Determination of association constants between enantiomers of orciprenaline and methyl-beta-cyclodextrin as chiral selector by capillary zone electrophoresis using a partial filling technique

The principles for the determination of conditional association constants of enantiomers by capillary zone electrophoresis employing a partial filling technique (PFT) using methyl-beta-cyclodextrin as chiral selector is presented. Orciprenaline was used as a model compound. Partial filling is a separation technique, where different lengths of the chiral selector solution are introduced into the capillary to a final zone length shorter than the effective length of the capillary, prior to application of the solutes. Lengthening of the separation zone results in improving enantioresolution in addition to decreasing electrophoretic mobility of the enantiomers, because of longer interaction time between the solute and chiral selector. The degree of the reduction in electromobility depends on the affinity of the solute to the chiral selector, i.e. strength of the complex formed between the solute and cyclodextrin. The decrease in the electrophoretic mobility with increasing length of the separation zone is used for determination of the association constant. The association constants of the enantiomers of orciprenaline and the chiral selector were evaluated from the slope of the plot, observed electrophoretic mobility versus the ratio between the length of the separation zone and the effective length of the capillary. It was found that the association constants were independent of the chiral selector concentration. The mean values were 110 M(-1) and 160 M(-1) for respective enantiomer. Constants obtained by a conventional CE technique were in good agreement with those from the PFT experiments. The highest enantioselectivityy was obtained when about 50% of the solute was distributed to the selector phase.     

Efficient applications of capillary electrophoresis-tandem mass spectrometry to the analysis of adrenoreceptor antagonist enantiomers using a partial filling technique

Throughout the separation of chiral basic drugs by capillary electrophoresis (CE) with neutral hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral selector, the sensitivity of detection can be improved by using tandem mass spectrometric (MS-MS) detection with a partial filling technique rather than with UV spectrometric detection. Prior to sample injection. the capillary was partly filled with HP-beta-CD dissolved in volatile ammonium formate buffer (pH 4, ionic strength 50 mM). The effects of modifying the HP-beta-CD concentration in the selector zone and the length of the separation zone on the enantioresolution and the signal-to-noise ratio of the pseudo-molecular MH+ ion were investigated. For a given selector zone length, as the concentration of the neutral cyclodextrin increases, the resolution between enantiomers becomes higher (the opposite of the behavior of the signal-to-noise ratio) and then reaches an optimum value. The decrease of the selector zone length lowered the resolution between the enantiomers but increased peak efficiencies and signal-to-noise ratio values. Accordingly, partial capillary filling at 80% (v/v) and 10 mM concentration of HP-beta-CD was selected as a suitable compromise between resolution and sensitivity of MS detection. Limits of detection for each adrenoreceptor antagonist enantiomer were 5 ng/ml (0.02 microM) in CE-MS-MS instead of 150 ng/ml (0.60 microM) in CE-UV, which enhances sensitivity by a factor of 30.     

Evaluation of balhimycin as a chiral selector for enantioresolution by capillary electrophoresis

The glycopeptide antibiotic balhimycin and its haloanalogue bromobalhimycin were evaluated as chiral selectors for enantioresolution by capillary electrophoresis. In order (i) to eliminate the adsorption of the glycopeptide antibiotics on the capillary wall, (ii) to shorten the separation time and (iii) to improve the detection sensitivity, a combined approach of the dynamic surface coating technique, the co-electroosmotic flow electrophoresis technique and the partial filling technique was employed for the enantioresolution of 16 acidic racemates. The effect of experimental parameters (plug length of the partial filling solution containing the chiral selector, selector concentration and buffer pH) on enantiorecognition was investigated. Furthermore, the enantiorecognition ability imparted by balhimycin, bromobalhimycin and vancomycin were compared. For most tested compounds, the highest enantiorecognition was obtained with balhimycin as chiral selector. Only in the case of the enantioresolution of tiaprofenic acid, vancomycin showed a superior enantiorecognition.     

