Role of capillary electrophoresis methods in the drug development process

In the past several years, capillary electrophoresis (CE) has generated considerable interest from pharmaceutical companies for control of both the chiral and achiral purity of bulk drugs and drug products. This paper evaluates the use of CE as: (1) a technique complementary to HPLC for the determination of peak homogeneity of a drug, (2) for determination of chiral purity, and (3) for determination of achiral purity. It would be greatly advantageous if CE could be used to determine both the chiral and achiral purity in a single assay. This investigation compares the results obtained for the separation of the enantiomers of duloxetine using several neutral cyclodextrins to those obtained using anionic cyclodextrins (sulfobutyl ether derivatives) as chiral selectors added to the separation buffer. In addition, it reports chiral separations obtained by using neutral cyclodextrins in a sulfonic acid-coated capillary column, which give a negatively charged capillary surface and electro-osmotic flow even in low pH buffers. The possible mechanism of separation is discussed. © 1996 Wiley-Liss, Inc.     

Comprehensive strategy for chiral separations using sulfated cyclodextrins in capillary electrophoresis

This review focuses on the emerging role of sulfated cyclodextrins in the capillary electrophoretic (CE) separation of chiral analytes. Since being introduced as enantioselective agents for CE in 1995, these anionic additives have continued to demonstrate remarkable application universality. The broad spectrum of chiral compounds successfully separated using this approach includes acidic, basic, neutral, and zwitterionic species. This impressive array of analyte structures is derived from a growing diversity of compound classes including pharmaceuticals, plant extracts, biomarkers, herbicides, alkaloids, fungicides, and metal ions. Moreover, literature reports highlight the minimal optimization required to achieve a successful separation. Based on these findings, sulfated cyclodextrins appear to be well suited for the development of a more universal, comprehensive separation strategy for chiral compounds. This review explores this proposition by beginning with the structure and migration properties of sulfated cyclodextrins, using applications to highlight the separating power of this technique and ending with a pragmatic, comprehensive separation strategy.     

Chiral separation of bioactive cyclic Mannich ketones by HPLC and CE using cellulose derivatives and cyclodextrins as chiral selectors

The chiral separation of cyclic Mannich ketones of potential pharmaceutical interest is investigated using HPLC and CE. These Mannich ketones show a marked antibacterial and antifungal activity. In HPLC, stationary phases containing cellulose derivatives or beta-cyclodextrin were used and in CE different cyclodextrins, such as beta-CD, gamma-CD, carboxymethyl-beta-CD and succinyl-beta-CD were added to the background electrolyte as chiral selectors.     

Dichloro-, dimethyl-, and chloromethylphenylcarbamate derivatives of cyclodextrins as chiral stationary phases for high-performance liquid chromatography

New dichloro-, dimethyl-, and chloromethylphenylcarbamate derivatives of cyclodextrins (CDs) were prepared and their enantiomeric recognition abilities were evaluated as chiral stationary phases (CSPs) in normal phase high-performance liquid chromatography (HPLC). The effects of the type of cyclodextrins, the nature and position of the substituents on the phenyl ring, binding mode and spacer on the chiral recognition were studied in detail. No marked change of chiral recognition abilities was established by reversing the binding side of CDs (i.e., by the narrower [primary] opening of the cone-shaped CD to silica gel with the wider [secondary] opening sides). This result indirectly proves the previously drawn conclusion about the minor role of inclusion phenomena in chiral recognition in this case. Nevertheless, chiral recognition of these CSPs toward some compounds critically depends on the type of CDs used. All CD derivatives described in this study show rather low enantiomeric resolving abilities compared with corresponding polysaccharide (cellulose and amylose) derivatives, although very high enantioselectivity of separation was observed for a few compounds, such as racemic flavanone and cyclopropanedicarboxilic acid dianilide. © 1996 Wiley-Liss, Inc.     

