Separation of etodolac enantiomers by capillary electrophoresis. Validation and application of the chiral method to the analysis of commercial formulations

Separation of etodolac enantiomers, which exhibit different biological activity and pharmacokinetic profiles, has been achieved using the randomly substituted (2-hydroxy)propyl-beta-cyclodextrin (HP-beta-CD) as chiral selector in capillary electrophoresis. The selection of this CD was made after screening of different CD derivatives of neutral and anionic nature. The effect on the enantioresolution of the buffer concentration and of the degree of substitution (DS) and concentration of the CD as well as of instrumental parameters, such as the capillary temperature and the separation voltage, were studied. The highest resolution of etodolac enantiomers was around 2.5 using 100 mM phosphate buffer (pH 7.0) with 20 mM HP-beta-CD (DS approximately 4.2) and UV detection at 225 (10) nm with a reference wavelength at 360 (50) nm. Validation of the chiral method in terms of selectivity, linearity, precision (instrumental repeatability, method repeatability, intermediate precision), and the limits of detection and quantitation allowed to evaluate its quality to the analysis of etodolac enantiomers in different pharmaceutical preparations containing racemic etodolac.     

Separation of enantiomers by capillary electrophoresis-mass spectrometry employing a partial filling technique with a chiral crown ether

Enantiomer separations were performed by capillary electrophoresis-mass spectrometry (CE-MS) with (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H4) as a chiral selector. In order to prevent the introduction of the nonvolatile chiral, selector, 18C6H4, into the nozzle of the CE-MS interface and/or the orifice plate, a partial filling technique was employed in this study. By the partial filling technique, the contamination caused by the nonvolatile chiral selector was avoided not only during the analysis but also during the washing of capillary with the separation solution prior to the run. Several racemic compounds having a primary amino group were successfully separated. Racemic 3-aminopyrrolidine and racemic alpha-amino-epsilon-caprolactam have no strong UV absorption, but such compounds were detected with a high sensitivity by MS detection. In this paper, the effects of the length of separation zone and those of the 18C6H4 concentration were described. As the length of the separation zone was longer or as the concentration of 18C6H4 was higher, the enantiomer resolution was enhanced more and more. However, the optimization of 18C6H4 concentration was practically enough to obtain the baseline separation.     

Chiral separation of four fluoroquinolone compounds using capillary electrophoresis with hydroxypropyl-beta-cyclodextrin as chiral selector

A capillary zone electrophoresis (CZE) investigation on the enantiomeric separation of lomefloxacin, gatifloxacin, pazufloxacin and ofloxacin was undertaken. Resolution of the enantiomers was achieved using hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as the chiral selector. Parameters influencing separation include cyclodextrin concentration, separational potential, pH and organic additive are discussed. A buffer consisting of 70 mM phosphate and 40 mM HP-beta-CD at pH 3.96 was found to be highly efficient for the separation of lomefloxacin, at pH 3.90 for gatifloxacin, at pH 5.04 for pazufloxacin and at pH 2.16 for ofloxacin. To the best of our knowledge, this is the first report on the enantiomeric resolution of lomefloxacin and gatifloxacin applying CE.     

Chiral separation of amino acids derivatized with fluoresceine-5isothiocyanate by capillary electrophoresis and laser-induced fluorescence detection using mixed selectors of beta-cyclodextrin and sodium taurocholate

Chiral separation of 20 pairs of amino acids derivatized with fluoresceine-5-isothiocyanate (FITC) by capillary electrophoresis and laser-induced fluorescence detection was studied using the mixture of beta-cyclodextrin (beta-CD) and sodium taurocholate (STC) as selector. Resolution was considerably superior to that obtained by using either beta-CD or STC alone. The molar ratio of beta-CD to STC of about 2:3 was found to be critical to achieve maximum separation. At this beta-CD-to-STC ratio, chiral separation occurred at really low total concentration of beta-CD and STC (<0.1 mM). Other impacting factors were investigated including the total concentration of beta-CD and STC, pH, and capillary conditioning procedure between two successive runs. Using a running buffer of 80 mM borate containing 20 mM beta-CD and 30 mM STC at pH 9.3, all of the 20 pairs of FITC-amino acid enantiomers were baseline resolved. The resolutions of the most pairs of the amino acid enantiomers (17 of 20) were higher than 3.0, only three pairs gave a resolution lower than 3.0 but higher than 1.90 (beta-phenylserine, pSer). The highest resolution reached 14.58 (Glu). Two derivatives of beta-CD, 2-hydroxypropyl-beta-CD (HP-beta-CD) and heptakis(2,6-di-O-methyl)-beta-CD (DM-beta-CD) were also explored. HP-beta-CD showed similar cooperative effect with STC, while DM-beta-CD together with STC led to poorer chiral separation.     

