Enantioseparation of chiral benzimidazole derivatives by electrokinetic chromatography using sulfated cyclodextrins

Baseline separation of 18 new substituted benzimidazole derivatives, potent AMP‐activated protein kinase (AMPK) activators, with one chiral center, was achieved by CD‐EKC using sulfated and highly sulfated CDs (SCDs and HS‐CDs) as chiral selectors. The influence of the type and concentration of the chiral selectors on the enantioseparations was investigated. The SCDs exhibit a very high enantioselectivity power since they allow excellent enantiomeric resolutions compared to those obtained with the neutral CDs. The enantiomers were resolved with analysis times around 6 min using 25 mM phosphate buffer at pH 2.5 containing either β‐S‐CD, HS‐β‐CD, HS‐γ‐CD (3 or 4% w/v) at 25°C, with a voltage of 20 kV. The apparent association constants of the inclusion complexes were calculated. The study of the solute structure‐enantioseparation relationships seems to show the high contribution of the interactions between the solutes phenyl ring and the CDs to the enantiorecognition process. The optimized method was briefly validated (LOD less than 1%) and the purity of enantiomers of compound 3 was determined. The enantiomer migration shows reversal order depending on the kind of CD.     

Chiral separation of lansoprazole and rabeprazole by capillary electrophoresis using dual cyclodextrin systems

Novel capillary electrophoresis methods using CDs as chiral selectors were developed and validated for the chiral separation of lansoprazole and rabeprazole, two proton pump inhibitors. Fourteen different neutral and anionic CDs were screened at pH 4 and 7 in the preliminary analysis. Sulfobutyl‐ether‐β‐CD with a degree of substitution of 6.5 and 10 at neutral pH proved to be the most suitable chiral selector for both compounds. Various dual CD systems were also compared, and the possible mechanisms of enantiomer separation were investigated. A dual selector system containing sulfobutyl‐ether‐β‐CD degree of substitution 6.5 and native γ‐CD proved to be the most adequate system for the separations. Method optimization was carried out using an experimental design approach, performing an initial fractional factorial screening design, followed by a central composite design to establish the optimal analytical conditions. The optimized methods (25 mM phosphate buffer, pH 7, 10 mM sulfobutyl‐ether‐β‐CD/20 mM γ‐CD, +20 kV voltage; 17°C temperature; 50 mbar/3 s injection, detection at 210 nm for lansoprazole; 25 mM phosphate buffer, pH 7, 15 mM sulfobutyl‐ether‐β‐CD/30 mM γ‐CD, +20 kV voltage; 18°C temperature; 50 mbar/3 s injection, detection at 210 nm for rabeprazole) provided baseline separation for lansoprazole (R_s = 2.91) and rabeprazole (R_s = 2.53) enantiomers with favorable migration order (in both cases the S‐enantiomers migrates first). The optimized methods were validated according to current guidelines and proved to be reliable, linear, precise, and accurate for the determination of 0.15% distomer as chiral impurity in dexlansoprazole and dexrabeprazole samples.     

Enantiomeric separation of R,S‐tolterodine and R,S‐methoxytolterodine with negatively charged cyclodextrins by capillary electrophoresis

The methods for separation of R,S‐tolterodine and R,S‐methoxytolterodine enantiomers using sulfated α‐, β‐CD and phosphated‐γ‐CD by CE in acidic BGE based on Tris/phosphate pH 2.5 buffer were developed. Sulfated α‐ and β‐CD allow anodic detection while phosphated‐γ‐CD allows only cathodic detection of the separated enantiomers. The influence of chiral selector (CS)'s concentration as well as the influence of composition and concentration of BGE on resolutions were studied. Reversal migration order of tolterodine and methoxytolterodine enantiomers was observed, when sulfated‐α‐ and sulfated‐β‐CD were used. The developed methods with all three studied CSs, were validated and compared. All proposed methods enable determination of 0.2% of S‐tolterodine as an optical impurity in pills, however the method with phosphated‐γ‐CD provided lower detection limit, better repeatability of peak areas and migration times, and also lower consumption of CS. Developed method employing phosphated‐γ‐CD that was applied for the determination of optical purity of R‐tolterodine in commercial pills.     