卡那霉素作为手性选择剂的毛细管电泳手性药物分离研究

建立了一种以天然易得的卡那霉素为手性添加剂，用毛细管区带电泳法快速分离市售对乙肝有良好治疗效果的药物联苯双脂衍生物的方法，拓宽了毛细管电泳中手性选择剂的范围，通过实验研究了卡那霉素、甲醇 含量PH值，磷酸盐缓冲体系和硼硝缓冲体系对手性分离的影响，以及三种有机溶剂（甲醇、乙晴、异丙醇）添加剂对手性分离的影响，结果表明，在含有3％卡那霉素，30mol/L，硼砂缓冲体系（PH＝8．0）添加30％异丙醇是最佳的分离条件。     

Simultaneous stereoselective analysis of tramadol and its main phase I metabolites by on-line capillary zone electrophoresis-electrospray ionization mass spectrometry

On-line combination of partial filling capillary electrophoresis and electrospray ionization mass spectrometry was demonstrated for the simultaneous enantioseparation of tramadol and its main phase I metabolites. The partial filling technique was efficient at avoiding MS contamination by the chiral selector. Different experimental factors were investigated, including the chiral selector nature and concentration, plug length as well as the separation temperature. The best enantioseparation of the investigated compounds was achieved with a coated polyvinyl alcohol capillary and a 40 mM ammonium acetate buffer, pH 4.0, adding sulfobutyl ether beta-cyclodextrin (2.5 mg/ml) as the chiral selector. The charged cyclodextrin not only allowed enantioseparation of tramadol and its metabolites, but also improved the selectivity of compounds with the same molecular mass. Finally, CE-electrospray ionisation-MS was successfully applied to the stereoselective analysis of tramadol and its main metabolites in plasma after a simple liquid-liquid extraction.     

Evaluation of quail egg white riboflavin binding protein as a chiral selector in capillary electrophoresis by applying a modified partial filling technique.

A preliminary evaluation of the enantioselective properties of quail egg yolk riboflavin binding protein (qRfBP) was carried out in capillary electrophoresis by using the complete filling technique. The most promising results obtained by this screening of nineteen chiral drugs were singled out with the aim of optimizing enantiomer separations by applying the partial filling technique, which allows operating at much higher protein concentrations without detection problems. The building of the separation zone in the partial filling technique has been modified in order to enable on-line monitoring, before each run, of the actual protein plug application velocity and, consequently, the building of a plug of the desired length. The electrophoretic conditions chosen gave opposite migration directions for the chiral selector and the analytes, with qRfBP migrating away from the detector. A polyvinyl alcohol-coated capillary was first totally filled with protein and the optimal plug length was obtained by further applying negative pressure together with positive voltage for the time needed. Separations of basic drugs were optimized by using protein concentrations ranging from 200 microM up to 900 microM and different plug lengths, while the running buffer pH (6.0), temperature (25 degrees C) and operating voltage (+20 kV) were kept constant. The enantioresolution of all solutes was affected by both the chiral selector concentration and protein plug length. Baseline separations were obtained for oxprenolol, prilocaine and bupivacaine.     

Simultaneous separation and enantioresolution of racemic local anesthetic drugs by capillary zone electrophoresis with Tween 20 and methyl-beta-cyclodextrin as selectors, employing a double plug technique

A new approach for simultaneous chiral and achiral separations by capillary zone electrophoresis is described. Two adjacent selector plugs, consisting of Tween 20 as an achiral and methyl-beta-cyclodextrin (CD) as a chiral selector, are employed and four related local anesthetics are used as model compounds. The principles of the partial filling technique, whereby the capillary is filled with the chiral selector solution followed by the micellar solution at different plug lengths and concentrations, prior to application of the solutes, was employed. During the run both capillary ends were dipped in a simple buffer, i.e., one without additives. The two separation media worked independently without any interaction. Separation of the solutes and their enantiomers was regulated by adjusting both the concentration and plug length (PL) of the micellar solution in the capillary, employing methyl beta-CD as chiral selector either at 38 or 76 mM. The solutes were separated on the basis of their affinity towards the micellar phase before they reached the methyl-beta-CD plug for enantioseparation. In the absence of the micellar plug, the enantiomers of prilocaine overlapped those of bupivacaine. The solutes and their enantiomers were completely separated by employing two adjacent plugs consisting of 100 mM Tween 20 solution (PL approximately 10 cm) and methyl-beta-CD solution at either 38 or 76 mM (PL approximately 30 cm).     