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.     

Application of capillary electrophoresis with field-amplified sample injection for the detection of new adrenoreceptor antagonist enantiomers in plasma in the low ng/mL concentration range

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 has been improved by using field-amplified sample injection (FASI). In the present work, this on-line stacking method has been used to detect low ng/mL levels of cationic enantiomers of a new adrenoreceptor antagonist in plasma. A systematic study of the parameters affecting on-line concentration of these enantiomers (nature of the preinjection plug, composition of sample solvent, injection times of water and sample plugs) has been performed enabling the detection sensitivity of antagonist enantiomers to be improved by 180 times compared with usual hydrodynamic injection. The quantification of each adrenoreceptor antagonist enantiomer in plasma samples was then performed in the 2-100 ng/mL (or 8-400 nM) concentration range after a solid-phase extraction step. Using this FASI-CE-UV procedure, the limit of quantification (LOQ) for each enantiomer was in the low ng/mL concentration range (3 ng/mL or 10 nM).     

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).     

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.     

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.     

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.     

Retention behaviour of peptides in capillary electrochromatography using an embedded ammonium in dodecacyl stationary phase

A highly water-soluble new cyclodextrin (CD) derivative 2-O-acetonyl-2-O-hydroxypropyl-beta-CD (2-AHP-beta-CD) was synthesized and tested as an effective chiral selector for the capillary zone electrophoretic resolution (Rs) of several basic and acidic analytes. The primary purpose of the research was to explore the capability of the 2-AHP-beta-CD as chiral selectors on comparison with the neutral CDs such as beta-CD, DM-beta-CD and HP-beta-CD. Substitution with 2-O-acetonyl-2-O-hydroxypropyl group at the secondary hydroxyl sites of the CD is aimed at influencing the magnitude and selectivity of analyte-CD interactions. The chiral resolution was strongly influenced by the concentration of the CDs and buffer pH. 2-AHP-beta-CD showed the best enantiomer resolution properties among the tested compounds, while the other CDs showed inferior or no performances at all.     

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).     

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).     

Effects of partial/asymmetrical filling of micelles and chiral selectors on capillary electrophoresis enantiomeric separation: generation of a gradient

The effect of several experimental parameters on enantiomeric separations in micellar capillary electrophoresis (MCE) was studied. A model separation system was tested. It was composed of an acidic phosphate buffer with sodium dodecyl sulfate (SDS) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as the chiral selector. A substituted angelicin was used as a chiral analyte. Changes in the concentration of SDS micelles/SDS monomers in the presence of HP-beta-CD and their impact on the enantioselective separation were investigated. Variation of the composition of electrolytes in the individual compartments of the separation system (inlet vial, capillary, and outlet vial) affected both the migration times and the resolution of the enantiomers. Current vs. time dependencies also were monitored during the separations. A mathematical model of electromigration in micellar systems with chiral selector present was proposed and a computer simulation was used to explain the observed phenomena and to confirm the generation of a CD/SDS-micelle concentration gradient under certain experimental conditions. This is the reported first attempt of a computer simulation of the complex, dynamic chiral environment of the CD-SDS-MCE system.     

