Cyclodextrin‐mediated capillary electrophoresis enantioseparation of dansylated β‐amino acids with bicyclo[2.2.2]octane,bicyclo[3.1.1]heptane and cyclopenta[d][1,2]oxazole core structures

The present study investigated the separation of bicyclic β‐amino acids with bicyclo[2.2.2]octane, bicyclo[3.1.1]heptane and cyclopenta[d][1,2]oxazole core structures by capillary electrophoresis using native cyclodextrins as well as neutral and charged derivatives as chiral selectors. The amino acids were derivatized with dansyl chloride to provide a UV chromophore. Separations were carried out at 20°C in a 48.5/40 cm, 50 µm fused‐silica capillary at an applied voltage of 20 kV. Fifty millimolar sodium phosphate background electrolytes pH 2.5 and 7.2 containing either 5 or 30 mg/mL of the CDs were used. For the majority of the investigated CDs, enantioseparations could only be achieved at pH 2.5 when the analytes are positively charged. Successful enantioseparations as negatively charged analytes at pH 7.2 were only observed for few compounds. In the case of methyl‐γ‐cyclodextrin, opposite enantiomer migration order was observed in pH 2.5 or 7.2 background electrolytes. Dependence of the enantiomer migration order on the size of the cavity of the cyclodextrins was also found. Furthermore, the degree of methylation of β‐cyclodextrin derivatives affected the migration order of several analyte enantiomers.     

Characterization of complexes between phenethylamine enantiomers and β‐cyclodextrin derivatives by capillary electrophoresis—Determination of binding constants and complex mobilities

To optimize chiral separation conditions and to improve the knowledge of enantioseparation, it is important to know the binding constants K between analytes and cyclodextrins and the electrophoretic mobilities of the temporarily formed analyte‐cyclodextrin‐complexes. K values for complexes between eight phenethylamine enantiomers, namely ephedrine, pseudoephedrine, methylephedrine and norephedrine, and four different β‐cyclodextrin derivatives were determined by affinity capillary electrophoresis. The binding constants were calculated from the electrophoretic mobility values of the phenethylamine enantiomers at increasing concentrations of cyclodextrins in running buffer. Three different linear plotting methods (x ‐reciprocal, y ‐reciprocal, double reciprocal) and nonlinear regression were used for the determination of binding constants with β‐cyclodextrin, (2‐hydroxypropyl)‐β‐cyclodextrin, methyl‐β‐cyclodextrin and 6‐O‐α‐maltosyl‐β‐cyclodextrin. The cyclodextrin concentration in a 50 mM phosphate buffer pH 3.0 was varied from 0 to 12 mM. To investigate the influence of the binding constant values on the enantioseparation the observed electrophoretic selectivities were compared with the obtained K values and the calculated enantiomer‐cyclodextrin‐complex mobilities. The different electrophoretic mobilities of the temporarily formed complexes were crucial factors for the migration order and enantioseparation of ephedrine derivatives. To verify the apparent binding constants determined by capillary electrophoresis, a titration process using ephedrine enantiomers and β‐cyclodextrin was carried out. Furthermore, the isothermal titration calorimetry measurements gave information about the thermal properties of the complexes.     

Enantioselective capillary electrophoresis for pharmacokinetic analysis of methadone and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine in equines anesthetized with ketamine and isoflurane

An enantioselective assay for the determination of methadone and its main metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine in equine plasma based on capillary electrophoresis with highly sulfated γ‐cyclodextrin as chiral selector and electrokinetic analyte injection is described. The assay is based on liquid/liquid extraction of the analytes at alkaline pH from 0.1 mL plasma followed by electrokinetic sample injection of the analytes from the extract across a buffer plug without chiral selector. Separation occurs cationically at normal polarity in a pH 3 phosphate buffer containing 0.16% (w/v) of highly sulfated γ‐cyclodextrin. The developed assay is precise (intra‐ and interday RSD < 4% and < 7%, respectively), is capable to determine enantiomer levels of methadone and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine in plasma down to 2.5 ng/mL, and was successfully applied to monitor enantiomer drug and metabolite levels in plasma of a pony that was anesthetized with racemic ketamine and isoflurane and received a bolus of racemic methadone and a bolus followed by constant rate infusion of racemic methadone. The data suggest that the assay is well suited for pharmacokinetic purposes.     