Enantioselective separations using capillary electrophoresis

The preconditions are outlined for enantioselective separations in capillary electrophoresis (CE) with chiral selectors as additives to the background electrolyte. Free solution capillary electrophoresis conditions are characterised by a single solution phase. Chiral separations are reviewed by selector type (chiral ligand exchange, cyclodextrins, crown ethers, glycoproteins) with the extensive studies on cyclodextrins grouped into sections on amino acids, pharmaceuticals, and speciality chemicals, optimisation, biological fluids, and quantitative aspects. In micellar electrokinetic capillary chromatography, enantioselective discrimination occurs by partition in a two-phase system, with a chiral micellar phase as selector. Optimum separation conditions can be readily predicted for a given selector–selectand combination, and absolute values of binding constants determined by CE. Advantages of CE in comparison with HPLC using a chiral stationary phase include robust, rapid assays and the use of small volumes of aqueous solutions; disadvantages include less favourable detection limits. © 1994 Wiley-Liss, Inc.     

Enantiomeric separation of bupropion enantiomers by electrokinetic chromatography: quantitative analysis in pharmaceutical formulations

The first CE method enabling the quantitation of the two enantiomers of bupropion was developed in this work. Electrokinetic chromatography (EKC) mode using cyclodextrins as chiral selectors was employed. A study on the enantiomeric separation ability of different neutral and anionic CDs was carried out. Sulfated-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, temperature, and separation voltage on the enantiomeric separation of bupropion was also studied. The use of 10 mM sulfated-beta-CD in 50 mM borate buffer (pH 9.0) with an applied voltage of 30 kV and a temperature of 30 degrees C enabled the separation of the enantiomers of bupropion with high resolution (Rs > 7) and short analysis time (approximately 3.5 min). Finally, the method was successfully applied to the quantitation of bupropion in two pharmaceutical formulations.     

Enantioselectivity of potentiometric sensors with application of different mechanisms of chiral discrimination

In the recent years, numerous successful applications of various chiral selectors in high performance separation methods have generated an increasing interest in the application of some of these compounds as electroactive species in potentiometric sensors. The objective of this work was to examine the enantioselectivy of several different sensors employing substituted cyclodextrins, example antibiotic teicoplanin and electrodeposited conductive polymers for various chiral analytes. Varying degrees of enantioselectivity were found for the ion-selective electrodes examined, depending on the chiral selector used and the target analyte.     

Separation of the enantiomers of some racemic nonsteroidal aromatase inhibitors and barbiturates by capillary electrophoresis

High-performance capillary electrophoresis (HPCE) and micellar electrokinetic capillary chromatography (MECC) were applied to the resolution of racemic nonsteroidal antiaromatase drugs and intermediates. Successful results were obtained in both modes using α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD), or 2,6-di-O-methyl-β-cyclodextrin (DM-β-CD) as chiral selectors. Depending on the structure of the solute, one of the cyclodextrins was generally better suited for resolution of the racemate. The basic solutes were analyzed under HPCE conditions, whereas the nonionizable compounds such as glutethimide (Doriden®) were analyzed in MECC mode. For the azole-type antiaromatase Fadrozole, both HPCE and MECC modes could be used to achieve the separation of the enantiomers. The influence of experimental factors such as pH, the presence of organic modifier, temperature, the micelle concentration, and the concentration of the chiral selector is also discussed on the basis of the results obtained with some chiral barbiturates. The possibility of analyzing the enantiomers directly in plasma samples was also demonstrated. © 1993 Wiley-Liss, Inc.     

Emerging subject for chiral separation science: cluster boron compounds

The structural chirality is an inherent feature of fully synthetic boron cluster compounds that sometimes exhibit unique biochemical effects. HPLC studies with zwitter-ionic cluster boron compounds and electrophoretic studies with boron cluster anions reveal that the chiral separability of these species is remarkably dissimilar to that of organic species, if uncharged cyclodextrins are used as chiral selectors. Furthermore, marked differences were found between the analytical characteristics of the chiral separations of the boron cluster species and those of the organic species with uncharged cyclodextrins. The present-day experimental database indicates that the rules valid for the chiral separations of the organic species cannot be applied to the chiral separations of the boron cluster species without experimental verification. The current extent of research work devoted to the investigation of chirality and chiral separations of boron cluster species is negligibly small in comparison with that devoted to the investigation of chirality and chiral separations of organic species. This makes difficult a reliable explanation of both the particularities observed in chiral separations of boron cluster species with cyclodextrins as chiral selectors and the strange effects related to these separations at the moment.     

