Evaluation of the enantioselectivity of glycogen-based dual chiral selector systems towards basic drugs in capillary electrophoresis

Several chiral reagents including cyclodextrins (CDs) and derivatives, crown ethers, proteins, chiral surfactants and polymers have been involved in dual selector systems for enantioseparation of a series of chiral compounds by capillary electrophoresis (CE). In comparison to the chiral reagents above-mentioned, there is no report concerning the use of polysaccharides in dual chiral CE system. In this paper we first investigate the enantioselectivity of polysaccharide-based dual selector systems towards some chiral drugs. During our recent work, glycogen belonging to the class of branched polysaccharides has been used as a novel chiral selector in CE. In this study, three glycogen-based dual chiral CE systems have been established for enantiomeric separations of several racemic basic drugs consisting of duloxetine, cetirizine, citalopram, sulconazole, laudanosine, amlodipine, propranolol, atenolol and nefopam. These three dual systems combined glycogen (neutral polysaccharide) with chondroitin sulfate A (CSA, ionic polysaccharide), β-CD and HP-β-CD, respectively. It was found that the dual system of glycogen/CSA exhibited good enantioselective properties toward the tested drugs. More importantly, compared to the single selector systems, synergistic effect was observed when glycogen was used with CSA for most of the analytes. This indicated the enhancement of enantioseparation observed for these analytes in glycogen/CSA system might be due to some favorable interaction effects between glycogen and CSA. Moreover, in order to evaluate the stereoselectivity of glycogen/CSA, the influences of buffer pH and selector concentration on enantioseparation of the studied drugs were also investigated.     

Evaluation of the enantioseparation capability of the novel chiral selector clindamycin phosphate towards basic drugs by micellar electrokinetic chromatography

To date, a series of chiral selectors have been utilized successfully in capillary electrophoresis (CE). Among these various chiral selectors, macrocyclic antibiotics have been demonstrated to represent powerful enantioselectivity towards many chiral compounds. Differing from macrocyclic antibiotics, the use of lincosamide antibiotics as chiral selectors has not been reported previously. In our recent work, clindamycin phosphate belonging to the group of lincosamides has been first used as a chiral selector in capillary zone electrophoresis (CZE). In this paper, a micellar electrokinetic chromatography (MEKC) method has been developed for the evaluation of enantioseparation capability of this novel chiral selector towards several racemic basic drugs. As observed during the course of this work, clindamycin phosphate allowed excellent separation of the enantiomers of nefopam, citalopram, tryptophan, chlorphenamine, propranolol and metoprolol, as well as partial enantioresolution of tryptophan methyl ester and cetirizine. In this MEKC chiral separation system, different types of anionic surfactants, organic additives and background electrolytes were tested, and satisfactory enantioseparations of basic drugs above-mentioned were achieved using sodium dodecyl sulfate (SDS) as the surfactant, isopropanol as the organic additive, and phosphate as the background electrolyte. Furthermore, both migration times and enantioseparation of the analytes were influenced by several experimental parameters such as pH of the BGE, clindamycin phosphate and SDS concentrations, phosphate and isopropanol concentrations, and applied voltage. Consequently, the effects of these factors on enantioseparations of the studied basic drugs were systematically investigated in order to evaluate the stereoselectivity of clindamycin phosphate in MEKC.     

Heptakis(6-amino-6-deoxy)-beta-cyclodextrin as a chiral selector for the separation of anionic analyte enantiomers by capillary electrophoresis

A hepta-substituted beta-cyclodextrin bearing seven amino groups, heptakis(6-amino-6-deoxy)-beta-cyclodextrin (per-6-NH2-beta-CD) was successfully used as a chiral selector for the enantioseparation of different anionic analytes. The running buffer pH and chiral selector concentration were the studied parameters crucial in achieving the maximum possible enantioresolution. Enantiomeric separation of a mixture of seven carboxybenzyl-amino acids was achieved in 24 min. Excellent resolution was obtained for carboxybenzyl-tryptophan (Rs = 11.2).     

