A screening strategy for the development of enantiomeric separation methods in capillary electrophoresis

Method development of enantiomeric separations in capillary electrophoresis (CE) is a time-consuming task, since finding the appropriate chiral selector is usually a "trial and error" process. It is impossible to predict the selectivity of a selector towards a certain enantiomer. Therefore, the affinity of all selectors has to be examined one at a time. In order to speed up this process, a strategy is proposed based on simple experimental design methodology. The approach includes first a screening in function of the pH to determine the optimal migration conditions followed by a selection of the right chiral selector by means of Taguchi designs. In the approach several variables, such as the type and concentration of cyclodextrin, the concentration of buffer electrolyte, and the percentage of organic modifier, are varied simultaneously to find initial separation conditions rapidly. The resulting initial separation conditions can be optimized in further steps to be more reproducible. We discuss the results of the approach when applied on a number of selected compounds that are recently in development at Johnson & Johnson--Pharmaceutical Research and Development. Parameters, such as quality of the separation and analysis time, are evaluated to determine initial separation conditions for each compound.     

Enantiomeric separation of chiral peptide nucleic acid monomers by capillary electrophoresis with charged cyclodextrins

Direct chiral separation of chiral peptide nucleic acid (PNA) monomers has been achieved for the first time by capillary electrophoresis (CE) with charged cyclodextrins as chiral selectors added to the electrophoretic buffer. Selectively modified 6-deoxy-6-N-histamino-beta-cyclodextrin and sulfobutyl ether-beta-CD were successfully used as chiral selectors for the enantiomeric separation of chiral monomers based on different aminoethylamino acids bearing thymine or adenine as nucleobases. Chiral separations were obtained at low selector concentrations (1-3 mM) with good enantioselectivity and resolution factors. Separations were optimized as a function of pH in order to exploit the effect of the electrostatic interactions between the oppositely charged selector and selectand. The method has been applied to the analysis of the enantiomeric excess of chiral monomers used for the solid phase synthesis of chiral PNA oligomers. CE chiral analysis showed that a very high enantiomeric purity was generally achieved in the synthesis of all monomers, except for histidine and aspartic acid based monomers in which ca. 10% of the "wrong" enantiomer was always present.     

Neutral cyclodextrins as chiral agents for enantiomeric separations of chromanes in capillary electrophoresis

Summary Six different cyclodextrins with varying cavity size and rim substitution were used as chiral agents for the enantiomeric separation of eight chromane compounds or analogues using capillary electrophoresis. It is shown that the cyclodextrin type and concentration have a large influence on the enantiomeric separation obtained for these compounds. A chiral resolution of 1.4 or better could be obtained for all the substances with either substituted heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin or unsubstituted γ-cyclodextrin as the chiral selector. The influence of the γ-cyclodextrin concentration, ionic strength and pH on the chiral separations was also investigated with a multivariate screening design. The detection limit and resolution of the present method allow determinations of the investigated compounds down to a chiral impurity of less than 0.1 % (area/area).     

Use of vancomycin as chiral selector in capillary electrophoresis. Optimization and quantitation of loxiglumide enantiomers in pharmaceuticals

Vancomycin has been used as chiral selector for the enantiomers separation of D, L-loxiglumide, a new drug proposed for the treatment of gastrointestinal pathology. The chiral selector, dissolved at very low concentration in the running buffer, filled only part of the capillary (polyacrylamide coated) and allowed chiral resolution in less than 12 min using a 50 mM phosphate buffer at pH 6. The partial separation technique allowed to obtain a detection limit of 0.5 μg/ml for each enantiomer avoiding the drop in sensitivity due to the strong UV absorption of vancomycin when present in the detector path. The effects of vancomycin concentration and buffer pH on enantiomers resolution have been studied in order to find the optimum experimental conditions for the chiral purity control of drug. The optimized method, using the internal standard, showed good reproducibility for both migration times and normalized peak area ratio and for linearity. Under the studied operating conditions it was possible to detect 0.2 % (w/w) of L-loxiglumide as a chiral impurity. Analysis of pharmaceutical preparations of D-loxiglumide did not reveal the presence of the impurity (L-isomer).     