Study of the enantioseparation capability of chiral dual system based on chondroitin sulfate C in CE

It has been reported that chiral dual system is able to improve the enantioseparation of enantiomers in many cases. Currently, the dual systems involved in CE chiral separation are mostly dual CDs systems, and the polysaccharides‐based chiral dual system was reported in only one paper. To the best of our knowledge, the use of chondroitin sulfate C (CSC)‐based dual system for enantiomeric separation has not been reported previously. Herein, four CSC‐based chiral dual systems, namely CSC/glycogen, CSC/chondroitin sulfate A (CSA), CSC/hydroxypropyl‐β‐CD (HP‐β‐CD), as well as CSC/β‐CD (β‐CD), were evaluated for the first time for their enantioseparation capability by CE in this paper. During the course of the work, the influences of chiral selector concentration and buffer pH values on enantioseparation in dual systems were systematically investigated. Under the optimized conditions, the dual system consisting of CSC and glycogen exhibited better separations toward nefopam, duloxetine, sulconazole, atenolol, laudanosine, and cetirizine enantiomers compared to the single CSC or glycogen system. The combination of CSC and HP‐β‐CD improved the separation of amlodipine and chlorphenamine enantiomers. However, no synergistic effect was observed in the CSC/CSA and CSC/β‐CD systems.     

Enantioseparation of hydroxyeicosatetraenoic acids by hydroxypropyl‐γ‐cyclodextrin‐modified micellar electrokinetic chromatography

Complete resolution of hydroxyeicosatetraenoic acid (HETE) enantiomers was achieved using hydroxypropyl‐γ‐cyclodextrin (HP‐γ‐CD)‐modified MEKC. The optimum running conditions were determined to be utilizing a 30 mM phosphate–15 mM borate buffer (pH 9.0) containing 30 mM HP‐γ‐CD and 75 mM SDS as the BGE, application of +30 kV as the effective voltage, and carrying out the experiment at 15°C. The eluents were detected at 235 nm. The method was used successfully for the simultaneous separations of (S)‐ and (R)‐enantiomers of regioisomeric 8‐, 11‐, 12‐, and 15‐HETEs. Subsequently, the optimized method was applied to evaluate the stereochemistry of 8‐ and 12‐HETEs from the marine red algae, Gracilaria vermiculophylla and Gracilaria arcuata, respectively. The 8‐HETE was found to be a mixture of 98% (R)‐enantiomer and 2% (S)‐enantiomer, while the 12‐HETE was a mixture of 98% (S)‐enantiomer and 2% (R)‐enantiomer. The present study demonstrates that the HP‐γ‐CD‐modified MEKC method is simple and sensitive and provides unambiguous information on the configuration of natural and synthetic HETEs.     

Enantioseparation of phenothiazines in cyclodextrin-modified capillary zone electrophoresis using sulfated cyclodextrins as chiral selectors

In this study, enantioseparations of five phenothiazines, including promethazine, ethopropazine, trimeprazine, methotrimeprazine, and thioridazine, in CD-modified CZE using dual CD systems consisting of randomly sulfate-substituted CD (MI-S-beta-CD) and a neutral CD as chiral selectors in a citrate buffer (100 mM) at pH 3.0 were investigated. The results indicate that MI-S-beta-CD is an excellent chiral selector for enantioseparation of ethopropazine. The enantiomers of promethazine can also be baseline-resolved with MI-S-beta-CD at concentrations in the range of 0.5-1.0% w/v. On the other hand, thioridazine and trimeprazine interact strongly with neutral CDs. As a result, the enantioselectivity of these two phenothiazines is remarkably and synergistically enhanced with increasing the concentration of neutral CDs in the presence of MI-S-beta-CD and simultaneous enantioseparations of these phenothiazines, except for methotrimeprazine, could favorably be achieved with the use of dual CD systems. Moreover, by varying the concentration of beta-CD or gamma-CD at a fixed concentration of MI-S-beta-CD (0.75% w/v) reversal of the enantiomer migration order of promethazine occurred. This may be attributable to the opposite effects of charged and neutral CDs on the mobility of the enantiomers of promethazine.     