Influence of substituent position and cavity size of the regioisomers of monocarboxymethyl‐α‐, β‐, and γ‐cyclodextrins on the apparent stability constants of their complexes with both enantiomers of Tröger's base

Enantiomers of Tröger's base were separated by capillary electrophoresis using 2^I‐O‐, 3^I‐O‐, and 6^I‐O‐carboxymethyl‐α‐, β‐, and γ‐cyclodextrin and native α‐, β‐, and γ‐cyclodextrin as chiral additives at 0–12 mmol/L for β‐cyclodextrin and its derivatives and 0–50 mmol/L for α‐ and γ‐cyclodextrins and their derivatives in a background electrolyte composed of sodium phosphate buffer at 20 mmol/L concentration and pH 2.5. Apparent stability constants of all cyclodextrin–Tröger's base complexes were calculated based on capillary electrophoresis data. The obtained results showed that the position of the carboxymethyl group as well as the cavity size of the individual cyclodextrin significantly influences the apparent stability constants of cyclodextrin–Tröger's base complexes.     

Quality by design-guided development of a capillary electrophoresis method for the simultaneous chiral purity determination and impurity profiling of tamsulosin

Analytical Quality by Design principles using the design of experiments were applied for the development of a capillary electrophoresis method for the determination of enantiomeric purity and chemically related impurities of tamsulosin. From initial scouting experiments, a dual cyclodextrin (CD) system composed of sulfated β-CD and carboxymethyl-α-CD was selected as the chiral selector. A fractional factorial resolution V+ design was used for the identification of the critical process parameters, while a face-centered central composite design and Monte Carlo simulations were employed for final optimization and defining the design space of the method. The experimental conditions of the working point were: 30 mM sodium phosphate buffer, pH 3.0, containing 40 mg/mL sulfated β-CD and 7 mg/mL carboxymethyl-α-CD, capillary temperature 18°C, applied voltage -23 kV. Following the assessment of robustness by applying a Plackett-Burman design, the method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guideline Q2(R1). The method allowed the quantification of the chiral impurity and three other related impurities at the 0.1 % level with acceptable accuracy and precision.     

Dispersive micro‐solid‐phase extraction combined with online preconcentration by capillary electrophoresis for the determination of glycopyrrolate stereoisomers in rat plasma

A simple and sensitive analytical method for four isomers of glycopyrrolate in rat plasma was developed using cation‐selective exhaustive injection‐sweeping cyclodextrin‐modified electrokinetic chromatography (CSEI‐Sweeping‐CDEKC) for online enrichment combined with dispersive micro‐solid‐phase extraction pretreatment. The CSEI‐Sweeping‐CDEKC was conducted on an uncoated fused silica capillary (40.2 cm × 75 μm) with an applied voltage of –20 kV. The electrophoretic analysis was carried out in 30 mM phosphate solution at pH 2.0 containing 20 mg/mL sulfated‐β‐cyclodextrin and 5% acetonitrile. Under these optimized conditions, the detection limit for racemic glycopyrrolate was found to be 2.0 ng/mL and this method could increase 495‐fold detection sensitivity compared with the traditional injection method. Additionally, the parameters that affected the extraction efficiency of dispersive micro‐solid‐phase extraction were also examined systematically. The glycopyrrolate isomers in rat plasma samples as low as 0.0625 μg/mL were able to be separated and detected by capillary electrophoresis with the aid of CSEI‐sweeping. The findings of this study show that the dispersive micro‐solid‐phase extraction pretreatment coupled with CSEI‐Sweeping‐CDEKC is a rapid and convenient method for analyzing glycopyrrolate isomers in rat plasma.     

