Simultaneous determination of dopa and carbidopa enantiomers by capillary zone electrophoresis

Dopa and carbidopa, components of the dual therapy for Parkinson's disease treatment, are both provided as single enantiomers, since their D-forms are inactive. To ensure the efficiency and safety of the therapy, these D-enantiomers, therefore, should be considered as impurities. In this paper, the enantioseparation power of different types of cyclodextrins, both neutral and charged ones, on dopa and carbidopa enantiomers was tested. Three methods of simultaneous separation of dopa and carbidopa enantiomers were developed, using highly sulfated beta-cyclodextrin and sulfated beta-cyclodextrin as chiral selector, in normal and reversed polarity mode. Two methods among these three were found sensitive enough for the quantitation of 0.1% D-enantiomers in L-forms (impurity level). After the optimization study, the best method was selected, using 16 mM sulfated beta-cyclodextrin in 15 mM sodium phosphate buffer pH 2.45, an uncoated fused-silica capillary (50 num inner diameter, 30 cm total length), and an applied voltage of -12 kV. This method is robust and efficient, with very high resolution for all peaks within a short analysis time of 10 min. Quantitatively, the method offers a limit of detection (LOD) of 0.2 nug/mL and a limit of quantitation (LOQ) of 0.5 nug/mL for both D-dopa and D-carbidopa, which is equivalent to 0.02% and 0.05% against the respective L-enantiomers. A linear relationship was found between the concentration of the analyte and the corresponding peak area in a range of 0.5-2.0 nug/mL.     

Simultaneous contactless conductivity detection and UV detection for the study of separation of tamsulosin enantiomers in discontinuous electrolyte systems by CE

This work shows the potential of using discontinuous electrolyte systems for the separation of tamsulosin enantiomers by CE. Sulfated beta-cyclodextrin was used as a chiral selector. In acidic electrolytes, sulfated beta-cyclodextrin migrates as an anion and the analyte (tamsulosin) migrates as a cation. Due to this, four experimental arrangements were proposed. These arrangements differ in composition of electrolytes in the inlet compartment, in the capillary and in the outlet compartment. The separation of tamsulosin enantiomers in acetate buffers with sodium and Tris counterions was studied. Simultaneous contactless conductivity detection and UV detection were used for the study of the separation mechanism in these systems. Mobilities of sulfated beta-cyclodextrin were used for the calculation of the time when the analyte migrates through the BGE zone with the selector. The simulation program Simul 4.0 was used for the calculations of the concentration profiles of the electrolyte components dependent on the time of the separation. The mechanism of enantioseparation in these arrangements was suggested.     

Enantioseparation of chiral sulfoxides and sulfinate esters by capillary electrophoresis

Forty-one chiral sulfoxides and sulfinate esters were separated using sulfated beta-cyclodextrin and carboxymethyl beta-cyclodextrin as chiral selectors. Binding constants of some analytes to both chiral selectors were measured in order to examine and help explain the observed migration behavior and enantioselectivity trends. Overall, sulfated beta-cyclodextrin separated a greater number of compounds, and had better separating capabilities than did carboxymethyl beta-cyclodextrin for these analytes. This was true even though all of the analytes showed much stronger binding to carboxymethyl beta-cyclodextrin than to sulfated beta-cyclodextrin. General procedures to optimize the separation, by varying pH, selector concentration, and organic modifier concentration were examined and discussed. Chiral selector concentration had the greatest effect on enantioseparation, with higher concentrations of selector giving better peak-to-peak separations. Organic modifier had an adverse affect on resolution, with increasing amounts giving lower mobility differences. Lastly, pH had only a minimal effect on separation.     