Separation of nucleoside phosphoramidate diastereoisomers by high performance liquid chromatography and capillary electrophoresis

Separations of five diastereoisomers of nucleoside phosphoramidate derivatives (pronucleotides) were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and CE method using anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using in a normal-phase methodology. Capillary electrophoresis was used as an alternative technique to HPLC for the separation of pronucleotides. The diastereoisomers were fully resolved with sulfated cyclodextrins at both BGE pH (2.5 and 6.2). Limits of detection and limits of quantification, calculated for both methods, are up to 200 times higher in CE separations than in HPLC separations. The analytical HPLC method was then applied in a preliminary study for the pronucleotide 1 quantification in cellular extract.     

Structural studies on the chiral selector capacity of cyclodextrin derivatives

Chromatographic separation of enantiomers to assure or enhance chiral purity is of considerable importance and can be achieved by the use of selectors of great structural variety. Cyclodextrins are an important and frequently used class, and they are multimodal selectors since multiple chiral interactions are possible by very different mechanisms. Here, the results of a preliminary examination on the possible value of computational molecular modeling approaches for the predictability of cyclodextrin selector effects for compounds that possess both geometrical and optical isomerism are presented. Interactions between various cyclodextrins and pyrethroic acids are modeled, interpreted, and compared to experimental capillary electrophoresis data.     

Evaluation of newly synthesized and commercially available charged cyclomaltooligosaccharides (cyclodextrins) for capillary electrokinetic chromatography

A highly new charged cyclodextrin (CD) derivatives, (6-O-carboxymethyl-2,3-di-O-methyl)cyclomaltoheptaoses (CDM-beta-CDs), was synthesized and characterized as anionic reagents for capillary electrophoresis (CE) in an electrokinetic chromatography mode of separation. Substitution with dimethyl groups at the secondary hydroxyl sites of the CD is aimed at influencing the magnitude and selectivity of analyte-CD interactions, while substitution by carboxymethyl groups at the primary hydroxyl sites provides for high charge and electrophoretic mobility. Full regioselective methylation at the secondary hydroxyl sites was achieved in this work, while substitution at the primary hydroxyl sites generated a mixture of multiply charged products. The separation performance of CDM-beta-CD was evaluated using a variety of analyte mixtures. The results obtained from commercially available negatively charged cyclodextrins, heptakis(2,3-di-O-methyl-6-O-sulfo)cyclomaltoheptaose (HDMS-beta-CD) and O-(carboxymethyl)cyclomaltoheptaose (CM-beta-CD) with an average degree of substitution one (DS 1), were compared to CDM-beta-CD using a sample composed of eight positional isomers of dihydroxynaphthalene. Four hydroxylated polychlorobiphenyl derivatives, a group of chiral and isomeric catchecins, and chiral binaphthyl compounds were also separated with CDM-beta-CD. The effect of adding neutral beta-cyclodextrin (beta-CD) into the running buffer containing charged cyclodextrins was investigated and provided evidence of significant inter-CD interactions. Under certain running buffer conditions, the charged cyclodextrins also appear to adsorb to the capillary walls to various degrees.     

Enantiomer separation of dihydropyridine calcium antagonists with cyclodextrins as chiral selectors: structural correlation

Native and substituted cyclodextrins (CDs) were used as chiral selectors both in high-performance liquid chromatography and capillary electromigration separations (HPCE and MEKC). Chromatographic data of five dihydropyridine calcium antagonists obtained on three β-CD chiral stationary phases in reversed-phase mode were compared with those of capillary electrophoresis using β-CDs in the presence and absence of sodium dodecyl sulfate (SDS). Competition of separated compounds with SDS molecules for penetration into the CD cavity can limit their necessary interaction with the chiral selector and consequently even preclude enantiomer separation. Some insight into this problem can be brough about by comparing the experimental data with computer-aided energy minimization of CD-solute and CD-SDS inclusion complexes.     