A cationic β‐cyclodextrin as a dynamic coating for the separation of proteins in capillary electrophoresis

A cationic cyclodextrin was used as dynamic coating for the capillary electrophoresis of a model mixture of proteins (i.e., ubiquitin, α‐lactoglobulin, cytochrome‐c, and myoglobin) as positively charged species in a fused silica capillary. An interesting feature of the coating is that by simple adjustment of the concentration of cyclodextrin added into the background electrolyte, a neutral or positively charged surface, which was beneficial in preventing protein adsorption at the inner capillary wall surface, was obtained. This is the first demonstration of a dynamic coating that yielded a neutral surface for protein separations in capillary electrophoresis. Based on electro‐osmotic flow measurements, addition of 0.05 to 0.10 mg/mL quaternary β‐cyclodextrin in a low pH electrolyte resulted in a neutral or positive surface (undetectable to very slow anodic electro‐osmotic flow). The coating approach afforded the electrophoretic separation of the mixture of proteins at positive polarity with good repeatability and separation performance.     

Surfactant‐assisted electromembrane extraction combined with cyclodextrin‐modified capillary electrophoresis for the separation and quantification of Tranylcypromine enantiomers in biological samples

Surfactant‐assisted electromembrane extraction coupled with cyclodextrin‐modified capillary electrophoresis was developed for the separation and determination of Tranylcypromine enantiomers in biological samples. This combination would provide a new strategy for selective and sensitive determination of target analytes. The addition of surfactant in the donor solution improved the analyte transport into the lumen of hollow fiber that resulted in an enhancement in the analytes migration into acceptor solution. Optimization of the variables, affecting proposed method, was carried out and best results were achieved with a 175 V potential as driving force of the electromembrane extraction, 2‐nitrophenyloctylether as the supported liquid membrane, donor solution containing 0.2 mM Triton X‐100 with pH 3 and 0.1 M HCl for acceptor solution. Then, the extract was analyzed using cyclodextrin‐modified capillary electrophoresis method for separation of Tranylcypromine enantiomers. The best results were obtained with a phosphate running buffer (100 mM, pH 2.0) containing 7% w/v hydroxypropyl‐α‐cyclodextrin. Under the optimum conditions, a low limit of detection (3.03 ng/mL), good linearity (R² > 0.9953), and relative standard deviations below 4.0% (n = 5) were obtained. Finally, this procedure was applied to determine the concentration of Tranylcypromine enantiomers in urine samples with satisfactory results.     

Preparative enantioseparation of loxoprofen precursor by recycling countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as a chiral selector

Recycling countercurrent chromatography was successfully applied to the resolution of 2‐(4‐bromomethylphenyl)propionic acid, a key synthetic intermediate for synthesis of nonsteroidal anti‐inflammatory drug loxoprofen, using hydroxypropyl‐β‐cyclodextrin as chiral selector. The two‐phase solvent system composed of n‐hexane/n‐butyl acetate/0.1 mol/L citrate buffer solution with pH 2.4 (8:2:10, v/v/v) was selected. Influence factors for the enantioseparation were optimized, including type of substituted β‐cyclodextrin, concentration of hydroxypropyl‐β‐cyclodextrin, separation temperature, and pH of aqueous phase. Under optimized separation conditions, 50 mg of 2‐(4‐bromomethylphenyl)propionic acid was enantioseparated using preparative recycling countercurrent chromatography. Technical details for recycling elution mode were discussed. The purities of both the S and R enantiomers were over 99.0% as determined by high‐performance liquid chromatography. The enantiomeric excess of the S and R enantiomers reached 98.0%. The recovery of the enantiomers from eluted fractions was 40.8–65.6%, yielding 16.4 mg of the S enantiomer and 10.2 mg of the R enantiomer. At the same time, we attempted to enantioseparate the anti‐inflammatory drug loxoprofen by countercurrent chromatography and high‐performance liquid chromatography using a chiral mobile phase additive. However, no successful enantioseparation was achieved so far.     

