究

采用新合成的荧光试剂咔唑-9-乙基氯甲酸酯作为柱前衍生试剂,利用毛细管区带电泳法对衍生氨基酸进行分离,考察了该试剂用于毛细管区带电泳法进行氨基酸分离的关键条件,实现了12种氨基酸的快速基线分离.     

Continuous on-line derivatization and selective separation of D-aspartic acid by a capillary electrophoresis system with a continuous sample introduction interface

A rapid and selective method is described for the separation of D-aspartic acid (D-Asp) using a continuous on-line derivatization system coupled to capillary electrophoresis (CE). D-Asp was derivatized using o-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC). By on-line derivatization, amino acid enantiomers were automatically and reproducibly converted to the UV-absorbing diastereomer derivatives which were separated by capillary zone electrophoresis (CZE) in the presence of 10 mmol/L beta-cyclodextrin (beta-CD). Under the investigated separation conditions, D-Asp is resolved from L-aspartic acid (L-Asp) and other amino acids in a standard mixture of amino acids. The separation could be achieved within 4 min and the sample throughput rate can reach up to 16 h(-1). The repeatability (defined as relative standard deviation, RSD) was 3.21%, 3.58% with peak area evaluation and 3.72%, 4.03% with peak height evaluation for L-Asp and D-Asp.     

Determination of amino acids in tobacco samples by capillary electrophoresis/indirect absorbance detection with isolation of the electrolysis compartment and p-Aminobenzoic acid as a background electrolyte.

A fast, convenient and sensitive method of capillary zone electrophoresis (CZE) and indirect UV detection was proposed for the determination of 16 amino acids. p-Aminobenzoic acid (PAB) was selected as a background electrolyte (BGE). An isolated cell included a BGE buffer part and an electrode buffer one, which were jointed with a glass frit. The isolated cell can prevent PAB from the electrode reaction and improve the stability of the detection baseline. The separation conditions of amino acids were investigated, such as different BGEs, BGE concentration, buffer pH and electroosmotic flow (EOF) modifiers. Under the selected separation conditions, 14 amino acid peaks could be separated in 12 min. The detection limits of the amino acids were in the range of 1.7 - 4.5 micromol/L. The isolated cell is suitable for reagents reacting on the electrodes in capillary electrophoresis. The proposed method has been successfully applied to the determination of the amino acids in tobacco samples.     

西他沙星差向异构体的毛细管电泳分离

以γ-环糊精和D-苯丙氨酸为手性选择剂,采用毛细管区带电泳成功地分离了新型抗菌素西他沙星差向异构体。考察了添加剂的种类、浓度和缓冲溶液的pH对毛细管电泳分离西他沙星的影响。分离电压为15 kV,选用60 cm(有效长度52.5 cm)×50 μm i.d.的石英毛细管,缓冲溶液组成为10 mmol/L KH2PO4-K2HPO4(pH 4.5),10 mmol/L CuSO4,20 mmol/L γ-环糊精和 10 mmol/L D-苯丙氨酸。实验结果表明,添加剂的种类和浓度是影响西他沙星手性分离的重要因素,只有当 D-苯丙氨酸、铜离子和γ-环糊精同时存在并达到一定浓度时,西他沙星差向异构体在毛细管电泳中才具有良好的分离效果。该方法可用于西他沙星差向异构体的定量分析。     

Absolute determination method using chirality for glufosinate and bialaphos by gamma-cyclodextrin modified capillary zone electrophoresis.

A novel absolute determination method using chirality without any calibration curves or comparison standards has been proposed for phosphorus-containing amino acid-type herbicides, glufosinate (D,L-GLUF) and bialaphos (BIAL). This method is based on a change in the enantiomeric ratio after the spiking of a known amount of the enantiomers with different enantiomeric ratios to a sample. D,L-GLUF was determined by adding a known amount of L-GLUF to the sample, derivatizing them with dansyl chloride, and measuring the ratio of the peak area of the D-isomer to that of the L-isomer by means of gamma-cyclodextrin modified capillary zone electrophoresis. The accuracy and precision of the method were evaluated using a synthetic sample. The mean values obtained for D- and L-GLUF agreed with the values taken within 1.6%; also the reproducibility was as good as less than 2.8%. The determination of BIAL was achieved by determining GLUF quantitatively produced by the acid hydrolysis of BIAL. The proposed methods were applied to the analysis of commercial herbicides and the validity and usefulness were evaluated.     

Carboxymethyl-β-cyclodextrin for Chiral Separation of Amino Acids Derivatized with Fluorescene-5-isothiocyanate by Capillary Electrophoresis and Laser-induced Fluorescence Detection

A method using carboxymethyl-β-cyclodextrin (CM-β-CD) as selector for chiral separation of amino acids by capillary electrophoresis and laser-induced fluorescence detection was studied. Resolution was better than that obtained by β-CD or HP-β-CD.     