Chiral separations of 1,3,4-thia- and 1,3,4-selenadiazine derivatives by use of non-aqueous capillary electrophoresis

Our aim was to establish suitable conditions for the chiral separation of 12 1,3,4-thia- and 1,3,4-selenadiazine derivatives; some of them were identified in screening tests as potential antituberculotics. To overcome possible problems with the water insolubility of most analytes, we profited by the advantages of non-aqueous capillary electrophoresis. Methanol, formamide, and a mixture of formamide with acetonitrile (1:2, v/v) were used as separation media. Hydroxyethyl-, hydroxypropyl-, and methyl-beta-cyclodextrin were applied as chiral selectors in concentrations of 200 mM. Besides the effect of these different electrophoretic media and selectors, we also investigated the consequences of using different electrolytes (25 mM ammonium acetate/1 M acetic acid and 25 mM citric acid/12.5 mM TRIS). Distinct differences of the separation factors in the different separation media were observed. Depending on structure characteristics of the analytes, we established clear classifications to these cyclodextrins (CD), which were most appropriate for the separation of the enantiomers of the particular analytes.     

Use of Sulfated Cyclofructan 6 and Sulfated Cyclodextrins for the Chiral Separation of Four Basic Pharmaceuticals by Capillary Electrophoresis

Sulfated cyclofructan 6 (S‐CF6) and sulfated cyclodextrins (S‐α‐, β‐, γ‐CDs) are highly selective chiral selectors for the enantioseparation of basic solutes. In this study, S‐CF6 was introduced for the enantiomeric separation of four basic pharmaceuticals (including tamsulosin, tiropramide, bupivacaine, and norephedrine) by capillary electrophoresis (CE), and the enantiomeric separation performance was compared with S‐α‐, β‐, γ‐CDs. The effects of the chiral selector type, chiral selector concentration, operating voltage, and column temperature were examined and optimized. Excellent resolutions were obtained for all solutes on these chiral selectors. Chirality 25:735–742, 2013. © 2013 Wiley Periodicals, Inc.     

Enantioseparation of beta-methyl-substituted amino acids with cyclodextrins by capillary zone electrophoresis

Direct capillary zone electrophoretic methods were developed for the separation of the enantiomers of unnatural beta-methyl-amino acids such as erythro- and threo-beta-methylphenylalanine, beta-methyltyrosine, beta-methyltryptophan and beta-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Capillary zone electrophoresis was carried out using sulfopropylated-alpha-CD (SP2-alpha-CD), sulfopropylated-beta-CD (SP2-beta-CD) both with a degree of substitution of 2 moles/mole cyclodextrin, and sulfopropylated-beta-CD (SP4-beta-CD) with a degree of substitution of 4moles/mole beta-cyclodextrin. The effects of selector and buffer concentrations, electrolyte pH and applied voltage were studied on the separation efficiency. Varying the electrophoretic conditions with application of 20 kV, hydrodynamic injection, unmodified silica capillary, three different buffers (borate, phosphate and acetate) and modified cyclodextrins as chiral selectors all compounds investigated are nearly baseline resolved. The elution sequence was determined in most cases.     

Gas chromatographic enantioseparation of unfunctionalized chiral alkanes: a challenge in separation science (overview, state of the art, and perspectives)

The chromatographic enantioseparation of small unfunctionalized chiral alkanes C*HR(1)R(2)R(3) (R = alkyl) represents a challenge in separation science. Because of the lack of any functional groups, enantiorecognition in the presence of a chiral selector is solely based upon weak enantioselective Van der Waals forces. Racemic alkanes containing seven and eight carbon atoms, i.e. 3-methylhexane (C7), 2,3-dimethylpentane (C7), 3-methylheptane (C8), 3,4-dimethylhexane (C8), 2,4-dimethylhexane (C8), 2,3-dimethylhexane (C8), and 2,2,3-trimethylpentane (C8) have been gas chromatographically enantioseparated on different modified cyclodextrins. The substitution pattern and cavity size of the cyclodextrin selectors have a pronounced effect on the degree of enantiorecognition observed. Thermodynamic parameters of enantiorecognition between four chiral alkanes and octakis(6-O-methyl-2,3-di-O-pentyl)-gamma-cyclodextrin (Lipodex G) have been determined. The possible role of molecular inclusion is indicated by the complete loss of enantioselectivity when the cyclodextrins are replaced by the corresponding linear dextrins ("acyclodextrins"). The enantioseparations of all seven chiral C7-C8 alkanes, six of them simultaneously, has been achieved on mixed binary selector systems whereby two different modified cyclodextrins are present in one gas chromatographic column. The smallest chiral (nonisotopically labeled) allene, i.e., 2,3-pentadiene, has been resolved gas chromatographically on a cyclodextrin selector.     