Enantioseparation of warfarin and its metabolites by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method with direct ultraviolet (UV)-absorbance detection is presented for the simultaneous enantiomeric separation of warfarin and its main metabolites, including warfarin alcohols, 4'-, 6-, and 7-hydroxywarfarin, using highly sulfated beta-cyclodextrin (HS-beta-CD) as the chiral selector. This chiral separation method was optimized in terms of the electrophoretic parameters, which included the concentration of HS-beta-CD used, the type and composition of organic modifier added to the background electrolyte (BGE) buffer, and the BGE buffer pH. Chiral separation of warfarin and its major metabolites was achieved with high resolution, selectivity, efficiency, repeatability, and reproducibility. This optimized chiral analysis of warfarin along with its metabolites was completed within a satisfactory electrophoresis time of 20 min.     

Rapid enantioseparation of 1-aminoindan by microchip electrophoresis with linear-imaging UV detection.

Chiral separations of 1-aminoindan (AI) by cyclodextrin electrokinetic chromatography (CDEKC) were investigated on microfluidic quartz chips. By using a microchip electrophoresis (MCE) instrument equipped with a linear-imaging UV detector, the separation process of the enantiomeric compounds was observed. When sulfated beta-cyclodextrin was employed as a chiral selector, the baseline separation of AI could be achieved within 1 min with a high repeatability. The relative standard deviation of the migration time was less than 6%. The fastest separation was achieved in 14 s, utilizing a separation length of only 6.1 mm. These results show that the MCE analysis employing a linear imaging UV detector has a significant potential for fast chiral analysis.     

Analysis of vancomycin and related impurities by micellar electrokinetic capillary chromatography. Method development and validation

A fast and highly selective micellar electrokinetic capillary chromatography (MEKC) method for quantitative analysis of vancomycin and related impurities is described. Among the tested surfactants, cetyltrimethylammonium chloride (CTAC) offered the best selectivity. Another important parameter, which strongly influenced the selectivity, was buffer pH. It was found that the selectivity increased with buffer pH decreasing from 9 to 5. Using Tris-phosphate buffer containing CTAC, satisfactory separation could be obtained in the pH range from 5.0 to 5.5. Excellent repeatability in terms of migration time and peak area could be obtained when the capillary was carefully washed between two runs. In order to obtain optimal conditions and to evaluate the method robustness, a central composite experimental design was carried out. The optimal conditions were: 44 cm length of fused-silica capillary with 50 microm ID, 120 mM Tris-phosphate buffer (pH 5.2) containing 50 mM CTAC, -15 kV applied voltage, UV detection at 210 nm, and a column temperature of 25 degrees C. Under the optimal conditions, more than 20 peaks could be separated within 8 min. The method has a linearity range from 0.004 to 1.2 mg/ml (concentration of vancomycin B, active component). The limit of detection (LOD) and limit of quantitation (LOQ) were 0.4 microg/mL vancomycin, equivalent to 0.3 microg/mL vancomycin B (0.04%) and 1.1 microg/mL vancomycin, equivalent to 0.9 microg/mL vancomycin B (0.1%), respectively.     

A comparison of phosphated and sulfated beta-cyclodextrins as chiral selectors for capillary electrophoresis

The enantioseparation capabilities of three different functionalized beta-cyclodextrins, two sulfated beta-cyclodextrins with 4 and 15 nominal degrees of substitution and a phosphated beta-cyclodextrin with 8 degrees of substitution, were compared. While anodic detection was used with both sulfated cyclodextrins, the phosphated cyclodextrin required cathodic detection suggesting either lower ionization of the phosphated cyclodextrin or generally lower affinity of the analytes for the phosphated cyclodextrin. The effects of several experimental parameters were evaluated with respect to enantioseparation. The degrees of substitution of the cyclodextrin, pH of the background electrolyte as well as the concentration of the functionalized beta-cyclodextrin, each had a significant influence on the successful enantiomeric separation of the chiral drugs investigated.     