Differential effects of organic modifiers on the enantioseparation of dimetindene maleate with carboxymethyl-beta-cyclodextrin in capillary electrophoresis

Methanol enhances the enantioresolution of dimetindene enantiomers with carboxymethyl-beta-cyclodextrin (CMCD) as chiral selector at a concentration below its optimal value. The same effect was observed with ethanol (EtOH), although less pronounced. On the other hand, the addition of isopropanol (IP) or acetonitrile (ACN) decreases the enantioseparation. To elucidate the underlying mechanisms of these observed effects, other neutral (beta-CD, hydroxypropyl-beta-CD, and trimethyl-beta-CD) as well as chargeable (carboxyethyl-beta-CD and succinyl-beta-CD) CD derivatives were also tested with MeOH as organic modifier. It can be concluded that the increased enantioresolution of dimetindene enantiomers was only noted with CMCD as chiral selector and a short-chain organic modifier containing an alcohol function. The slight deprotonation of CMCD at pH 3.0 was only partly responsible for the high enantioselectivity and the 'favourable' effect of MeOH or EtOH. An important feature that can be concluded from these results is that for this particular analyte approximately the same resolution can be obtained with a lower CMCD concentration and the addition of some MeOH, compared to a MeOH free buffer.     

Enantioseparations of hydrobenzoin and structurally related compounds in capillary zone electrophoresis using heptakis(2,3-dihydroxy-6-O-sulfo)-beta-cyclodextrin as chiral selector and enantiomer migration reversal of hydrobenzoin with a dual cyclodextrin system in the presence of borate complexation

We investigated the enantioseparations of racemic hydrobenzoin, together with benzoin and benzoin methyl ether, in capillary electrophoresis (CE) using the single-isomer heptakis(2,3-dihydroxy-6-O-sulfo)-beta-cyclodextrin (SI-S-beta-CD) as a chiral selector in the presence and absence of borate complexation and enantiomer migration reversal of hydrobenzoin with a dual CD system consisting of SI-S-beta-CD and beta-CD in the presence of borate complexation at pH 9.0 in a borate buffer. The enantioselectivity of hydrobenzoin increased remarkably with increasing SI-S-beta-CD concentration and the enantioseparation depended on CD complexation between hydrobenzoin-borate and SI-S-beta-CD. The (S,S)-enantiomer of hydrobenzoin-borate complexes interacted more strongly than the (R,R)-enantiomer with SI-S-beta-CD. The enantiomers of hydrobenzoin could be baseline-resolved in the presence of SI-S-beta-CD at a concentration as low as 0.1% w/v, whereas the three test analytes were simultaneously enantioseparated with addition of 0.3% w/v SI-S-beta-CD or at concentrations >2.0% w/v in a borate buffer and 0.5% w/v in a phosphate background electrolyte at pH 9.0. Compared with the results obtained previously using randomly sulfated beta-CD (MI-S-beta-CD) in a borate buffer, enantioseparation of these three benzoin compounds is more advantageously aided by SI-S-beta-CD as the chiral selector. The enantioselectivity of hydrobenzoin depended greatly on the degree of substitution of sulfated beta-CD. Moreover, binding constants of the enantiomers of benzoin compounds to SI-S-beta-CD and those of hydrobenzoin-borate complexes to SI-S-beta-CD were evaluated for a better understanding of the role of CD complexation in the enantioseparation and chiral recognition. Enantiomer migration reversal of hydrobenzoin could be observed by varying the concentration of beta-CD, while keeping SI-S-beta-CD at a relatively low concentration. SI-S-beta-CD and beta-CD showed the same chiral recognition pattern but they exhibited opposite effects on the mobility of the enantiomers.     

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.     

Enantioselective separation in capillary electrophoresis using a novel mono-6-propylammonium-β-cyclodextrin as chiral selector

The chiral resolving ability of a novel single-isomer cationic β-cyclodextrin (CD), mono-6^A-propylammonium-6^A-deoxy-β-cyclodextrin chloride (PrAMCD), as a chiral selector in capillary electrophoresis (CE) is reported in this work for the enantioseparation of hydroxy, carboxylic acids and amphoteric analytes. The effect of chiral selector concentration on the resolution was studied. Good resolutions were achieved for hydroxy acids. Optimum resolutions were obtained even at 3.5 mM CD concentration for carboxylic acids. The electrophoretic method showed good linearity and reproducibility in terms of migration times and peak areas, which should make it suitable for routine analysis. In addition, baseline chiral separation of a six-acid mixture was achieved within 20 min. PrAMCD proved to be an effective chiral selector for acidic analytes.     