Enantiomeric separation of glycidyl tosylate by CE: Application to the study of catalytic asymmetric epoxidation of allyl alcohol

A CE method using CDs as chiral selectors was developed and validated to achieve the separation of glycidyl tosylate enantiomers originated by in situ derivatization of glycidol enantiomers obtained in asymmetric epoxidation of allyl alcohol with chiral titanium‐tartrate complexes as catalysts. The effects of the nature, pH and concentration of the buffer, the nature and concentration of chiral selector, the addition of SDS, methanol, ethanol or 2‐propanol, the capillary temperature, the effective capillary length and the applied voltage on the chiral resolution of glycidyl tosylate enantiomers were investigated. The best separation conditions were achieved using a Tris‐borate buffer mixture (50 and 25 mM, respectively) at pH=9.3 with a dual CD system consisting of 2.5% succinyl‐β‐CD and 1.0% β‐CD w/v at 15°C. A baseline separation (resolution～2.0) of the glycidyl tosylate enantiomers was obtained in a relatively short time (less than 12 min). Satisfactory results were obtained in terms of linearity (r>0.99) and intermediate precision (RSD below 8.5%). The LOD and LOQ were 3.0 and 10.0 mg/L, respectively, and the recoveries ranged from 99.8 to 108.8%. Finally, the method was applied to the determination of the enantiomeric excess and the yield obtained in the asymmetric epoxidation of allyl alcohol employing chiral titanium‐tartrate complexes as catalysts after an in situ derivatization of glycidol enantiomers to glycidyl tosylate.     

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.     

The Use of Dual Cyclodextrin Chiral Selector Systems in the Enantioseparation of Pharmaceuticals by Capillary Electrophoresis: An Overview

Cyclodextrin (CD) derivatives are the most efficient and frequently used chiral selectors (CSs) in capillary electrophoresis (CE). There are situations when the use of a single CD as CS is not enough to obtain efficient chiral discrimination of the enantiomers; in these cases, sometimes this problem can be resolved using a dual CD system. The use of dual CD systems can often dramatically enhance enantioseparation selectivity and can be applied for the separation of many analytes of pharmaceutical interest for which enantioseparation by CE with another CS systems can be problematic. Usually in a dual CD system an anionic CD is used together with a neutral one, but there are situations when the use of a cationic CD with a neutral one or the use of two neutral CDs or even two ionized CDs can be an efficient solution. In the current review we present general aspects of the use of dual CD systems in the analysis of pharmaceutical substances. Several examples of applications of the use of dual CD systems in the analysis of pharmaceuticals are selected and discussed. Theoretical aspects regarding the separation of enantiomers through simultaneous interaction with the two CSs are also explained. Finally, advantages, disadvantages, potential and new direction in this chiral analysis field are highlighted.     

CE‐LIF chiral separation of aspartic acid and glutamic acid enantiomers using human serum albumin and sodium cholate as dual selectors

Sodium cholate (SC), β‐CD, hydroxypropyl (HP)‐β‐CD, HSA, and the dual mixtures of them were evaluated for the analysis of aspartic acid (Asp) and glutamic acid (Glu) enantiomers fluorescently tagged with 5‐(4,6‐dichloro‐s‐triazin‐2‐ylamino) fluorescein (DTAF) by CE with LIF detection. Among the investigated chiral selectors and the dual selector systems, the dual selector systems of HSA and SC resulted to be the most useful chiral selectors allowing relatively high chiral resolution. Several experimental parameters such as chiral reagent type and concentration, buffer concentration, and pH, type and concentration of organic modifier were studied in order to find the optimum conditions for the chiral resolution of the two derivatized amino acids in their enantiomers. The effect of different variables that affect derivatization (time, temperature, pH, and DTAF concentration) was studied. Under optimum conditions, the analytes were separated in a short 10.5 min analysis time, and the RSDs for migration time and peak area were less than 0.12 and 2.8%, respectively. The method was applied for the analysis of compound amino acids injection without interference from other amino acids in the sample matrices observed.     