Enantiomeric separation of furan derivatives and fused polycycles by cyclodextrin-modified micellar capillary electrophoresis

The enantiomeric separations of highly hydrophobic furan derivatives and polycycles were performed and optimized using CD-modified micellar CE. The most effective chiral selector for the enantiomeric separation of these analytes was hydroxypropyl-gamma-CD. The effects of CD and SDS concentration and organic modifier were examined in order to optimize the separation conditions. The ratio of CD to surfactant concentration affected the enantiomeric separation significantly, with increases in the derivatized CD concentration generally enhancing resolution. Addition of an organic solvent modifier to the run buffer served to increase the analytes' solubility and enhance the separation efficiency. A highly acidic pH was necessary to effectively suppress the EOF when operating in the reverse polarity mode.     

Enantiomeric separation and determination of the enantiomeric impurity of armodafinil by capillary electrophoresis with sulfobutyl ether-β-cyclodextrin as chiral selector

A selective capillary electrophoresis method using sulfobutyl ether-β-cyclodextrin as a chiral selector was developed and validated for the determination of the enantiomeric impurity of (R)-modafinil, i.e., armodafinil. Several parameters were optimized for a satisfactory enantioresolution, including the type and concentration of chiral selector and organic modifier, pH of background electrolyte (BGE), capillary temperature. The finally adopted condition was: 20 mmol/L phosphate buffer at pH 7.5, containing 20 mmol/L sulfobutyl ether-β-cyclodextrin and 20% methanol, at temperature of 25 °C. A good resolution of 3.3 for the two enantiomers of modafinil was achieved by applying the optimal conditions. The limit of detection (LOD) and limit of quantification (LOQ) of (S)-modafinil were 1.25 μg/mL and 2.50 μg/mL, respectively. The established method was also proven to display good selectivity, repeatability, linearity and accuracy. Finally, the method was used to investigate the enantiomeric purity of armodafinil in bulk samples.     

Enantioseparation of warfarin and its metabolites by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method with direct ultraviolet (UV)-absorbance detection is presented for the simultaneous enantiomeric separation of warfarin and its main metabolites, including warfarin alcohols, 4'-, 6-, and 7-hydroxywarfarin, using highly sulfated beta-cyclodextrin (HS-beta-CD) as the chiral selector. This chiral separation method was optimized in terms of the electrophoretic parameters, which included the concentration of HS-beta-CD used, the type and composition of organic modifier added to the background electrolyte (BGE) buffer, and the BGE buffer pH. Chiral separation of warfarin and its major metabolites was achieved with high resolution, selectivity, efficiency, repeatability, and reproducibility. This optimized chiral analysis of warfarin along with its metabolites was completed within a satisfactory electrophoresis time of 20 min.     

Enantiomeric separation of ketoconazole and terconazole antifungals by electrokinetic chromatography: Rapid quantitative analysis of ketoconazole in pharmaceutical formulations

EKC using a neutral CD as chiral selector was applied in this work to the development of a method enabling the enantiomeric separation of ketoconazole and terconazole antifungals. The influence of different experimental conditions such as temperature, CD concentration, pH, and nature and concentration of the buffer on the enantiomeric resolution of the compounds studied was investigated. The use of 10 mM heptakis-(2,3,6-tri-O-methyl)-beta-CD in a 100 mM phosphate buffer (pH 3.5) with a temperature of 15 degrees C allowed the separation of the enantiomers of ketoconazole and terconazole with high resolution (R(s) > 2.0). The rapid separation of ketoconazole enantiomers with an analysis time less than 3 min was carried out after fitting some experimental parameters. The developed method was applied to the determination of ketoconazole in different pharmaceutical formulations.     