Enantioseparation of dihydropyridine derivatives by means of neutral and negatively charged beta-cyclodextrin derivatives using capillary electrophoresis

Employing capillary electrophoresis, the racemates of 29 acidic, neutral and basic dihydropyridines (DHPs) were separated by means of neutral and negatively charged cyclodextrins (CDs). Whereas the enantiomers of the acidic DHPs could be resolved with neutral CDs, mostly alpha- and beta-CD, the enantiomers of the neutral DHPs were only baseline-separated using the sulfobutyl ether-substituted beta-CD. Working in reversed polarity mode (detector at the anode) improved the peak shape and the resolution of the enantiomers. The racemates of the DHP bearing a secondary or tertiary amine function in the side chain at position 3 could be separated by using either the neutral gamma-CD or negatively charged CDs. The poor peak shape found with anionic CDs could be improved by the addition of methanol. The combination of gamma-CD and sulfated beta-CD allowed the detection of the minor enantiomer of lercanidipine (24) at less than 1% w/w.     

Use of vancomycin as chiral selector in capillary electrophoresis. Optimization and quantitation of loxiglumide enantiomers in pharmaceuticals

Vancomycin has been used as chiral selector for the enantiomers separation of D, L-loxiglumide, a new drug proposed for the treatment of gastrointestinal pathology. The chiral selector, dissolved at very low concentration in the running buffer, filled only part of the capillary (polyacrylamide coated) and allowed chiral resolution in less than 12 min using a 50 mM phosphate buffer at pH 6. The partial separation technique allowed to obtain a detection limit of 0.5 μg/ml for each enantiomer avoiding the drop in sensitivity due to the strong UV absorption of vancomycin when present in the detector path. The effects of vancomycin concentration and buffer pH on enantiomers resolution have been studied in order to find the optimum experimental conditions for the chiral purity control of drug. The optimized method, using the internal standard, showed good reproducibility for both migration times and normalized peak area ratio and for linearity. Under the studied operating conditions it was possible to detect 0.2 % (w/w) of L-loxiglumide as a chiral impurity. Analysis of pharmaceutical preparations of D-loxiglumide did not reveal the presence of the impurity (L-isomer).     

Capillary electrophoretic behaviors of pharmacologically active xanthones from Securidaca inappendiculata with beta-cyclodextrin as a buffer additive

The capillary electrophoretic (CE) behaviors of ten xanthones in the presence of beta-cyclodextrin (CD) are investigated, and apparent analyte-selector binding constants between beta-CD and the xanthones in the CE running buffer are calculated to elucidate the migration order. Also, the separation selectivity with beta-CD additive is compared with that of sulfated beta-CD additive. It is indicated that beta-CD can greatly change the separation selectivity of xanthones, and the electrophoretic behaviors of xanthones are rather different when using beta-CD from that when using sulfated beta-CD as an additive.     

Enantiomeric Separation of Two Antiparkinsonian Drugs by Electrokinetic Chromatography Using Dextran Sulfate

Anionic polysaccharide dextran sulfate (DxS) was successfully employed as chiral selector for the enantioseparation of two antiparkinsonian drugs, including rotigotine and trihexyphenidyl (THP), by electrokinetic chromatography (EKC). The enantioseparation was performed under normal and reversed polarity modes and reversed enantiomer migration order was achieved under two modes. The parameters affecting the chiral separation, such as buffer pH, DxS concentration, organic additive, and temperature were investigated and optimized. Reversed polarity mode provided better separation for the two drugs. The optimized conditions for the enantioseparation under reversed polarity mode were 2.0% (w/v) DxS, 10 mM phosphate buffer, pH 2.5 with an applied voltage of ?30 kV at 25 °C. Direct UV detection was performed at 200 nm. Under the optimal conditions, rotigotine and THP enantiomers were enantioresolved in 40 min with the resolution of 2.0 and 5.8, respectively. The analytes could be enantioseparated using DxS of molecular mass 1,000,000 or 500,000. It was inferred that the electrostatic, hydrophobic, and steric interactions may be involved in the chiral separation mechanism in this study.     