Use of various β‐cyclodextrin derivatives as chiral selectors for the enantiomeric separation of ofloxacin and its five related substances by capillary electrophoresis

A capillary electrophoretic method for the enantioseparation of ofloxacin and its five related substances (potential impurities, indicated as impurities B–F) was developed using β‐cyclodextrin derivatives as chiral selectors. To our knowledge, there are no previous studies about using capillary electrophoresis for the separation of impurities B–D. Six β‐cyclodextrin derivatives including cationic (piperidine‐ and cyclohexylamine‐), neutral (dimethyl‐ and hydroxypropyl‐), and anionic (carboxymethyl‐ and sulfated‐) β‐cyclodextrin derivatives were tested and operational parameters such as buffer pH and concentration of β‐cyclodextrin derivatives were investigated. The best resolutions were all obtained with anionic β‐cyclodextrin derivatives: ofloxacin, impurities C–F could be best resolved with carboxymethyl‐β‐cyclodextrin at satisfactory resolutions of 8.27, 9.98, 5.92, 8.49 and 6.78, respectively, while for impurity B, a particularly impressive resolution value, up to 21.38, was observed using sulfated‐β‐cyclodextrin. The enhancement of enantioseparation observed for the tested analytes using anionic β‐cyclodextrin derivatives might be due to some favorable interaction between selectors and analytes. Given the fact that the selection of chiral selector depends on the structures of analytes, with the help of structural similarities and differences of the analytes, the structure–separation relationship was further discussed.     

Chiral separation of lenalidomide by liquid chromatography on polysaccharide‐type stationary phases and by capillary electrophoresis using cyclodextrin selectors

Complementary techniques were applied for the investigation of the chiral recognition and enantiomeric resolution of lenalidomide using various cyclodextrins and polysaccharides as chiral selectors. The high‐performance liquid chromatography enantioseparation of the anticancer drug was achieved using polysaccharide‐type chiral stationary phases in polar organic mode. Elution order and absolute configuration were elucidated by combined circular dichroism spectroscopy and time‐dependent density functional theory calculations after the isolation of pure enantiomers. Chiral selector dependent and mobile‐phase dependent reversal of the enantiomer elution order was observed, and the nonracemic nature of the lenalidomide sample was also demonstrated. Eight anionic cyclodextrins were screened for their ability to discriminate between the uncharged enantiomers by using capillary electrophoresis. Only two derivatives presented chiral interactions, these cases being interpreted in terms of apparent stability constants and complex mobilities. The best results were delivered by sulfobutylether‐β‐cyclodextrin, where quasi‐equal stability constants were recorded and the enantiodiscrimination process was mainly driven by different mobilities of the transient diastereomeric complexes. The optimized high‐performance liquid chromatography (Chiralcel OJ column, pure ethanol with 0.6 mL/min flow rate, 40°C) and capillary electrophoresis methods (30 mM sulfobutylether‐β‐cyclodextrin, 30 mM phosphate pH 6.5, 12 kV applied voltage, 10°C) were validated for the determination of 0.1% (R)‐lenalidomide as a chiral impurity, which could be important if a racemic switch is achieved.     

Photoirradiation surface molecularly imprinted polymers for the separation of 6‐O‐α‐d‐maltosyl‐β‐cyclodextrin

Photoirradiation surface molecularly imprinted polymers for the separation of 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin were synthesized using functionalized silica as a matrix, 4‐(phenyldiazenyl)phenol as a light‐sensitive monomer, and 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin as a template. Fourier transform infrared spectroscopy results indicated that 4‐(phenyldiazenyl)phenol was grafted onto the surface of functionalized silica. The obtained imprinted polymers exhibited specific recognition toward 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin. Equilibrium binding experiments showed that the photoirradiation surface molecularly imprinted polymers obtained the maximum adsorption amount of 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin at 20.5 mg/g. In binding kinetic experiments, the adsorption reached saturation within 2 h with binding capacity of 72.8%. The experimental results showed that the adsorption capacity and selectivity of imprinted polymers were effective for the separation of 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin, indicating that imprinted polymers could be used to isolate 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin from a conversion mixture containing β‐cyclodextrin and maltose. The results showed that the imprinted polymers prepared by this method were very promising for the selective separation of 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin.     