Enantiomeric separation of gemfibrozil chiral analogues by capillary electrophoresis with heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin as chiral selector

The enantiomeric separation of gemfibrozil chiral analogues was performed by capillary zone electrophoresis (CZE). Resolution of the enantiomers was achieved using heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TM-beta-CD) as chiral selector dissolved into a buffer solution. In order to optimize the separation conditions, type, pH and concentration of running buffer and chiral selector concentration were varied. For each pH value, the optimum chiral selector concentration that produced the resolution of the isomers was found. The migration order of labile diastereoisomers formed was valued at the optimum experimental conditions by adding a pure optical isomer to the racemic mixture. Data from 1H NMR studies confirmed host-guest interaction between TM-beta-CD and 5-(2,5-dimethylphenoxy)-2-ethylpentanoic acid sodium salt. The hypothesized stoichiometry host:guest was 1:1. An apparent equilibrium constant (Ka) was estimated monitoring the chemical shift variation as a function of TM-beta-CD concentration. Salt effect on complexation equilibrium constant was also investigated.     

Fast enantioseparation of arylglycine amides by capillary electrophoresis with highly sulfated-beta-cyclodextrin as a chiral selector

Nine racemic arylglycine amides were synthesized and successfully enantioseparated by capillary electrophoresis (CE) using highly sulfated beta-cyclodextrin (HS-beta-CD) as a chiral selector. Baseline enantioseparation of the analytes was obtained around neutral pH but not in the acidic conditions that are commonly used. HS-beta-CD content, buffer pH, type and concentration, and organic modifier concentration were studied and optimized for fast and efficient separation. A chiral CE separation system composed of 1.5% (w/v) HS-beta-CD, 0 to 10% (v/v) methanol and 20 mM 3-(N-morpholino)propanesulfonic acid at pH 6.5 was shown suitable for baseline enantioseparation of the mentioned amides within 6 min, including simultaneous enantioseparation of three positional isomer series (methyl-, methoxyl or chloro-substituted). By using this system, D-enantiomers migrated ahead of the L-enantiomers and the enantiomeric resolution order of arylglycine amides was more or less parallel to the pK(a), order of the analytes.     

Cyclodextrin-mediated micellar electrokinetic chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the enantiomer separation of racemorphan in human urine.

Micellar electrokinetic chromatography (MEKC) was successfully and conveniently applied to the chiral separation with the addition of cyclodextrins (CDs) as chiral selector to the running buffer. Chiral separation depended on the type of CD; in particular, beta-CD was effective for the chiral separation of racemorphan. We investigated the optimal conditions of type and concentration of CD as chiral selector for the routine enantiomeric separation of racemorphan with good reproducibility. The effects of other parameters such as buffer pH and detection wavelength were also investigated to obtain the optimum conditions for the enantiomeric separation of racemorphan. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for confirmation of racemorphan. The optimal conditions for enantiomeric separation of the racemorphan were as follows: 50 mM borate buffer at pH 9.4 with 50 mM SDS, 10 mM beta-CD and 20% 1-propanol, 57 cm x 50 microns fused-silica capillary column, and UV detection at 192 nm. Based on the developed method, racemorphan in human urine was also separated and determined using solid-phase extraction and MEKC.     

Enantiomeric separation of synthetic 2,3-dihydroxy-3-phenylpropionate compounds by beta-cyclodextrin-modified capillary electrophoresis

A new capillary electrophoretic method was developed for enantiomeric separation and optical impurity analysis of three synthetic 2,3-dihydroxy-3-phenylpropionate compounds using native beta-cyclodextrin (beta-CD) as chiral selector and borate as a background electrolyte. The separation was carried out in uncoated capillary (58.5 cm x 75 microm I.D., effective length 48.5 cm). The results showed that beta-CD as the chiral selector exhibited good enantioselectivity and the baseline separation was obtained at pH 9.8, 200 mM borate buffer containing 1.7% beta-CD at applied voltage 15 kV and capillary temperature 20 degrees C within 15 min. The precision of each tested compound was less than 1.0% at migration time and 5.0% in corrected peak area and the accuracy of the method was in the range of 98.7-105%. Furthermore, the developed method was successfully applied to the determination of the undesirable trace (2S,3R)-(+)-form impurity in the synthetic (2R,3S)-(-)-2,3-dihydroxy-3-phenylpropionate samples.     

