Separation of neutral and basic enantiomers by cyclodextrin electrokinetic chromatography using anionic cyclodextrin derivatives as chiral pseudo-stationary phases

Separations of neutral and basic racemates were performed using five different anionic cyclodextrin (CD) derivatives as chiral selectors, viz. carboxymethylated β-CD, β-CD phosphate sodium salt, sulfobutyl ether β-CD sodium salt, carboxymethylated γ-CD, and γ-CD phosphate sodium salt. For the separation of neutral racemates, an untreated fused silica capillary was employed and various neutral racemates were successfully separated. Since the pH of the buffer affected the electroosmotic flow (EOF), the resolution was improved by changing the buffer pH. A polyacrylamide coated capillary was employed for the separation of basic racemates to suppress EOF and to prevent adsorption of cationic analyte on the capillary surface. By choosing an appropriate type and concentration of anionic CD, about 40 basic racemates were successfully separated. Some rough binding constants of basic analytes with an anionic β-CD were measured to discuss the optimum concentration of the CD. The migration direction was dependent on the binding constants and the concentration of the CD. The analyte strongly bound to the anionic CD migrated towards the anode but the weakly bound one moved towards the cathode. Anionic γ-CDs were also very useful for the separation of basic enantiomers. Five neutral CDs were employed as chiral selectors to compare selectivity between charged and neutral CDs, and eleven racemates could only be resolved using anionic CDs. The separation of some basic racemates in human plasma was also described. The direct injection of plasma samples was possible for some enantiomers that did not interact strongly with plasma proteins.     

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.     

Brush-type chiral stationary phase for enantioseparation of acidic compounds. Optimization of chiral capillary electrochromatographic parameters

The capillary electrochromatographic separations of three acidic enantiomers (carprofen, coumachlor and warfarin) were studied on a capillary column packed with 5 microm (3R,4S)-Whelk-O 1 chiral stationary phase. The influence of several experimental parameters (mobile phase pH, type of background electrolyte, acetonitrile ratio, temperature, applied voltage and ionic strength) on electroosmotic flow velocity, retention factor, selectivity factor, efficiency, resolution and effectiveness of chiral separation was evaluated. It was notable that the optimum resolution of the acidic enantiomers was achieved at pH 3.0 phosphate buffer, suggesting that capillary electrochromatography in the ion-suppressed mode can be applied for chiral separations of a range of acidic compounds.     

Enhancement of second-migrating enantiomer peak symmetry of basic drugs by using dual-cyclodextrin system in capillary electrophoresis

Today, chiral separations of cationic drugs by capillary electrophoresis are generally carried out by adding negatively charged cyclodextrins (CDs) to the running buffer while anionic or neutral drug separations require the use of dual-CD systems (mixtures of neutral and charged CDs). Chiral separation of some basic drugs (idazoxan, efaroxan, milnacipran) has been studied by using mixtures of sulfated-beta-CD (S-beta-CD) and hydroxypropyl-gamma-CD (HP-gamma-CD). The influence of the following parameters (nature and concentration of neutral CD, concentration of S-gamma-CD) on many separation factors (electrophoretic mobility, selectivity, efficiency, asymmetry factor, resolution) demonstrated that dual-CD systems are useful for chiral separation of basic drugs in order to improve the symmetry of the second-migrating enantiomer. Indeed, the neutral CD reduces the extent of electromigration dispersion by mobility tuning. Finally, the 0.5 mg/mL S-beta-CD/5 mg/mL HP-gamma-CD dual system has allowed the chiral separation of idazoxan, efaroxan and milnacipran enantiomers in less than 9 min.     

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.     

Enantiomeric separation by capillary electrochromatography. I. Chiral separation of dansyl amino acids and organochlorine pesticides on a diol-silica dynamically coated with hydroxypropyl-beta-cyclodextrin

In this work, a commercially available diol-silica stationary phase was converted in situ to a chiral stationary phase by dynamically coating it with hydroxypropyl-beta-cyclodextrin (HP-beta-CD). This stationary phase was shown useful for the capillary electrochromatography (CEC) separation of neutral and anionic enantiomers such as some organochlorine pesticides and dansyl amino acids, respectively. The inclusion of HP-beta-CD in the mobile phase to produce the in situ chiral stationary phase allowed the rapid separation of the anionic dansyl amino acid enantiomers at relatively low electroosmotic flow (EOF). The formation of host-guest complexes between the dansyl amino acids and the neutral HP-beta-CD in the mobile phase lowered the actual charge-to-mass ratios of the anionic solutes, thus speeding up their transport by the EOF across the packed capillary column. Several parameters affecting enantioseparation were investigated, including the concentration of HP-beta-CD, ionic strength, pH, and organic modifier content of the mobile phase.     