Combination of capillary electrophoresis and molecular modeling to study the enantiomer affinity pattern between β‐blockers and anionic cyclodextrin derivatives in a methanolic and water background electrolyte

In order to have deep insights into the mechanisms of enantiomer affinity pattern in both aqueous and non‐aqueous systems, an approach combining capillary electrophoresis and molecular modeling was undertaken. A chiral β‐blocker; acebutolol, was enantioseparated in aqueous capillary electrophoresis and non‐aqueous capillary electrophoresis using two anionic β‐cyclodextrin derivatives. The enantiomer affinity pattern of acebutolol was found to be opposite when an aqueous background electrolyte was replaced with non‐aqueous background electrolyte in the presence of heptakis(2,3‐di‐O‐acetyl‐6‐sulfo)‐β‐cyclodextrin but remained the same in the presence of heptakis(2,3‐di‐O‐methyl‐6‐sulfo)‐β‐cyclodextrin. Molecular docking of acebutolol into two β‐cyclodextrin derivatives indicated two distinct binding modes called ‘up’ and ‘down’ conformations. After structure optimization by molecular dynamics and energy minimization, both enantiomers of acebutolol were preferred to the ‘up’ conformation with heptakis(2,3‐di‐O‐methyl‐6‐sulfo)‐β‐cyclodextrin while ‘down’ conformation with heptakis(2,3‐di‐O‐acetyl‐6‐sulfo)‐β‐cyclodextrin. The further calculation of the complex energy with solvent effect indicated that heptakis(2,3‐di‐O‐acetyl‐6‐sulfo)‐β‐cyclodextrin had higher affinity to S‐acebutolol than R‐acebutolol in non‐aqueous capillary electrophoresis while it showed better binding to R‐acebutolol in aqueous capillary electrophoresis. However, the heptakis(2,3‐di‐O‐methyl‐6‐sulfo)‐β‐cyclodextrin bound better to R‐acebutolol in both aqueous and non‐aqueous capillary electrophoresis, implying that the binding mode played more important role in chiral separation of heptakis(2,3‐di‐O‐methyl‐6‐sulfo)‐β‐cyclodextrin while the solvent effect had prevailing impact on heptakis(2,3‐di‐O‐acetyl‐6‐sulfo)‐β‐cyclodextrin.     

Field‐amplified sample injection combined with CE for the enantiodetermination of cathinones in urine samples

This work presents a capillary electrophoresis methodology for the enantiodetermination of cathinones in urine employing a liquid–liquid extraction sample pretreatment. The cathinones were enantioseparated by adding a mixture of 8 mM 2‐hydroxypropyl β‐cyclodextrin and 5 mM β‐cyclodextrin to the background electrolyte, which consists of 70 mM of monosodium phosphate aqueous solution at pH 2.5. Field‐amplified sample injection was used as preconcentration strategy to improve the sensitivity. We studied various parameters that affect this stacking strategy, in particular, the sample solvent and its pH, the presence or absence of a low conductivity solvent plug introduced before the sample injection, the nature and volume of this plug, and the voltage and time of the electrokinetic injection of the sample. The optimum conditions were achieved by injecting a plug of isopropanol:H₂O 50/50 at 50 mbar for 5 s prior to the electrokinetic injection of the sample prepared in an aqueous solution of HCl 10^?6 M. The sensitivity enhancement factors were from 562 to 601 in terms of peak area and from 444 to 472 in terms of peak height. The method was validated by analyzing spiked urine samples, obtaining a linear range of 25 to 1000 ng/mL and limits of detection ranging from 15 to 45 ng/mL.     