Enantioseparation of chiral aromatic acids by multiple dual mode counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin as chiral selector

This work concentrates on extending the utilization of multiple dual mode (MDM) counter‐current chromatography in chiral separations. Two aromatic acids, 2‐(6‐methoxy‐2‐naphthyl)propionic acid (NAP) and 2‐phenylpropionic acid (2‐PPA), were enantioseparated by MDM counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as chiral selector. The two‐phase solvent systems consisting of n‐hexane/ethyl acetate 0.1 mol/L phosphate buffer pH 2.67 containing 0.1 mol/L HP‐β‐CD (7.5:2.5:10 for NAP and 7:3:10 for 2‐PPA, v/v/v) were used. Conventional MDM and modified MDM were compared according to peak resolution under current separation mechanism. The influence of elution time after the first‐phase inversion and number of cycles for MDM were investigated. Peak resolution of NAP and 2‐PPA increased from 0.62 to 1.05 and 0.72 to 0.84, respectively, using optimized MDM conditions. Being an alternative elution method for counter‐current chromatography, MDM elution greatly improved peak resolution in chiral separations.     

Capillary electrophoretic enantioseparation of m‐nisoldipine using two different β‐cyclodextrins

The methods for the enantioseparation of m‐nisoldipine, a new 1,4‐dihydropyridine calcium ion antagonist, were developed. The elaborated methods of m‐nisoldipine enantiomers separation were successfully performed using an anionic CD–sulfobutyl ether‐β‐CD (SBE‐β‐CD) or carboxymethyl‐β‐CD as chiral selector. However, the results indicated that SBE‐β‐CD was a better chiral selector for enantioseparation of the neutral m‐nisoldipine. Furthermore, comparing the two SBE‐β‐CDs, the derivative with a higher degree of substitution (DS) of 7.0 induced better enantioresolution than the one with low DS (4.0). In addition, possible chiral recognition mechanisms of dihydropyridines were discussed.     

Separation of brombuterol enantiomers in capillary electrophoresis with cyclodextrin‐type chiral selectors and investigation of structure of selector‐selectand complexes using nuclear magnetic resonance spectroscopy

The major goal of this study was to determine the affinity pattern of brombuterol (BB) enantiomers toward various cyclodextrins (CD) and to evaluate the potential of NMR spectroscopy for understanding fine mechanisms of interactions between CDs and BB enantiomers. Separation of BB enantiomers was performed in a fused‐silica capillary using a phosphate buffer, pH 2.5, at the room temperature in the normal polarity mode. It was shown once again that CE in combination with NMR spectroscopy represents a very sensitive tool for studies of affinity patterns and structure of CD complexes with chiral guests. Although opposite affinity patterns of BB enantiomers were observed toward native β‐ and γ‐CDs, no significant differences between the structures of the complexes of these two CDs with BB were detected by NMR spectroscopy. In contrary to this, the opposite affinity pattern of BB enantiomers toward β‐CD and its two sulfated derivatives, heptakis (2,3‐O‐diacetyl‐6‐sulfo)‐β‐CD (HDAS‐β‐CD) and heptakis (2‐O‐methyl‐3,6‐di‐O‐sulfo)‐β‐CD (HMDS‐β‐CD) was associated with major differences in the structure of the complexes. In addition, it was shown again that HMDS‐β‐CD provides separation of enantiomers without formation of inclusion‐type complex with the chiral analyte.     