Separation and Determination of Cold Medicine Ingredients by Capillary Zone Electrophoresis Using Sulfated β‐Cyclodextrin as an Electrolyte Modifier and Chiral Selector

Capillary zone electrophoretic separations of cold medicine ingredients, including acetaminophen (AC), dextromethorphan (DMF) and racemates of phenylpropanolamine (PPA) and chlorpheniramine maleate (CPM) using randomly sulfated‐β‐CD (S‐β‐CD) as an electrolyte modifier and a chiral selector were investigated. The results indicate that S‐β‐CD is an excellent chiral selector and a suitable electrolyte modifier as well for the separation of those cold medicine ingredients. Influences of S‐β‐CD concentration and buffer concentration on the separation were examined. Baseline separation of these cold medicine ingredients with 1.0 % (w/v) S‐β‐CD could be simultaneously and successfully achieved within 11.8 minutes. In addition, S‐β‐CD could also act as a chiral selector for enantioseparation of PPA and CPM. A high enantioselectivity was obtained for these two analytes. Linear relationships between the peak area and its concentration for the calibration curves of AC and DMF were obtained (correlation coefficients: 0.9987 for AC, 0.9965 for DMF, respectively). The relative standard deviations of the migration time and peak area of AC and DMF were 0.19, 2.44 % and 0.34, 2.99 %, respectively. Detection limits were 0.93 and 2.57 μg/mL for AC and DMF, respectively. Recoveries of AC and DMF ranging between 102.42 and 97.28 % were observed. The proposed method was successfully applied to the determination of AC and DMF in cold medicines.     

Chiral separation of dansyl amino acids in capillary electrophoresis using mono-(3-methyl-imidazolium)-beta-cyclodextrin chloride as selector

Enantioseparations of fourteen dansyl amino acids were achieved by using a positively-charged single-isomer beta-cyclodextrin, mono-(3-methyl-imidazolium)-beta-cyclodextrin chloride, as a chiral selector. Separation parameters such as buffer pH, selector concentration, separation temperature, and organic modifier were investigated for the enantioseparation in order to achieve the maximum possible resolution. Chiral separation of dansyl amino acids was found to be highly dependent on pH since the degree of protonation of these amino acids can alter the strength of electrostatic interaction and/or inclusion complexation between each enantiomer and chiral selector. In general, the chiral resolution of dansyl amino acids was enhanced at higher pH, which indicates that the carboxylate group on the analytes may interact with the imidazolium group of cationic cyclodextrin. For most analytes, a distinct maximum in enantioresolution was obtained at pH 8.0. Moreover, the chiral separation can be further improved by careful tuning of the separation parameters such as higher selector concentration (e.g. 10 mM), lower temperature, and addition of methanol. Enantioseparation of a standard mixture of these dansyl amino acids was further achieved in a single run within 30 min.     

Enantioseparation of novel COX-2 anti-inflammatory drugs by capillary electrophoresis using single and dual cyclodextrin systems

A capillary electrophoresis method was developed for the enantioseparation of three novel cyclooxygenase-2 (COX-2) inhibitor drugs (E-6259, E-6036 and E-6087) with anti-inflammatory and analgesic activities using sulfobutyl ether-beta-cyclodextrin (SBE-beta-CD) as a chiral selector. The use of 50 mM sodium tetraborate at pH 9.2 with 30% v/v methanol, containing 7.1 mM SBE-beta-CD, as a background electrolyte (BGE) allowed the complete enantioseparation of the three neutral racemic mixtures (resolution = 2.4, 3.0 and 8.7, respectively) and their corresponding metabolites (oxidation products) in a single run. Migration times were shortened with some loss of enantioresolution by adding 1.75 mM dimethyl-beta-cyclodextrin (DM-beta-CD) to the previous BGE (dual CD system). The reversal of the migration order of E-6259 enantiomers in the dual CD system was also studied. Furthermore, the addition of DM-beta-CD to the BGE introduced a new chemoselectivity in the system that allowed E-6259 to be separated from the structurally similar compound E-6036.     

Simultaneous enantioseparation of four beta2-agonists by capillary electrophoresis with cyclodextrin additives. Study of the enantioselective mechanism

The simultaneous capillary electrophoretic enantioseparation of adrenergic beta(2)-agonists enantiomers (trantinterol, mabuterol, clenbuterol, bambuterol) was studied with beta-cyclodextrin, ethyl-beta-CD, methyl-beta-CD, hydroxypropyl-beta-CD, and hydroxyethyl-beta-CD as chiral selector. The type and concentration of the chiral selector and buffer pH played a very important role in the enantioseparation of the analyzed compounds. Hydroxypropyl-beta-CD was found to be the most effective complexing agent and allowed excellent chiral/achiral resolutions compared to the other CDs. The simultaneous enantioseparation of four beta(2)-agonists was achieved using 100 mM citric acid-10 mM Na(2)HPO(4) buffer at pH 2.5 containing 120 mM hydroxypropyl-beta-CD with an applied voltage of 20 kV. Method validation in terms of repeatability, linearity, and limits of detection and quantification was performed. The effect of structural features of analytes on R(s) and t(m) was studied. Complexation binding constants for the interactions between the four compounds and three different CDs were evaluated for elucidating the enantioseparation mechanism. It was found that very small differences in the chemical structure of the analytes resulted in significant changes in stereoselective recognition.     