Chiral separation of benzothiazole derivatives of amino acids using capillary zone electrophoresis

A method for the separation of enantiomers of leucine and phenylalanine benzothiazole derivatives as potential antimicrobial agents was developed using capillary zone electrophoresis with a dual cyclodextrin (CD) system. The best resolution of enantiomers was achieved in 100 mmol/L phosphate background electrolyte (pH 3.5) with the dual CD system consisting of 10 mmol/L of β‐CD with 10 mmol/L of 2‐hydroxypropyl‐β‐cyclodextrin for leucine derivative and 10 mmol/L of 2‐hydroxypropyl‐γ‐cyclodextrin for phenylalanine derivative, respectively. Under the optimal conditions, the highest enantioresolution of 1.25 was achieved in a noncoated‐fused silica capillary at 17°C and 24 kV applied voltage.     

Determination of brompheniramine enantiomers in rat plasma by cation‐selective exhaustive injection and sweeping cyclodextrin modified electrokinetic chromatography method

A method consisting of cation‐selective exhaustive injection and sweeping (CSEI‐sweeping) as online preconcentration followed by a cyclodextrin modified electrokinetic chromatography (CDEKC) enantioseparation has been developed for the simultaneous determination of two brompheniramine enantiomers in rat plasma. In this method, analytes were electrokinetically injected at a voltage of 8 kV for 80 s in a fused‐silica capillary. Prior to the injection, the capillary was rinsed with 50 mM phosphate buffer of pH 3.5, followed by a plug of a higher conductivity buffer (150 mM phosphate pH 3.5, 20 psi, 6 min) and a plug of water (0.5 psi, 5 s). Separation was carried out applying –20 kV in 50 mM phosphate buffer, pH 3.5, containing 10% v/v ACN and 30 mg/mL sulfated‐β‐cyclodextrin (S‐β‐CD). Analytical signals were monitored at 210 nm. The detection sensitivity of brompheniramine enantiomers was enhanced by about 2400‐fold compared to the normal injection mode (hydrodynamic injection for 3 s at 0.5 psi, with a BGE of 50 mM phosphate buffer containing 20 mg/mL S‐β‐CD at pH 3.5), and LLOQ of two enantiomers were both 0.0100 μg/mL. In addition, this method had fairly good repeatability and showed promising capabilities in the application of stereoselective pharmacokinetic investigations for brompheniramine enantiomers in rat.     

Chiral separation of 12 pairs of enantiomers by capillary electrophoresis using heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector and the elucidation of the chiral recognition mechanism by computational methods

Chiral separation of 12 pairs of basic analyte enantiomers including oxybutynin, bambuterol, tradinterol, clenbuterol, clorprenaline, terbutaline, tulobuterol, citalopram, phencynonate, fexofenadine, salbutamol, and penehyclidine was conducted by capillary electrophoresis using a single‐isomer anionic β‐cyclodextrin derivative, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector. Parameters influencing separation were studied, including background electrolyte pH, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin concentration, buffer concentration, and separation voltage. A background electrolyte consisting of 50 mM Tris‐H₃PO₄ and 6 mM heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin at pH 2.5 was found to be highly efficient for the separation of most enantiomers, with other conditions of normal polarity mode at 10 kV, detection wavelength of 210 nm using hydrodynamic injection for 3 s. Under the optimal conditions, baseline resolution (>1.50) for 11 pairs of enantiomers and somewhat lower resolution for penehyclidine enantiomers (1.17) were generated. Moreover, the possible mechanism of separation of clenbuterol, oxybutynin, salbutamol, and penehyclidine was investigated using a computational modeling method.     