Combined use of hydroxypropyl‐β‐cyclodextrin and ionic liquids for the simultaneous enantioseparation of four azole antifungals by CE and a study of the synergistic effect

A CE method employing a dual system of hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) and ionic liquids (ILs) has been developed for the simultaneous enantioseparation of four azole antifungals for the first time. In this study, three different types of ILs were employed as modifiers and among them dodecyl trimethyl ammonium chloride was found to be the most effective. The effects of the concentration, cations, and anions of ILs on the enantioseparation were investigated. With the developed dual system, all the enantiomers were well separated in resolutions of 3.8, 3.5, 2.8, and 2.5 for miconazole, econazole, ketoconazole, and itraconazole, respectively. The interactions between dodecyl trimethyl ammonium chloride and HP‐β‐CD were also studied using a neutral polyacrylamide coated capillary and ¹H NMR spectroscopy to further explore the synergistic effect involved. It was found that ILs improved the enantioseparation not only by changing the EOF, but also by interactions with HP‐β‐CD that could change its ability of forming inclusion complex with the enantiomers.     

Simultaneous Chiral Separation of Four H1-Antihistamines by Capillary Zone Electrophoresis Using a Dual Cyclodextrin System

Capillary zone electrophoresis employing a dual cyclodextrin (CD) system, consisting of anionic sulfobutylether-β-CD and native β-CD, was developed for the simultaneous chiral separation of four H1-antihistamine racemates (brompheniramine, chlorpheniramine, cetirizine and promethazine). A cost-effective screening using different native and derivatized, neutral and ionized CDs as chiral selectors was performed to find suitable derivatives for the dual CD system. Under the optimized conditions consisting of 25 mM phosphate background electrolyte at pH 7.0, a combination of 15 mM SBE-β-CD and 10 mM β-CD as chiral selectors, +25 kV applied voltage and 20 °C system temperature, the baseline chiral separation of all racemates was accomplished in less than 8 min. The method proved to be suitable for routine analysis, since it provided satisfactory results during sensitivity, linearity and repeatability studies.

     

New single isomer negatively charged β‐cyclodextrin derivatives as chiral selectors in capillary electrophoresis

To improve resolution power of chiral selector and enantiomeric peak efficiency in CE, single isomer negatively charged β‐CD derivatives, mono(6‐deoxy‐6‐sulfoethylthio)‐β‐CD (SET‐β‐CD) bearing one negative charge and mono[6‐deoxy‐6‐(6‐sulfooxy‐5,5‐bis‐sulfooxymethyl)hexylthio]‐β‐CD (SMHT‐β‐CD) carrying three negative charges, were synthesized. The structure of these two β‐CD derivatives was confirmed by ¹H NMR and MS. SET‐β‐CD and SMHT‐β‐CD successfully resolved the enantiomers of several basic model compounds. SMHT‐β‐CD provided for a significantly greater enantioseparation than SET‐β‐CD at lower concentrations. This appears to be due to the higher binding affinity of SMHT‐β‐CD to the model compounds and the wider separation window resulting from an increased countercurrent mobility of the selector. Overall, the new chiral selectors provided enantioseparations with high peak efficiency while avoiding peak distortion due to polydispersive and electrodispersive effects. The information obtained from an apparent binding constant study suggested that the enantioseparation of the model compounds followed the predictions of charged resolving agent migration model and that the observed degree of enantioseparation difference were due to the magnitude of differences in both enantiomer‐chiral selector binding affinities (ΔK) and the mobilities of the complexed enantiomers (Δμ_c).     

Capillary Zone Electrophoresis Resolutions of 2,4‐Dinitrophenyl Labeled Amino Acids Enantiomers by N‐MethylatedAmino‐β‐Cyclodextrins

Abstract

Capillary zone electrophoresis resolutions of 2,4‐dinitrophenyl labeled amino acids (DNP‐AAs) enantiomers using three N‐methylated amino‐β‐cyclodextrins (CDs) [6^I‐deoxy‐6^I‐monomethylamino‐β‐CD (M‐A‐βCD), 6^I‐deoxy‐6^I‐dimethylamino‐β‐CD (diM‐A‐βCD), 6^I‐deoxy‐6^I‐trimethylammonium‐β‐cyclodextrin (triM‐A‐βCD)] as chiral selectors were investigated. These cationogenic selectors, differing in ionization and steric properties, exhibited clear differences in their enantioselectivities.