Chiral separation of gemifloxacin in sodium-containing media using chiral crown ether as a chiral selector by capillary and microchip electrophoresis

Chiral crown ether, (+)-(18-crown-6)-tetracarboxylic acid (18C6H(4)), is an effective chiral selector for resolving enantiomeric primary amines owing to the difference in affinities between 18C6H(4) and each of the amine enantiomers. In addition to the destacking effect of sodium ion in the sample solution, the strong affinity of sodium ion to the polyether ring of crown ether is unfavorable to chiral capillary electrophoresis using 18C6H(4) as a chiral selector. In this report, the chiral separation of gemifloxacin dissolved in a saline sample matrix using 18C6H(4) was investigated. Adding a chelating agent, ethylenediaminetetraacetic acid (EDTA), to the run buffer greatly improved the separation efficiencies and peak shapes. The successful chiral separation of gemifloxacin in a urinary solution was demonstrated for both capillary and microchip electrophoresis.     

Competitive binding of a Tris run buffer with chiral crown ether in chiral capillary electrophoresis

In capillary electrophoresis of primary amine racemates using (+)-(18-crown-6)-tetracarboxylic acid (18C6H4) as a chiral selector, chiral recognition emanates from the differences in the complex formation between 18C6H4 and the two protonated amine enantiomers. The presence of buffer constituents such as tris(hydroxymethyl)aminomethane (Tris) or Na+, capable of forming complexes with 18C6H4, is thus detrimental to the chiral separation of primary amines. Such a competitive binding of buffer constituents was studied by comparing the electrophoretic mobilities of racemic analytes obtained in Tris/citric acid and triethylamine/citric acid buffers. We developed a simple fitting method to determine the competitive binding constant and applied it to the Tris buffer system. The competitive binding constant of Tris with 18C6H4 obtained at pH 3.0 was 27 +/- 4.     

Enantioseparation of amino acid derivatives by capillary zone electrophoresis using vancomycin as chiral selector

The separation of racemic derivatized amino acids (N-acetyl) into their enantiomers was achieved using capillary zone electrophoresis employing vancomycin as a chiral selector. Due to the strong absorption properties of the chiral selector at the low wavelengths used, the partial-filling countercurrent method was adopted in order to improve method sensitivity. In the separation system studied, the chiral selector filled only a part of the capillary and, due to the appropriate selection of the pH, was moving in the opposite direction of the analytes keeping the detector free from absorbing compounds. The effect of several experimental parameters on the enantioresolution of analytes was studied, e.g., vancomycin concentration (0-5 mM), pH of the background electrolyte (pH 4-7), capillary temperature (15-35 degrees C), and the presence of an organic modifier in the run buffer (methanol or ethanol or n-propanol). N-Acetyl glutamic acid, serine, cystine, tyrosine, and proline were all baseline-resolved into their enantiomers and the enantioresolution factor (R(s)) was increased by raising the vancomycin concentration. pH 4 allowed the baseline resolution of the five studied analytes in the presence of 2.5 mM of chiral selector and an increase in pH caused a decrease of R(s).     

Partial filling technique in capillary electrophoresis for the separation of phenolic isomers with sulfonatocalix[4]arene as a selector

p-Sulfonatocalix[4]arene was used as a selector in capillary electrophoresis to separate phenolic positional isomers. To avoid the detection interference caused by the high UV absorption of calixarene, the partial filling technique was applied. The operation variables, including buffer, separation voltage, the concentration of the selector and the plug length of the selector zone, were systematically optimized. The detection limits of mass were in the range of 0.07-0.28 pg. Molecular modeling was used to explain the interaction between calixarene and phenolic isomers.     

Chiral counter-current chromatography of gemifloxacin guided by capillary electrophoresis using (+)-(18-crown-6)-tetracarboxylic acid as a chiral selector

(+)-(18-crown-6)-tetracarboxylic acid (18C6H4) has been known as a highly efficient chiral selector for resolving primary amine enantiomers in capillary electrophoresis (CE). We investigated the chiral separation of gemifloxacin using 18C6H4 in analytical counter-current chromatography (CCC). The separation conditions for CE, including the binding constant, pH, and run buffer constituents, provided a helpful guideline for chiral CCC. A successful separation of gemifloxacin enantiomers could be achieved using a two-phase solvent system composed of 1-butanol-ethyl-acetate-bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane acetate buffer with a small amount of 18C6H4. The hydrophobicity of the solvent system and the 18C6H4 concentration were varied to optimize the chiral separation.     