Electrophoretic enantioseparation of profens in water-methanol solutions using eremomycin as a chiral selector

The possibility of the electrophoretic enantioseparation of profens in water-methanol solution of eremomycin was demonstrated. The effect of the methanol content, phosphate buffer pH, and chiral selector concentration on enantioresolution was studied. Water-methanol background electrolyte was shown to provide better reproducibility of migration times and baseline stability compared to aqueous buffers. The time of analysis in nonaqueous conditions is less than 15 min.     

Enantioseparation of phenothiazines in CD-modified CZE using single isomer sulfated CD as a chiral selector

Enantioseparations of five chiral phenothiazines in CD-modified CZE using the single isomer sulfate-substituted beta-CD (heptakis(2,3-dihydroxy-6-O-sulfo)-beta-CD, SI-S-beta-CD) and dual CD systems consisting of SI-S-beta-CD and a neutral CD as chiral selectors in a citrate buffer at pH 3.0 were investigated. The results indicate that SI-S-beta-CD is an excellent chiral selector for enantioseparation of promethazine. The enantiomers of trimeprazine were well separated, while those of ethopropazine could also be baseline-resolved with SI-S-beta-CD. With dual CD systems, especially with hydroxypropyl-beta-CD (HP-beta-CD) as neutral CD, the enantioselectivity of thioridazine and ethopropazine was considerably enhanced. Effective enantioseparation of phenothiazines, except for methotrimeprazine, could thus be favorably and simultaneously achieved. Moreover, reversal of the enantiomer migration order of ethopropazine and thioridazine occurred by varying the concentration of gamma-CD in the presence of SI-S-beta-CD. These phenomena may be attributable to the opposite effects of sulfated beta-CD and gamma-CD on the mobility of the enantiomers of ethopropazine and of thioridazine. Comparative studies on the enantioseparations of phenothiazines with single CD and dual CD systems containing SI-S-beta-CD and randomly sulfate-substituted beta-CD (MI-S-beta-CD) were made.     

Comparison of vancomycin-based stationary phases with different chiral selector coverage for enantioselective separation of selected drugs in high-performance liquid chromatography

Two vancomycin-based chiral stationary phases (CSPs) with different coverage of the chiral selector vancomycin (Chirobiotic V and Chirobiotic V2) were compared. beta-Blockers and profens, as structurally diverse groups of drugs, were chosen as analytes. Retention and enantioseparation of beta-blockers were studied in reversed-phase (RP) and polar-organic (PO) separation modes. Higher retention and better enantioresolution were obtained on the CSP with higher coverage of vancomycin in the both separation modes. Baseline separation of four beta-blockers (eight enantiomers) in the PO mode was achieved on the Chirobiotic V2 column within 15 min. The enantioseparation of profens did not bring so excellent and easy to interpret results. Higher retention of profens on the Chirobiotic V2 column was not always accompanied by an improvement of their chiral separation in the RP mode. The polar-organic mode was not suitable for these derivatives at all. The most interesting result was obtained with flobufen; its chiral center is further away from the rigid part of the molecule, which mostly causes difficulties in enantioselective recognition. Nevertheless, the enantiomers of flobufen were shown to be much better (baseline) resolved on the CSP with lower coverage of the chiral selector (Chirobiotic V).     

Chiral ligand exchange micellar electrokinetic chromatography using borate anion as a central ion

Three compounds having 1,2-diol structure (1-phenyl-1,2-ethanediol, 3-phenoxy-1,2-propanediol, and 3-benzyloxy-1,2-propanediol) were enantioseparated by ligand exchange MEKC using (5S)-pinanediol (SPD) as a chiral selector and borate anion as a central ion together with SDS. When (S)-1,2-propanediol, (S)-1,2,4-butanetriol, or (S)-3-tert-butylamino-1,2-propanediol were used as the chiral ligand instead of SPD, these three compounds were not enantioseparated. When borate was replaced with 2-aminoethane-1-sulfonate or N-cyclohexyl-3-aminopropanesulfonate, no chiral separation was achieved. Therefore, the hydrophobic interaction between the chiral selector and the chiral analytes within the transient diastereomeric complex may play an important role in the enantioseparation achieved by the proposed method.     