The differences in enantioresolution observed under identical acid‐base conditions (pH 5.2), providing comparable effective charges/mobilities of the CDs, e.g., excellent separations of single enantiomeric couples (triM‐A‐βCD, M‐A‐βCD), multicomponent mixtures of enantiomers (M‐A‐βCD), and mixtures of positional isomers (M‐A‐βCD, diM‐A‐βCD), indicated the importance of structural parameters (different degrees of methylation) of the studied chiral selectors in the separation mechanism.

The differences in enantioresolution observed under various acid base conditions (pH 5.2 and 9.6), providing significant differences of effective charges/mobilities of CDs, e.g., a dramatic decrease in enantioresolution as well as achiral resolution with uncharged M‐A‐βCD and preserved resolution with permanently charged triM‐A‐βCD, indicated the importance of charge of the studied chiral selectors in the separation mechanism.

The present study clearly showed that the studied CD derivatives have great potential as chiral selectors in capillary zone electrophoresis separations of DNP‐AAs and that their effective use is related to the character of the analyte (structure, hydrophobicity) as well as to working conditions (pH).     

Separation and Migration Behavior of Dichlorophenols in β‐Cyclodextrin‐Modified Capillary Zone Electrophoresis

The separation and migration behavior of six isomeric dichlorophenols (DCPs) in cyclodextrin‐modified capillary zone electrophoresis (CD‐CZE) using a phosphate‐borate buffer at alkaline pH with β‐CD and hydroxypropyl‐β‐CD (HP‐β‐CD) as electrolyte modifiers were investigated. The influence of buffer pH and the concentration of β‐cyclodextrins were examined. The results indicate that baseline separation of six isomeric DCPs can be achieved with addition of β‐CD concentration in the range of 2.0‐10 mM or HP‐β‐CD concentration in the range of 4.0‐10 mM at pH 10.0. Binding constants of DCPs to β‐CDs were evaluated for a better understanding of the interaction of DCPs with β‐CDs.     

The degree of substitution affects the enantioselectivity of sulfobutylether‐β‐cyclodextrin chiral stationary phases

Three chiral stationary phases were prepared by dynamic coating of sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) with different degrees of substitution, onto strong anion‐exchange stationary phases. The enantioselective potential and stability of newly prepared chiral stationary phases were examined using a set of structurally different chiral analytes. Measurements were performed in RP‐HPLC. Mobile phases consisted of methanol/formic acid, pH 2.10, and methanol/10 mM ammonium acetate buffer, pH 4.00, in various volume ratios. SBE‐β‐CDs with degrees of substitution (DS) 4, 6.3, and 10 proved suitable for the enantioseparation of 14, 11, and 8 analytes, respectively. The SBE‐β‐CD DS 4 based chiral stationary phase enabled the enantioseparation of the nearly all basic and neutral compounds. Chiral stationary phases with higher sulfobutylether‐β‐cyclodextrin substitution (especially DS 10) yielded higher enantioresolution values for acidic compounds.     

Determination of the binding constants of modafinil enantiomers with sulfated β‐cyclodextrin chiral selector by capillary electrophoresis using three different linear plotting methods

Binding constants for the enantiomers of modafinil with the negatively charged chiral selector sulfated‐β‐CD (S‐β‐CD) using CE technique is presented. The calculations of the binding constants employing three different linearization plots (double reciprocal, X‐reciprocal and Y‐reciprocal) were performed from the electrophoretic mobility values of modafinil enantiomers at different concentrations of S‐β‐CD in the BGE. The highest inclusion affinity of the modafinil enantiomers were observed for the S‐enantiomer–S‐β‐CD complex, in agreement with the computational calculations performed previously. Binding constants for each enantiomer–S‐β‐CD complex at different temperatures, as well as thermodynamic parameters for binding, were calculated. Host–guest binding constants using the double reciprocal fit showed better linearity (r²>0.99) at all temperatures studied (15–30°C) and compared with the other two fit methods. The linear van't Hoff (15–30°C) plot obtained indicated that the thermodynamic parameters of complexation were temperature dependent for the enantiomers.