Comparative studies of various run buffers for chiral capillary electrophoresis using chiral crown ether as a chiral selector

In the capillary electrophoretic separation of primary amine enantiomers using (+)-(18-crown-6)-tetracarboxylic acid (18C6H4) as a chiral selector, the presence of run buffer constituents such as tris(hydroxymethyl)aminomethane (Tris) or Na+ competing with analytes for 18C6H4, diminishes the effectiveness of 18C6H4. In order to determine appropriate buffer systems for 18C6H4, various run buffer cationic components including Tris, 1,3-bis[tris(hydroxymethyl)methylamino]propane, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, triethanolamine, tetramethylammonium, and Na+ were compared. Quantitative studies of the effects of the competitive constituents were carried out by measuring the electrophoretic mobilities of histidine as a function of the 18C6H4 concentration. We also derived a simple equation to estimate the optimal chiral selector concentration for a maximum mobility difference in the presence of a competitive inhibitor.     

Electrokinetic partial filling technique as a powerful tool for enantiomeric separation of DL-lactic acid by CE with contactless conductivity detection

A modified partial filling method for chiral separation of DL-lactic acid as the model chiral compound with vancomycin chloride as the chiral selector was developed by CE with contactless conductivity detection. Electrokinetic partial filling technique (EK-PFT) was used as an alternative method to the conventional hydrodynamic partial filling method. EK-PFT, in contrast to the hydrodynamic partial filling technique, allowed the removal of the chloride counterions from the chiral selector which otherwise led to poor sensitivity in conductivity detection. The baseline separation of DL-lactic acid as the model analyte was achieved in 5 min in a polyacrylamide-coated capillary. The best resolution was achieved by electrokinetic partial filling of vancomycin cations from the injection solution containing 5 mmol/L oxalate L-histidinium at pH 4.5 with 10 mmol/L vancomycin chloride. Computer simulation was used to explain the observed phenomena in the boundary between the inject vial and the capillary during the EK-PFT of vancomycin cations.     

Fast enantiomeric separation with vancomycin as chiral additive by co-electroosmotic flow capillary electrophoresis: increase of the detection sensitivity by the partial filling technique

A fast and sensitive method is described by using vancomycin as a chiral additive for enantiomeric separation by capillary electrophoresis (CE). In order to overcome disadvantages associated with use of vancomycin as chiral additive in CE, several strategies including the dynamic coating technique, the co-electroosmotic flow technique, and the partial filling technique were employed sequentially in this method. Using the polycationic polymer hexadimethrine bromide (HDB) as a buffer additive, the capillary wall was dynamically coated with a thin film formed by the adsorbed HDB. Consequently, the adsorption of vancomycin onto the capillary wall was minimized via electrostatic repulsion between the coating of the capillary wall and the vancomycin molecule. In addition, the reversed electroosmotic flow (from cathode to anode) produced by the positively charged capillary wall migrates in the same direction of negatively charged analytes (co-electroosmotic flow electrophoresis). Thereby the electrophoretic mobility of negatively charged analytes were drastically accelerated leading to a short separation time of less than 3.4 min. The separation time was further reduced by the use of a short-end-injection technique. For example, the analysis time was achieved by as short as 55 s for a baseline separation of dansyl-alpha-amino-n-butyric acid. Concurrently, the partial filling technique was used to avoid the loss of detection sensitivity caused by the presence of vancomycin in the running buffer. The effect of several parameters, such as HDB concentration, buffer pH, plug length of the chiral selector, concentration of the chiral selector and applied voltage, on enantioselectivity were investigated toward optimization. Besides the advantage of a very short separation time, the method is characterized by high detection sensitivity, high selectivity, and high efficiency.     

Method Development of Enantiomer Separations by Affinity Capillary Electrophoresis,Cyclodextrin Electrokinetic Chromatography and Capillary Electrophoresis-Mass Spectrometry

 Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection     

Development of stereoselective nonaqueous capillary electrophoresis system for the resolution of cationic and amphoteric analytes