Enantioseparation of 3,4-dihydroxyphenylalanine and 2-hydrazino-2-methyl-3-(3,4-dihydroxyphenyl)propanoic acid by capillary electrophoresis using cyclodextrins

The enantiomeric separations of 3,4-dihydroxyphenylalanine (dopa) and 2-hydrazino-2-methyl-3-(3,4-dihydroxyphenyl)propanoic acid (carbidopa) by capillary electrophoresis were studied using several native, neutral and anionic cyclodextrins as chiral additives and uncoated fused-silica capillaries. The effect of the type and concentration of the cyclodextrin added to 20 mM phosphate buffer (pH 2.5) on enantioseparation and migration times was studied. A high resolution value of 15.63 was obtained for dopa enantiomers with a buffer containing 20 mM single isomer, heptakis(2,3-diacetyl-6-sulfato)-beta-cyclodextrin. The enantiomers of carbidopa were separated using 20 mM carboxymethyl-beta-cyclodextrin as a chiral resolving agent. Both methods allowed the determination of 0.1% of the D-enantiomer (second migrating) in the presence of the L-enantiomer (first migrating) of dopa and carbidopa with a good precision. These methods also gave good results in terms of precision for both peak area, migration time, linearity and accuracy.     

Enantioselectivity of monolithic silica stationary phases immobilized with different concentrations cellulose tris (3,5-dimethylphenylcarbamate), analyzed with different mobile phases in capillary electrochromatography

The 3,5-dimethylphenylcarbamate derivatives of cellulose bearing 3-(triethoxysilyl)propyl residues were immobilized in a capillary format onto a monolithic silica support by intermolecular polycondensation of the triethoxysilyl groups. The resulting columns were used for chiral separations using capillary electrochromatography. The effects of the synthesizing solvent, the selector coating procedure, the chiral selector concentration onto the silica monolith and the mobile phase pH value, on the separation of enantiomers were studied. The column-to-column reproducibility and stability also were evaluated. A test set of 14 chiral substances, including acidic, neutral, bifunctional and basic compounds, was used to investigate the effects of the factors mentioned above. Twelve pairs of enantiomers showed enantioselectivity at some of the different conditions tested. The column-to-column repeatability was satisfactory, and the prepared columns were stable under the adopted analysis conditions.     

应用四甲基氢氧化铵控制电渗流的毛细管电泳方法分离多沙唑嗪及其中间体的对映体

建立了毛细管电泳手性分离多沙唑嗪中间体对映体的方法,并同时分离了多沙唑嗪对映体。考察了不同种类季铵盐对电渗流及分离的影响,其中四甲基氢氧化铵(TMB)能有效控制电渗流并提高组分的分离度。实验还考察了其他因素,如pH值、分离电压和磷酸二氢钠浓度对分离的影响。所用的毛细管为40 cm(有效长度30 cm)×50 μm,缓冲液为12 mmol/L β-环糊精、30 mmol/L TMB、60 mmol/L 磷酸二氢钠(pH 2.2),分离电压为20 kV。在此条件下多沙唑嗪及其中间体的对映体均达到了基线分离。实验结果表明,一些用β-环糊精不能完全分离的对映体通过加入TMB控制电渗流能达到满意的分离效果。     

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.     