Separation and quantitation of the four stereoisomers of itraconazole in pharmaceutical formulations by electrokinetic chromatography

The four stereoisomers of itraconazole were resolved for the first time by EKC using a CD as chiral selector. A study on the enantiomeric separation ability of different neutral CDs was carried out. Heptakis-2,3,6-tri-O-methyl-beta-CD was shown to provide the highest values for the enantiomeric resolution. The influence of some experimental conditions, such as pH, chiral selector concentration, and temperature, on the enantiomeric separation was also studied. The use of a 100 mM phosphate buffer (pH 2.5), 30 mM in heptakis-2,3,6-tri-O-methyl-beta-CD together with an applied voltage of 30 kV and a temperature of 20 degrees C enabled the separation of the enantiomers of itraconazole with high resolutions (Rs > 3.0). Finally, the method was validated and successfully applied to the quantitation of itraconazole in three pharmaceutical formulations.     

Evaluation of the enantioselectivity of carbon nanoparticles‐modified chiral separation systems using dextrin as chiral selector by capillary electrokinetic chromatography

Nanoparticles (NPs) can be used as pseudostationary phases (PSPs) in EKC, which is similar to the use of micelle additives as applied in MEKC. To date, the use of NPs to enhance enantiomeric separation by EKC with β‐CD or its derivative as chiral selector has been reported only in two papers. However, to the best of our knowledge, there has been no prior effort to use NPs for achieving enantioseparation with polysaccharides as chiral selector. This paper describes for the first time the use of carbon nanoparticles (CNPs) as PSPs to modify chiral separation system employing dextrin as chiral selector for the enantioseparations of several basic drugs in capillary EKC. Three different types of CNPs, including carbogenic nanoparticles (NPs), carboxylated single‐walled carbon nanotubes, and carboxylated multiwalled carbon nanotubes, were used as running buffer additives, respectively. The potential of the PSPs and the effects of dextrin concentration, buffer pH, and buffer concentration on the enantioseparations were evaluated. Four pairs of tested enantiomers were successfully resolved in less than 15 min with the resolution values in the range of 1.41–4.52 under optimized conditions. Compared to the buffer without NPs, the introduction of NPs into the buffer enhanced the separation of the enantiomers.     

毛细管区带电泳对手性药物盐酸美西律和盐酸异博定的对映体分离

应用环糊精-毛细管区带电泳体系对手性药物盐酸美西律和盐酸异博定的对映体分离进行了研究。结果表明, 在所研究的手性选择剂α-环糊精, β-环糊精, 二甲基-β-环糊精, 羟丙基β-环糊精和γ-环糊精中, 羟丙基β-环糊精对所研究的手性药物分离效果较好。对盐酸美西律和盐酸异博定的最佳羟丙基-β-环糊精浓度分别为30mmol/L和9mmol/L, 最佳缓冲溶液浓度为100mmol/L Tris-H3PO4(pH2.3)。向缓冲溶液中加入0.05%羟丙基纤维素(HPLC)可改善分离。盐酸美西律获得了接近基线的手性分离, 而盐酸异博定亦获得了较好的分离。     

Determination of fenfluramine enantiomers in pharmaceutical formulations by capillary zone electrophoresis

Summary Capillary zone electrophoresis has been used for the enantiomeric separation of racemic ortho-fenfluramine and meta-fenfluramine employing a phosphate buffer at pH 2.5 added with cyclodextrins. The cyclodextrin type and concentration strongly influenced the chiral resolution. The uncharged β-cyclodextrin polymer gave enantiomeric resolution of both ortho and meta isomers, while γ-cyclodextrin was a good chiral selector for only ortho-fenfluramine; heptakis-2,3,6-tri-O-methyl-β-cyclodextrin permitted base line separation of meta-fenfluramine enantiomers but only partial resolution of racemic ortho-fenfluramine. The optimized electrophoretic method was applied to the quantitative analysis of 1-meta-fenfluramine (minor component in the mixture) and d-meta-fenfluramine in a commercial pharmaceutical formulation. Good reproducibility for migration time and corrected peak areas (R.S.D. <0.8% and <1.2%, respectively) was achieved and the presence of the minor component of the mixture was found to be in accord to previous determinations performed by other analytical methods.     