Development and validation of a cyclodextrin‐modified capillary electrophoresis method for the enantiomeric separation of vildagliptin enantiomers

The enantiomers of vildagliptin, an orally available and selective dipeptidyl‐peptidase‐4 inhibitor used for the treatment of type II diabetes, have been separated by CD‐modified CZE, using uncoated fused‐silica capillary. After screening 13 negatively charged CD derivatives as potential chiral selectors, sulfobutyl‐ether‐α‐CD (SBE‐α‐CD) was selected for the enantioseparation. For the optimization, a factorial analysis study was performed by orthogonal experimental design. Six experimental factors were chosen as variable parameters: temperature, applied voltage, chiral selector and BGE concentrations, pH, and the parameters of the hydrodynamic injection. The optimized system still was not considered final as the second peak (S‐enantiomer) migrated too close to the EOF, resulting in a potential inaccuracy during the determination of the chiral impurity. To fine‐tune the method “one factor at a time” variation approach was applied. The final method (applying 15°C capillary temperature, 40 mbar × 4 s hydrodynamic injection, 25 kV voltage in 75 mM acetate‐Tris buffer [pH 4.75] containing 20 mM SBE‐α‐CD as chiral selector) was validated according to the ICH guideline. RSD percentage of the resolution value, migration times, and corrected peak areas were below 5% during testing repeatability and intermediate precision. LOD and LOQ values were found to be 2.5 and 7.5 μg/mL, respectively. The method is considered linear in the 7.5–180 μg/mL range for the R‐enantiomer. The robustness of the method was justified using Plackett–Burmann statistical experimental design.     

Capillary electrophoresis separation of peptide diastereomers that contain methionine sulfoxide by dual cyclodextrin‐crown ether systems†

A dual‐selector system employing achiral crown ethers in combination with cyclodextrins has been developed for the separation of peptide diastereomers that contain methionine sulfoxide. The combinations of the crown ethers 15‐crown‐5, 18‐crown‐6, Kryptofix® 21 and Kryptofix® 22 and β‐cyclodextrin, carboxymethyl‐β‐cyclodextrin, and sulfated β‐cyclodextrin were screened at pH 2.5 and pH 8.0 using a 40/50.2 cm, 50 μm id fused‐silica capillary and a separation voltage of 25 kV. No diastereomer separation was observed in the sole presence of crown ethers, while only sulfated β‐cyclodextrin was able to resolve some peptide diastereomers at pH 8.0. Depending on the amino acid sequence of the peptide and the applied cyclodextrin, the addition of crown ethers, especially the Krpytofix® diaza‐crown ethers, resulted in significantly enhanced chiral recognition. Keeping one selector of the dual system constant, increasing concentrations of the second selector resulted in increased peak resolution and analyte migration time for peptide‐crown ether‐cyclodextrin combinations. The simultaneous diastereomer separation of three structurally related peptides was achieved using the dual selector system.     

Development and validation of a chiral capillary electrophoresis method for assay and enantiomeric purity control of pramipexole

A rapid method for the enantioseparation of pramipexole and its R‐enantiomer has been developed by capillary electrophoresis. The influence of chemical and instrumental parameters was investigated including the type and concentration of chiral selectors, buffer composition and pH, co‐ions, applied voltage, capillary length and temperature. Optimal separation conditions were obtained using a 50 mM phosphate buffer (pH 2.8) containing 25 mM carboxymethyl‐β‐cyclodextrin on a fused‐silica capillary. Online UV detection was performed at 262 nm. A voltage of 25 kV was applied, and the capillary temperature was kept at 25°C. Hydrodynamic injection was performed at 3.45 kPa for 5.0 s. The separation of enantiomers was achieved in <6.5 min. The method was further validated in terms of stability of solutions, selectivity, linearity (both pramipexole and R‐enantiomer, R²>0.995), LOD and LOQ (0.91 and 2.94 μg/mL, respectively), repeatability (RSD<1.5%) and accuracy (pramipexole, 100.4%; R‐enantiomer, 100.5%). The proposed method was then applied to two kinds of pramipexole dihydrochloride monohydrate commercially available tablets, immediate release tablets (1.50 and 0.125 mg) and sustained release tablets (0.52 mg), to quantify the main component in the tablets. The amount of distomer could be quantified in bulk sample materials.