Capillary electrophoresis for monitoring dityrosine and 3-bromotyrosine synthesis

Protein oxidation affects the structure of many amino acids. Variants of tyrosine are increasingly important in medical and food sciences. The synthesis of standards is essential for monitoring the disease state of patients with various illnesses and the quality of a number of food products. A method for monitoring standard synthesis of dityrosine and 3-bromotyrsoine from tyrosine using capillary electrophoresis (CE) is presented. Optimum separation was achieved using an isoelectric buffer consisting of 100mM iminodiacetic acid (IDA)+75 mM lauryl sulfobetaine (SB 3-12)+0.02% hydroxypropyl methylcellulose (HPMC) in a 27 cm x 75 microm capillary at 22 kV and 45 degrees C. Using these conditions the tyrosine adducts could be easily separated in less than 4 min. The resolution of the CE method was similar to HPLC separations, but analysis time was distinctly shorter.     

Determination of preservatives in soft drinks by capillary electrophoresis with ionic liquids as the electrolyte additives

A capillary electrophoresis method for separating preservatives with various ionic liquids as the electrolyte additives has been developed. The performances for separation of the preservatives using five ionic liquids with different anions and different substituted group numbers on imidazole ring were studied. After investigating the influence of the key parameters on the separation (the concentration of ionic liquids, pH, and the concentration of borax), it has been found that the separation efficiency could be improved obviously using the ionic liquids as the electrolyte additives and tested preservatives were baseline separated. The proposed capillary electrophoresis method exhibited favorable quantitative analysis property of the preservatives with good linearity (r² = 0.998), repeatability (relative standard deviations ≤ 3.3%) and high recovery (79.4–117.5%). Furthermore, this feasible and efficient capillary electrophoresis method was applied in detecting the preservatives in soft drinks, introducing a new way for assaying the preservatives in food products.     

The use of a highly sulfated cyclodextrin for the simultaneous chiral separation of amphetamine-type stimulants by capillary electrophoresis

We investigated the simultaneous chiral separation of nine amphetamine type stimulants (dl-norephedrine, dl-norpseudoephedrine, dl-ephedrine, dl-pseudoephedrine, dl-amphetamine, dl-methamphetamine, dl-methylenedioxyamphetamine (MDA), dl-methylenedioxymethamphetamine (MDMA), and dl-methylenedioxyethylamphetamine (MDEA)) by capillary electrophoresis using highly sulfated gamma-cyclodextrin (SU(XIII)-gamma-CD) as a chiral selector. Three different approaches using SU(XIII)-gamma-CD with 50 mM phosphate background electrolyte were designed; (I) high CD concentration (10 mM SU(XIII)-gamma-CD) at neutral pH (pH 7.0) in the normal polarity mode, (II) low CD concentration (1.0 mM) at low pH (pH 2.6) in the normal polarity mode and (III) high CD concentration at low pH (pH 2.6) in the reversed-polarity mode. In mode (II), the effects of adding three neutral CDs (beta-CD, dimethyl-beta-CD and hydroxypropyl-beta-CD) were also investigated. The best separation was obtained after optimizing mode (III) as follows: run buffer of 10 mM SU(XIII)-gamma-CD with 50 mM phosphate background electrolyte at pH 2.6, applied voltage of -12 kV and capillary temperature of 15 degrees C.     

Continuous on-line derivatization and determination of amino acids by a microfluidic capillary electrophoresis system with a continuous sample introduction interface

An automatic, rapid and continuous on-line derivatization system coupled to microfluidic capillary electrophoresis (CE) for the determination of amino acids using o-phthaldialdehyde/N-acetyl-l-cysteine (OPA/NAC) as the derivative agents has been developed. By on-line derivatization, amino acids were automatically and reproducibly converted to the UV-absorbing derivatives, which were separated by capillary zone electrophoresis (CZE). Optimization of derivatization and separation condition was carried out to achieve both good sensitivity and separation efficiency. The separation could be achieved within 4 min and sample throughput rate can reach up to 16 h⁻¹. The repeatability (defined as relative standard deviation, R.S.D.) was 2.56, 2.85, 3.24 and 3.60% with peak area evaluation and 2.93, 3.12, 4.20 and 4.91% with peak height evaluation for arginine (Arg), phenylalanine (Phe), serine (Ser) and glycine (Gly), respectively. The limits of detection (S/N=3) were 10.46, 13.14, 34.39 and 44.79 μmol/l for Arg, Phe, Ser and Gly, respectively. Major advantages of the proposed method include improved precision and efficient automation of the derivatization by the FI system and the enhanced sampling frequencies by the combined FI-CE system.     

Capillary electrophoretic separation of enantiomers in a high-pH background electrolyte by means of the single-isomer chiral resolving agent octa(6-O-sulfo)-gamma-cyclodextrin

The new, alkali-stable, single-isomer, sulfated gamma-cyclodextrin, the sodium salt of octa(6-O-sulfo)-gamma-cyclodextrin (OS) was used for the first time to separate the enantiomers of non-ionic, acidic, basic and ampholytic analytes by capillary electrophoresis in high-pH aqueous background electrolytes. The effective mobilities and separation selectivities were found to follow trends similar to those observed earlier in acidic aqueous background electrolytes. OS proved to be a broadly applicable chiral resolving agent and afforded adequate peak resolution values with short separation times for a number of non-ionic, weak acid, weak base and ampholytic analytes.