Rapid stereoselective separations of amphetamine derivatives with highly sulfated gamma-cyclodextrin

The highly sulfated gamma-CD (HS-gamma-CD) is a chiral selector widely used in CE for the enantioseparation of pharmaceutical compounds. This paper investigated different approaches to reduce the stereoselective analysis time of amphetamine (AT) derivatives according to the chiral selector concentration in the BGE. With high HS-gamma-CD concentration, tested analytes were separated in 3.5 min as anionic complexes with short-end injection technique in reversed polarity mode. However, this procedure presented some limitations in terms of efficiency and resolution, excessive Joule heating and poor compatibility with MS detection. With low HS-gamma-CD concentration, compounds were separated as cations. Conventional approaches to reduce CE analysis time demonstrated critical resolution between some analytes. Therefore, the use of the partial-filling technique compatible with MS detection was carried out. Under optimized conditions, the analysis time for the chiral separation of seven AT like compounds was reduced to 6 min. Moreover, sensitivity of CE-MS was sufficient for the determination of ATs in plasma following a simple liquid-liquid extraction.     

Enantiomeric separation of dihydroxyphenylalanine (DOPA), methyldihydroxyphenylalanine (MDOPA) and hydrazinomethyldihydroxyphenylalanine (CDOPA) by using capillary electrophoresis with sulfobutyl ether-beta-cyclodextrin as a chiral selector

Capillary electrophoresis (CE) was successfully applied to the enantiomer resolution of racemic structurally related compounds, namely dihydroxyphenylalanine (DOPA), methyldihydroxyphenylalanine (MDOPA) and hydrazinomethyldihydroxyphenylalanine (CDOPA). The chiral resolution was performed in an untreated fused-silica capillary by using a phosphate buffer at pH 2.5 or 3.0 supplemented with sulfobutylated beta-cyclodextrin (SBE-CD). Resolution was strongly influenced by the concentration of the chiral selector added to the background electrolyte. In fact, 2-5 mM of SBE-CD enabled the resolution of DOPA and MDOPA enantiomers, while CDOPA optical isomers were resolved by using either 0.5 mM or 6-20 mM of SBE-CD. The latter separation conditions (reversed polarity mode) made it possible to obtain inversion of migration order.     

Determination of antihistamines in pharmaceuticals by capillary electrophoresis.

Capillary electrophoresis with on-column UV detection at 214 nm was used to separate a group of nine antihistamines. All these compounds were satisfactorily separated within ca. 6 min using a mixed carrier system containing sodium dodecyl sulphate with beta-cyclodextrin and tetrabutylammonium hydrogensulphate as modifiers. The application of the method to the determination of the amount of antihistamines present in commercial pharmaceutical samples was demonstrated. In addition, the migration behaviour of the antihistamines in the mixed carrier system was examined.     

酸性药物的反向电渗流高效毛细管电泳分离分析研究

以水杨酸、乙酰水杨酸为代表药物,采用十六烷基三甲基溴化铵（ＣＴＡＢ）为电渗流改向剂,考察了酸性药物在反向电渗流高效毛细管电泳体系中的分离行为,并对其中影响迁移时间、峰形及柱效的诸多因素进行了系统研究。研究结果表明,以阳离子表面活性剂作为电渗流改向剂的反向电渗流高效毛细管电泳体系能显著加快酸性药物的分析速度。对于ＣＴＡＢ与酸性药物相互作用导致峰形展宽、柱效降低的现象,可通过加入合适的有机添加剂（如β－环糊精或乙腈）加以改善。     

Capillary Electrophoresis for the Simultaneous Determination of Metals by Using Ethylenediamine Tetraacetic Acid as Complexing Agent and Vancomycin as Complex Selector