卡替诺尔和氟西汀对映体的高效毛细管电泳分离

考察了以羧甲基－β－（环糊精－β－CD）、β－环糊清（β－CD）、羟丙基－β－环糊精（HP－β－CD）、二甲基－β－环糊精（DM－β－CD)为手性选择剂,在50mmol/L醋酸三乙胺缓冲溶液中分离卡替诺尔和氟西汀对映体。该文还通过考察手性选择剂的浓度、背景电解质的酸度、背景电解质的类型等因素对映体手性分离的影响,对分离条件进行了优化,初步探讨了手性识别机理。实验结果表明：用约4mmol/L的CM－β－CD分离氟西汀和卡替诺尔对映体,能使对映体达到良好分离,不仅节约了分析成本,也简化了分析过程。     

Enantiomer separation by CEC

Three chiral compounds were successfully separated in a short time with two enantiomer separation models on packed-capillary electrochromatography (CEC). (i) 75 μm I.D. capillaries were packed with 5 μm β-cyclodextrin (βCD) chiral stationary phase (CSP). Effects of voltage, pH and concentration of organic modifier on electroosmotic flow (EOF) and chiral separations were investigated systematically. Enantiomers of a neutral compound (benzoin) and a neutral drug (mephenytoin) were separated within a short time with high efficiency. Efficiency of 32 000 theoretical plates per meter and resolution (R8) of 1.42 were achieved for enantiomers of benzoin using a βCD packed column with 6.2crn packed length. Efficiency of 45 000 theoretical plates per meter andR8 of 3.40 were obtained for enantiomers of mephenytoin. Especially, the enantiomer separation of mephenytion was performed in just 3.4 min with R₈ of 2.60. (ii) 75 μm I.D. capillary was packed with octadecylsilica particles (ODs). Chiral separation of a basic drug, propranolol, was studied with chiral agent, via addition of the dimethyl-β-cyclodextrin (DM β-CD) directly into the mobile phase on this column. Baseline separation and efficiency of 81 000 theoretical plates per meter were achieved for propranolol. Project supported by the Natural Science Foundation of Liaoning Province, China, the National Natural Science Foundation of China (Grant No.29875030), and the Excellent Young Scientist Award from the National Natural Science Foundation of China. (Grant No.29725512).     

Optimisation of a Headspace Solid-Phase Micro- Extraction Procedure for the Determination of 2,4,6-Trichloroanisole and Various Related Compounds in Cork Washing Waste Water by Use of Gas Chromatography-Mass Spectrometry

This paper reports the use of an anionic cyclodextrin, heptakis(2,3-di-O-methyl-6-O-sulfato)-β-cyclodextrin (HDMS-β-CD), for chiral separations of pharmaceutical enantiomers by nonaqueous capillary electrophoresis (NACE). Enantiomer resolution was affected mainly by HDMS-β-CD concentration and the acidity of the background electrolyte (BGE). The effects of capillary length and applied voltage on enantiomer resolution were also investigated. Results showed that in a methanol solution of 20 mM phosphoric acid, 10 mM sodium hydroxide, and 10 mM HDMS-β-CD, seven anticholinergic drugs were separated to baseline but no chiral separation was obtained for three other similar drugs. NACE is suitable for routine, rapid separation of the enantiomers of pharmaceutical compounds.     

Enantioselective separation of the sunscreen agent 3-(4-methylbenzylidene)-camphor by electrokinetic chromatography: Quantitative analysis in cosmetic formulations

3-(4-Methylbenzylidene)-camphor (MBC) is a chiral sunscreen agent used in cosmetic products. In this work, the enantioseparation of MBC has been performed by EKC and applied to the analysis of the MBC enantiomers in cosmetic creams. Different experimental conditions (type and concentration of the chiral selector, temperature, and sample solvent) have been optimized. Due to the neutral nature of this compound, anionic CD derivatives were investigated as chiral selectors. Carboxymethylated-beta-CD (CM-beta-CD) showed the highest chiral separation power, observing that a 15 mM concentration of this CD at a working temperature of 15 degrees C enabled to obtain the highest enantioresolution. However, under these conditions, tailing of peaks obtained for the enantiomers was observed. The addition of increasing concentrations of the neutral alpha-CD to CM-beta-CD at a 15 mM concentration in a 100 mM borate buffer at pH 9.0 improved the enantiomeric separation and decreased peak tailing. The use of DMF for the total dissolution of the cosmetic creams, and methanol:water (1:1 v/v) for appropriate dilution enabled to observe good shape and size for the peaks of the MBC enantiomers. After optimizing a method for the preconditioning of the capillary, the analytical characteristics of the chiral separation method for the analysis of MBC were investigated. Linearity, LODs and LOQs, precision (instrumental repeatability, method repeatability, intermediate precision), accuracy, and selectivity were evaluated. The method was applied to analyze MBC enantiomers contained in two commercial cosmetic creams containing racemic MBC and to study the skin absorption of this compound with time.     