Determination of fenfluramine enantiomers in pharmaceutical formulations by capillary zone electrophoresis

Summary Capillary zone electrophoresis has been used for the enantiomeric separation of racemic ortho-fenfluramine and meta-fenfluramine employing a phosphate buffer at pH 2.5 added with cyclodextrins. The cyclodextrin type and concentration strongly influenced the chiral resolution. The uncharged β-cyclodextrin polymer gave enantiomeric resolution of both ortho and meta isomers, while γ-cyclodextrin was a good chiral selector for only ortho-fenfluramine; heptakis-2,3,6-tri-O-methyl-β-cyclodextrin permitted base line separation of meta-fenfluramine enantiomers but only partial resolution of racemic ortho-fenfluramine. The optimized electrophoretic method was applied to the quantitative analysis of 1-meta-fenfluramine (minor component in the mixture) and d-meta-fenfluramine in a commercial pharmaceutical formulation. Good reproducibility for migration time and corrected peak areas (R.S.D. <0.8 % and <1.2 %, respectively) was achieved and the presence of the minor component of the mixture was found to be in accord to previous determinations performed by other analytical methods.     

3-苯基乳酸的手性毛细管电泳拆分

考察了环糊精种类、环糊精浓度、缓冲溶液pH、分离电压、温度等因素对3-苯基乳酸手性分离的影响,并对分离条件进行了优化。结果表明,采用区带毛细管电泳技术,以0.03 mol/L的羟丙基-β-环糊精为手性选择剂,0.1 mol/L的磷酸缓冲溶液(pH 5.5)为电泳缓冲溶液,26 kV的分离电压,在25 ℃下可使3-苯基乳酸对映体达到基线分离,分离度为1.51。     

Determination of the enantiomeric purity of 2-(2′-carboxy-3′-phenylcyclopropyl)glycines by chiral capillary electrophoresis

Summary A fast and practical chiral capillary electrophoretic method has been developed for determination of the enantiomeric purity of 2-(2′-carboxy-3′-phenylcyclopropyl)glycine (PCCG) compounds. In particular, the isomer PCCG-13, a potent selective and competitive antagonist at phospholipaseD-coupled metabotropic glutamate receptors (mGluRs), was completely resolved from its enantiomer PCCG-15 by use of dimethyl-β-cyclodextrin (DMCD) as chiral selector. pH 9.0 running buffer containing 2-amino-2-methyl-1,3-propanediol (AMPD; 100mM) was a suitable medium enabling resolution of the enantiomers in a short (32.5 cm total length) poly(vinyl alcohol) (PVA)-coated capillary. Because of the suppression of the electroosmotic flow and the good peak shape, baseline resolution of the enantiomers was obtained by use of the optimum concentration of chiral selector. For quantitative purposes at impurity levels, high sample loading was required and adequate separation was obtained in the presence of 80 mM DMCD. This CE method enabled quantification of 0.3% m/m of undesired enantiomer in PCCG-13; the samples analyzed, obtained from enantioselective synthesis, proved to be of high enantiomeric purity.     

Enantioseparation of citalopram analogues with sulfated β‐cyclodextrin by capillary electrophoresis

Capillary electrophoresis methods were developed for the enantiomeric separation of 27 citalopram analogues. Sulfated β‐cyclodextrin was the most broadly selective and useful chiral selector. The separations of most of the citalopram analogue compounds reported in this work have not been reported previously. Excellent enantiomeric separations were obtained for 26 out of 27 compounds, and most of the separations were achieved within 10 min. The effects of chemical parameters such as chiral selector types, buffer types, chiral selector and buffer concentrations, buffer pH and organic modifiers on the separation were investigated. The influence of analyte structure on separation also was examined and discussed.