Effects of alcohols on CE enantioseparation of basic drugs with native gamma-CD as chiral selector

The effects of alcohol on the CE enantioseparation of selected basic drugs with gamma-CD as the chiral selector was investigated. The enantioseparation behavior of the analytes with gamma-CD in the absence and presence of different alcohols specifically methanol, ethanol, 2-propanol (IPA), and 2-methyl-2-propanol (TBA), the relationship of enantiomeric resolution (R(s)) values with either hydrophobicity or bulkiness of the alcohols, as well as the effect of these alcohols on interaction of the analytes with gamma-CD were studied. Results showed that hydrophobicity and/or bulkiness of alcohols have an influence on the enantioresolution of most of the analytes based on the relatively high correlation coefficients (R) obtained between R(s) versus log P and between R(s) versus ovality (i.e., parameter to indicate bulkiness of a molecule). Comparison of the values of the average binding constants obtained for each enantiomeric pair in the presence and absence of 5% IPA showed that alcohols can increase, decrease, or give a minimal effect on the analyte-gamma-CD interaction depending on the analyte. Furthermore, the significant enhancement in the enantioresolution of both propranolol and pindolol in the presence of either IPA or TBA led to the baseline enantioresolution of both drugs using 35 mM gamma-CD.     

环糊精毛细管区带电泳分离手性药物对映体

分别以2种天然环糊精(β、γ-环糊精)、2种常用的电中性环糊精衍生物(羟丙基-β-环糊精、二甲基-β-环糊精)和3种新型荷电环糊精衍生物(高取代磺酸基α、β、γ-环糊精)作为毛细管区带电泳手性添加剂,研究了环糊精的类型对6种手性药物对映体分离的影响.2种天然环糊精对所研究的手性药物均无手性识别能力,而环糊精经过衍生化后手性识别能力得到了很大的提髙,尤其是高取代磺酸基β-环糊精使6种手性药物均得到了基线分离.还考察了缓冲溶液的pH值和有机添加剂对手性分离的影响.     

Enantioseparation of chiral sulfoxides and sulfinate esters by capillary electrophoresis

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

Computer simulation of the enantioselective separation of weak bases in an online capillary electrophoresis based microanalysis configuration comprising sulfated cyclodextrin as selector

Computer simulation was utilized to characterize the electrophoretic processes occurring after reactant mixing in an online assay format used for monitoring the enantioselective N‐demethylation of ketamine to norketamine in the presence of highly sulfated γ‐cyclodextrin (HS‐γ‐CD). The incubated reaction mixture (at pH 7.4 and without chiral selector) is bracketed by a low pH BGE containing 2% HS‐γ‐CD as chiral selector, thereby forming a discontinuous buffer system. Upon power application, simulation provides insight into the formation of moving boundaries and new zones together with the prediction of the behavior of ketamine and norketamine enantiomers. The analytes first migrate cationically in a zone electrophoretic manner until they come in contact with HS‐γ‐CD upon which enantioseparation is initiated. Complexation has a focusing effect and the electrophoretic transport becomes reversed, that is, toward the anode. Simulation revealed that the initial conditions for the chiral separation, including buffer components concentrations, pH, and ionic strength, are different than those in the BGE. As a consequence thereof, the experimentally determined complexation parameters for the BGE were unable to correctly describe the migration behavior of the analytes in this column section. An increase in the input binding constants by a factor of two to four, as a result of the decreased ionic strength, resulted in simulation data that agreed with experimental findings.     

Chiral recognition of imperanene enantiomers by various cyclodextrins: a capillary electrophoresis and NMR spectroscopy study

The enantiomers of imperanene, a novel polyphenolic compound of Imperata cylindrica (L.), were separated via cyclodextrin-modified capillary electrophoresis. The anionic form of the analyte at pH 9.0 was subject to complexation and enantioseparation CE studies with neutral and charged cyclodextrins. As chiral selectors 27 CDs were applied differing in cavity size, sidechain, degree of substitution (DS) and charge. Three hydroxypropylated and three sulfoalkylated CD preparations provided enantioseparation and the migration order was successfully interpreted in each case in terms of complex mobilities and stability constants. The best enantioresolution (R(S) =?1.26) was achieved using sulfobutyl-ether-γ-CD (DS ～4), but it could be enhanced by extensive investigations on dual selector systems. After optimization (CD concentrations and pH) R(S) =?4.47 was achieved using a 12.5 mM sulfobutyl-ether-γ-CD and 10 mM 6-monodeoxy-6-mono-(3-hydroxy)-propylamino-β-cyclodextrin dual system. The average stoichiometry of the complex was determined with Job's method using NMR-titration and resulted in a 1:1 complex for both (2-hydroxy)propyl-β- and sulfobutyl-ether-γ-CD. Further NMR experiments suggest that the coniferyl moiety of imperanene is involved in the host-guest interaction.