A new separation system of capillary electrophoresis for the simultaneous determination of metals by using ethylenediamine tetraacetic acid （EDTA） as complexing agent and employing vancomycin as complex selector was described. The Z-shape cell capillary electrophoresis was used to enhance the sensitivity for the determination of the complexes of Cu（Ⅱ）, Ni（Ⅱ）, Co（Ⅱ） and Fe（Ⅲ） with EDTA. The partial filling method （co-current mode） was used in order to increase the selectivity of the electrophoretic method, meanwhile vancomycin was not present at the detector path during the detection of metal-EDTA complexes. The vancomycin concentration, phosphate concentration and pH of the buffer strongly influenced mobility, resolution and selectivity of the studied analytes. Under the optimal condition, the relative standard deviations （n=5） of the migration time and the peak area were less than 3.14% and 7.35%, respectively. Application of the Z-shape cell capillary electrophoresis method with UV detection and vancomycin loading led to the reliable determination of these metal ions in tap water and the recoveries were 97%-101%. The detection limits based on a signal to noise ratio of 3 ： 1 were found in the range of 2-10 μg·L^-1.     

New possibilities of micellar electrokinetic chromatography and microemulsion electrokinetic chromatography in the determination of catechols and catecholamines in natural samples

New possibilities were revealed and evaluative characteristics were obtained for different versions of capillary electrophoresis with UV detection, namely, micellar electrokinetic chromatography with normal and reversed polarity and microemulsion electrokinetic chromatography, used for determining catechols and catecholamines in green and black tea and in urine.     

Affinity chromatography on vancomycin coupled to reversed phase liquid chromatography/ electrospray-ion trap mass spectrometry for the screening of combinatorial libraries

Summary The combination affinity chromatography-mass spectrometry can be drastically improved by introducing a reversed phase column prior to the mass spectrometric detection. The interactions of the macrocyclic antibiotic vancomycin with oligopeptides were used to illustrate the performance of the technique. A library of 36 peptides was successfully screened and the active compounds identified by electrospray MS⁽ⁿ⁾. The strong affinity of compounds ending with (D)-alanine and with (D)-alanine or an aromatic (D)-amino acid in the penultimate position with vancomycin was confirmed.     

Enantioselective separation of epoxides by capillary electrophoresis employing sulfated beta-cyclodextrin as chiral selector

A capillary electrophoresis method was developed for the enantioseparation of epoxide compounds. Sulfated beta-cyclodextrin was employed as a chiral selector. Phosphate-triethanolamine buffer showed a chiral separation effect when employing charged sulfated beta-cyclodextrin. The effect of pH, triethanolamine concentration and sulfated beta-cyclodextrin concentration on the resolution was studied. Methanol was tested as an organic modifier. Several other epoxides were successfully separated by the proposed method.     

New preconditioning strategy for the determination of inorganic anions with capillary zone electrophoresis using indirect UV detection

It is widely accepted that preconditioning procedures are indispensable in capillary electrophoresis in order to achieve reproducibility of migration times and peak areas. Several preconditioning strategies have been employed for electrophoretic determinations of inorganic anions using indirect UV detection including simple flushing with buffer or alkaline or acid pre-rinsing followed by flushing with electrolyte. We investigated the influence of various preconditioning strategies on the reproducibility of migration times and peak areas of inorganic anions. The electrolyte systems for indirect UV detection were based on pyromellitic acid and chromic acid respectively as UV absorbing probes and hexamethonium hydroxide as electroosmatic flow modifier. Preconditioning agents under investigation were electrolyte buffer, NaOH, HCl and the free acids of the UV absorbing probes. Investigations showed that reproducibility of migration times and peak areas can be significantly improved by acid pre-rinsing using the corresponding acid of the UV absorbing probes compared to preconditioning by flushing the capillary with buffer. In contrast to acid pre-rinsing using hydrochloric acid no interfering signals within the migration time window of inorganic anions under investigation can be observed. The optimized preconditioning procedure yields relative standard deviations of migration times less than 0.25% (n=10). Relative standard deviations of corrected peak areas were below 5% applying acid preconditioning using pyromellitic acid.