Chiral separation of β‐blockers by MEEKC using neutral microemulsion: Analysis of separation mechanism and further elucidation of resolution equation

Based on the investigation of the effect of microemulsion charge on the chiral separation, a new chiral separation method with MEEKC employing neutral microemulsion was established. The method used a microemulsion containing 3.0% (w/v) neutral surfactant Tween 20 and 0.8% (w/v, 30 mM) dibutyl l ‐tartrate in 40 mM sodium tetraborate buffer to separate the enantiomers of β‐blockers. The effect of major parameters on the chiral separation was investigated. The applied voltage had little effect on the resolution, but the chiral separation could be improved by suppressing the EOF. Nine racemic β‐blockers obtained relatively good enantioseparation after appropriate concentrations of tetradecyl trimethyl ammonium bromide were added into the microemulsion to suppress the EOF. These results were explained based on the analysis of the separation mechanism of the method and deduced separation equations. The resolution equation of the method was further elucidated. It was found that the fourth term in the resolution equation, an additional term compared to the conventional resolution equation for column chromatography, represents the ratio of the relative movement distance between the analyte and microemulsion droplets relative to the effective capillary length. It can be regarded as a correction for the effective capillary length. These findings are significant for the development of the theory of MEEKC and the development of new chiral MEEKC method.     

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.     

Synthesis and application of clindamycin succinate as a novel chiral selector for capillary electrophoresis

A novel chiral selector, clindamycin succinate, was synthesized and first used as a chiral selector in capillary electrophoresis (CE). The chiral resolution ability of this kind of clindamycin derivation was studied by CE using some racemic drugs as model analytes. From the experimental results, it was found that both resolution and selectivity of the selector were dependent on the following parameters: concentration of chiral selectors, pH of the running buffer, temperature of the capillary column, applied voltage and organic modifier used. The results show that the chiral selector possesses high resolution toward some racemic drugs, including ofloxacin, chlorphenamine, tryptophan, propranolol, sotalol and metoprolol. Excellent chiral resolution of these tested drugs was achieved under the optimal conditions of 50 mM clindamycin succinate, 10% MeOH v/v, 50 mM Tris buffer, pH 4.0, at 22 kV and 20 °C within 25 min.     

Enantioseparation of cetirizine by sulfated-beta-cyclodextrin-mediated capillary electrophoresis

A sulfated beta-cyclodextrin (sulfated beta-CD)-mediated capillary electrophoresis method is described for the enantioseparation of cetirizine using achiral cefazolin as an internal standard. The enantioseparation of the drug was performed in a borate buffer (5 mM, pH 8.7) with 1% sulfated beta-CD (w/v) as chiral selector at 10 kV. Several parameters affecting the separation were studied, including the pH and the concentration of borate buffer and chiral selector. Under optimized conditions, a baseline separation of two enantiomers was achieved in less than 7 min. Using cefazolin as an internal standard (IS), the linear range of the method for the determination of levocetirizine was over 1.0 to 50.0 microg/mL; the detection limit (signal-to-noise ratio = 3) of levocetirizine was 0.5 microg/mL. The method allowed the enantioseparation of cetirizine in bulk samples and enantiomeric purity evaluation of levocetirizine (R-enantiomer) in pharmaceutical tablets (Xyzal), and it was also found to be suitable for enantioseparation in human plasma.     

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.     

Separation and determination of norepinephrine, epinephrine and isoprinaline enantiomers by capillary electrophoresis in pharmaceutical formulation and human serum

A capillary electrophoresis method with ultraviolet (UV) detection was developed and optimized for the enantiomer separation of norepinephrine (NE), epinephrine (EP) and isoprenaline (IP) using dual cyclodextrins (CDs) of 2-hydroxypropyl-beta-CD (HP-beta-CD) and heptakis (2,6-di-o-methyl)-beta-CD (DM-beta-CD) as chiral selectors. Optimal separation was obtained using a running buffer of 50mM phosphate containing 30mM HP-beta-CD and 5mM DM-beta-CD at pH 2.90 and a field strength of 20kV in 45cmx75mum (40cm effective length) uncoated capillary. The UV absorbance detection was set at 205nm. A 0.1% (w/w) polyethylene glycol or 0.1% (v/v) acetonitrile was used to enhance the detection sensitivity. There was a wide and excellent linear calibration graph for each enantiomer in the range 1.0x10(-3) to 1.0x10(-6)M and the detection limit (S/N=3) was found from 8.5x10(-7) to 9.5x10(-7)M. The method has been applied for the determination of isoprenaline in isoprenaline hydrochloride aerosol and to the analysis of serum samples. The recoveries of NE and EP in serum and IP in drug were ranged from 90 to 110%. The relative standard deviations of all the analyte peaks were less than 2.8% for migration time and less than 4.8% for peak area.