Sodium maleopimaric acid as pseudostationary phase for chiral separations of amino acid derivatives by capillary micellar electrokinetic chromatography

A new type of chiral surfactant, sodium maleopimaric acid (SMA), was synthesized, and employed for the enantioselective micellar electrokinetic chromatographic (MEKC) separation of amino acid enantiomers derivatized with naphthalene-2,3-dicarboxaldehyde (NDA-D/L-AAs). The effect of the surfactant concentration, type and concentration of the BGE, and buffer pH on the resolution was studied, and optimized conditions were used to evaluate the ability of this new surfactant to perform chiral separations toward NDA-D/L-AAs by MEKC. Enantiomeric separations of NDA-D/L-AAs were achieved with a running buffer consisting of 100 mM borate (pH 9.5) and 20 mM SMA in a 58.5 cm length x 50 microm id capillary. Under the conditions selected, two pairs of tested amino acid enantiomers including NDA-D/L-trptophan (Trp) and NDA-D/L-kynurenine (Kyn) were resolved.     

毛细管电动色谱带电环糊精拆分N-FMOC氨基酸对映体

 将负电性磺丁基 -β -环糊精手性添加剂应用于毛细管电泳氨基酸对映体的拆分研究中 ,对 8种氨基酸对映体与 9-芴甲基氧基甲酰氯 (FMOC-Cl)生成的衍生物进行了分离 ,其中 5种得到了基线分离。考察了背景电解质 p H值及磺丁基 -β -环糊精的浓度对 N-FMOC氨基酸对映体拆分的影响。     

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.     

Enantiomeric separation of enzymatic hydrolysis products of dihydropyrimidinone methyl ester with cationic cyclodextrin by capillary electrophoresis

The achiral separation of dihydropyrimidinone (DHP) methyl ester and its corresponding carboxylic acid and the chiral separation of their respective enantiomers were achieved in a single analysis using capillary electrophoresis (CE) with quaternary ammonium-beta-cyclodextrin (QA-beta-CD) as a chiral buffer additive. Separation of the DHP methyl ester from the corresponding carboxylic acid was achieved because the acid was negatively charged at pH 8.3 of the running buffer and the ester is neutral. Upon the addition of QA-beta-CD, the enantiomers of the acid and ester were well resolved before and after the electroosmotic flow, respectively. In addition, the minor DHP methyl ester enantiomer (R isomer) was well separated from several impurities. This CE system was used to monitor the progress of a bioresolution reaction that utilizes an enzyme to convert the R isomer of the ester to its corresponding acid. The quantities of all four enantiomers can be determined using a single set of CE conditions. In addition, it is demonstrated that samples can be directly injected into the capillary without sample pretreatment due to the fact that the coating of the cationic CD on the capillary surface prevents adsorption of the positively charged enzyme. The effects of other experimental parameters such as type of CDs, concentration of CDs, pH, temperature, and the preconditioning of capillary were also studied.     

Separation of enantiomers by capillary electrophoresis using pentosan polysulfate

Pentosan polysulfate, a semisynthetic polysaccharide, was employed as a chiral run buffer additive in capillary electrophoresis. Twenty-eight racemic analytes were resolved. The separations were successful only at low pH when the analytes were significantly protonated. This suggests that ionic interactions were the dominant associative interactions between the anionic pentosan polysulfate and the positively charged analytes. Compared to other linear, carbohydrate-based chiral selectors (i.e., chondroitin sulfates, heparin and dextran sulfate) pentosan polysulfate has some characteristics common of anionic polysaccharides; yet it has several differences in its structure and properties which account for its unusual enantioselectivity. The effects of pH, concentration of phosphate buffer, concentration of pentosan polysulfate and the type and concentration of organic modifier on the enantiomeric separations were investigated. The optimization of these separations were dependent on the nature of the analytes and could be achieved by the proper choice of experimental conditions.