A stereoselective ion-pair nonaqueous capillary electrophoresis (NACE) method employing the partial filling technique with N-derivatized amino acids, e.g., (R)- and (S)-3,5-dinitrobenzoyl-leucine (DNB-Leu), as chiral selector for the separation of "pseudoenantiomeric" cinchona alkaloid derivatives and other structurally related basic compounds like the enantiomers of mefloquine is presented. Originating from NACE with cinchona alkaloid derivatives as chiral counterions, this method was developed by application of the reciprocity principle of chiral recognition, which was proven to be valid for stereoselective ion-pair capillary electrophoresis (CE). A variety of basic and amphoteric selectands (SAs) could be well resolved. Thereby, the separation was primarily based on stereoselective ion-pair formation of corresponding SA stereoisomers and mobility differences of free and complexed (ion-paired) SAs. Additionally, in the case of diastereomeric SAs, naturally existing mobility differences between the diastereomers played also a role, but was shown by control experiments with racemic DNB-Leu and without selector (SO) to be of minor contribution to overall separation selectivity. Due to its simplicity, speed, and good reproducibility, the established method can be utilized for fast screening of cationic as well as amphoteric chiral compounds, and therefore is a valuable tool in the development of new chiral selectors and chiral stationary phases. Small sample amounts of the SO (4-5 mg) and only analytical amounts of SAs are needed, and about 20-50 compounds per day can be tested.     

Enantiomeric separation of gemfibrozil chiral analogues by capillary electrophoresis with heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin as chiral selector

The enantiomeric separation of gemfibrozil chiral analogues was performed by capillary zone electrophoresis (CZE). Resolution of the enantiomers was achieved using heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TM-beta-CD) as chiral selector dissolved into a buffer solution. In order to optimize the separation conditions, type, pH and concentration of running buffer and chiral selector concentration were varied. For each pH value, the optimum chiral selector concentration that produced the resolution of the isomers was found. The migration order of labile diastereoisomers formed was valued at the optimum experimental conditions by adding a pure optical isomer to the racemic mixture. Data from 1H NMR studies confirmed host-guest interaction between TM-beta-CD and 5-(2,5-dimethylphenoxy)-2-ethylpentanoic acid sodium salt. The hypothesized stoichiometry host:guest was 1:1. An apparent equilibrium constant (Ka) was estimated monitoring the chemical shift variation as a function of TM-beta-CD concentration. Salt effect on complexation equilibrium constant was also investigated.     

Development of a capillary electrophoresis method for the enantioselective estimation of primaquine in pharmaceutical formulations

A capillary electrophoresis (CE) method has been developed that allows the separation and estimation of primaquine enantiomers using hydroxypropyl-gamma-cyclodextrin (HP-gamma -CD) as a chiral selector. The influence of chemical and instrumental parameters on the separation, such as type and concentration of CD, buffer concentration, buffer pH, applied voltage, capillary temperature, and injection time, were investigated. Good separation of the racemic mixture of primaquine was achieved using a fused-silica capillary (52.5 cm effective length x 50 microm id) and a background electrolyte composed of tris-phosphate buffer solution (50 mM, pH 2.5) containing 15 mM HP-gamma-CD as a chiral selector. The recommended applied voltage, capillary temperature, and injection time were 15 kV, 25 degrees C, and 6 s, respectively. Within-day and interday reproducibility of peak area and migration time gave relative standard deviation values ranging from 1.05-3.30%. Good recoveries (range of 96.8-104.9%) were obtained from the determination of placebos that were spiked with 0.25-1.00 mg/L primaquine. The proposed CE method was successfully applied to the assay of primaquine diphosphate in pharmaceutical formulations (tablets).     

In-situ grown metal organic framework synergistic system for the enantioseparation of three drugs in open tubular capillary electrochromatography

Metal organic frameworks have received great attention as the chiral stationary phase for racemic drug separation because of their fascinating structures and properties. However, the most homochiral metal organic frameworks were constructed by rare and precious chiral organic ligands. In this work, an achiral metal organic framework, together with a natural chiral selector carboxymethyl β-cyclodextrin built a synergistic separation system in the open tubular capillary electrochromatography. The novel coated columns were developed by inducing metal organic framework nanoparticles to grow on the imidazolyl functional capillary inner wall. The baseline separations of hydroxychloroquine, ofloxacin, and atenolol were achieved in the synergistic separation system. The effects of the concentration of chiral selector, pH, voltage, and the concentration of organic additives were studied. Compared with chiral selector auxiliary bare capillary, the resolutions of three drugs were remarkably improved. The relative standard deviations for the retention time of intraday (n = 6), interday (n = 6), and column-to-column were less than 2.1, 2.6, and 5.2%, respectively. These results demonstrate that affordable synergistic separation systems are prospective for racemic drug enantioseparation in capillary electrochromatography.