Chiral separation of salbutamol and bupivacaine by capillary electrophoresis using dual neutral cyclodextrins as selectors and its application to pharmaceutical preparations and rat blood samples assay

An attempt for the simultaneous separation of salbutamol (Sal) and bupivacaine (Bup) enantiomers was performed by capillary elecytrophoresis with a dual mixture of neutral cyclodextrins as chiral selector. The influence on the separation of several parameters such as buffer composition, pH, the concentration ratio of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) to dimethyl-beta-cyclodextrin (DM-beta-CD) was investigated. A better separation was obtained for Sal and Bup with the CD mixtures compared to the use of HP-beta-CD or DM-beta-CD alone. The best simultaneous separation of Sal and Bup enantiomers was achieved with a 20 mM HP-beta-CD and 20 mM DM-beta-CD at pH 2.5 in a triethanolamine (TEA)/phosphate buffer. This method-utilized chlorphenamine (Chl) as an internal standard was found to be linear in the range 0.5-100 microg/mL and 0.5-150 microg/mL for Sal and Bup enantiomers, respectively. The limits of detection for both enantiomers of Sal and Bup were 0.18 and 0.24 microg/mL, respectively. The proposed method was applied to monitor Sal and Bup enantiomers concentration change in rat blood samples obtained from a male rat after celiac doses administration 0-30 min of Sal and Bup racemate. The method could also be used to determine Sal enantiomers in a pharmaceutical aerosol.     

Chiral separation of catechin by capillary electrophoresis using mono‐, di‐, tri‐succinyl‐β‐cyclodextrin as chiral selectors

The chiral separation of (±)‐catechin was investigated by capillary electrophoresis using characterized succinyl‐β‐cyclodextrins (Suc‐β‐CDs) with one to three degree of substitution values. The effects of nature and concentration of Suc‐β‐CDs and running buffer pH on the migration time and resolution of (±)‐catechin are discussed. All three kinds of Suc‐β‐CDs show a clear baseline separation of (±)‐catechin in capillary electrophoresis. Mono‐Suc‐β‐CD effectively separated (±)‐catechin, and additional substituted CDs (di‐ and tri‐Suc‐β‐CD) were capable of chiral separation at a broad pH range. The optimum running conditions were found to be 100 mM borate buffer (pH 9.8) containing 5 mM mono‐Suc‐β‐CD with no methanol organic modifier. Chirality, 2009. © 2009 Wiley‐Liss, Inc.     

Enantioseparation of citalopram enantiomers by capillary electrophoresis: Method development through experimental design and computational modeling

Citalopram (CIT) is a frequently used modern antidepressant that inhibits selectively serotonin reuptake in the brain. It has a chiral center in its structure and is used in therapy as both racemic mixture and pure enantiomer as its pharmacological effect is almost entirely associated with S-CIT. The aim of this study was the development of a simple and rapid capillary electrophoresis (CE) method for the separation and quantification of CIT enantiomers. To establish the optimum chiral selector, several native and derivatized, neutral, and ionized cyclodextrins (CDs) were examined at different pH levels. An experimental design strategy was adopted for method optimization; a fractional factorial design was applied for screening purposes to identify significant experimental factors followed by a face-centered central composite design used for optimization purposes. Computational modeling was used to obtain information on the interaction energy and the geometry of the complexes to aid in the understanding of chiral separation mechanism. The best results were obtained when using a 25-mM phosphate buffer at pH 7.0, 3-mM CM-β-CD as chiral selector, 17.5°C temperature, 15-kV voltage, and 50 mbar/s hydrodynamic injection. The separation time was fast, below 3 min, and the migration order was S-CIT followed by R-CIT. The analytical performance of the method was verified in terms of precision, linearity, accuracy, sensibility, and robustness, and the method was applied for the determination of CIT enantiomers from pharmaceutical preparations.