Method development strategies for the enantioseparation of drugs by capillary electrophoresis using cyclodextrins as chiral additives

General strategies for the development of capillary electrophoretic methods for the enantiomeric separation of basic, acidic or neutral drugs were developed. For all kinds of compounds, the use of a buffer made of 100 mM phosphoric acid adjusted to pH 3 with triethanolamine and containing anionic and/or uncharged cyclodextrin (CD) derivatives as chiral selectors was recommended. Two different optimization schemes depending on the acidic or basic character of the analytes, were elaborated. For most basic compounds present in cationic form at pH 3, enantiomeric separation could be achieved in the normal polarity mode. Different beta-cyclodextrin derivatives were first tested at a given concentration. Five derivatives were found to be particularly useful for enantioseparations in capillary electrophoresis (CE): the anionic carboxymethyl-beta-CD (CMCD) and sulfobutyl-beta-CD (SBCD) and the neutral dimethyl-beta-CD (DMCD), trimethyl-beta-CD (TMCD) and hydroxypropyl-beta-CD (HPCD). After selection of the most suitable CD, its concentration was optimized with respect to chiral resolution. If necessary, a further improvement in resolution could often be obtained for the enantiomers of cationic solutes by increasing the buffer pH from 3 to 5 using CMCD as chiral additive. Another possible alternative for enhancement in chiral resolution was the addition of metharlol or cyclohexanol to the buffer. For acidic drugs, essentially present in uncharged form at pH 3, and for neutral solutes, anionic CD derivatives such as SBCD or CMCD were first tested at a given concentration in the reversed polarity mode. Dual systems, based on the simultaneous addition of a charged CD (SBCD or CMCD) and a neutral CD (TMCD or DMCD), could then be investigated for resolution improvement. After optimization of the CD concentrations, the use of dual systems with CMCD at pH 5 could also be tested if necessary, especially for very weak acidic and neutral drugs. By applying these optimization strategies, 48 of the 50 drugs examined as model compounds could be fully enantioseparated by CE in short analysis times (usually less than 10 min).     

What makes people study more? An evaluation of factors that affect self-paced study

The separation of chiral compounds by capillary electrophoresis (CE) is a very interesting field of research in different areas such as pharmaceutical, environmental, agricultural analysis etc. The separation of two enantiomers can be achieved in CE using a chiral environment interacting with the two analytes on forming diastereoisomers with different stability constants and thus different mobilities. A wide number of chiral selectors have been employed in CE and among them glycopeptide antibiotics exhibited excellent enantioselective properties towards a wide number of racemic compounds. Vancomycin, ristocetin A, rifamycins, teicoplanin, kanamycin, streptomycin, fradiomycin, and two vancomycin analogues, added to the background electrolyte (BGE), are the antibiotics studied by CE running the separation in untreated and/or coated fused-silica capillary. Due to adsorption and absorption phenomena, some drawbacks can be expected when using bare fused-silica capillary, e.g., changes of electroosmotic flow (EOF), broaden peaks, reduced efficiency and low sensitivity. Coated capillary and counter current mode can be the solution to overcome the above mentioned problems. This review surveys the separation of enantiomers by CE when macrocyclic antibiotics are used as chiral selector. The enantioselectivity can be easily controlled modifying several parameters such as antibiotic type and concentration, pH, ionic strength and concentration of the background electrolyte, organic modifier etc. The paper also presents a list of the latest chiral separations achieved by CE where antibiotics were used as chiral selector.     

Evaluation of learning and memory dysfunction and histological findings in rats with chronic stage contusion and diffuse axonal injury

Enantiomer separations of various acidic racemates were performed by capillary electrophoresis (CE) using a commercial cationic beta-cyclodextrin (beta-CD), quaternary ammonium beta-CD (QA-beta-CD), and a commercial amphoteric beta-CD (AM-beta-CD) as chiral selectors. Eleven acidic racemates were successfully separated using QA-beta-CD by changing the CD concentration and the buffer pH. These enantiomer separations were compared with the results using five neutral CD derivatives. Although most racemates were separated with some of the neutral CDs, relatively high CD concentrations were required to obtain baseline separations. In contrast, when QA-beta-CD was employed, the enantiomer separations were successful at low concentrations below 5 mM. Enantiomers of five acidic racemates and ten dansylated amino acids (Dns-amino acids) were separated using AM-beta-CD. Although the baseline separation of racemic 4-chloromandelic acid was not achieved with either QA-beta-CD or five neutral CDs, AM-beta-CD showed complete resolution. Furthermore, the simultaneous enantiomer separation of eight Dns-amino acids was also achieved with AM-beta-CD. Both QA-beta-CD and AM-beta-CD were analyzed by CE and mass spectrometry (MS) in order to identify their compositions because they consisted of a mixture having different degrees of substitution. QA-beta-CD consisted of six components having from one to six quaternary ammonium groups. The composition of AM-beta-CD, however, was very complicated and could not be identified.     

Angioplasty and primary stenting of the subclavian, innominate, and common carotid arteries in 83 patients

A new macrocyclic of the bis(benzylisoquinoline) alkaloid family, d-(+)-tubocurarine chloride (DTC), has been evaluated as a chiral selector for the separation of optical isomers of organic carboxylates using capillary electrophoresis (CE). The pertinent physicochemical properties, such as absorption spectrum, isoionic point, and solution conformation, of DTC were determined. The effects of varying such experimental parameters as DTC concentration, pH, and methanol content in the running buffer were assessed. CE separation of the enantiomers of 18 different compounds was achieved using DTC as the chiral selector under optimized background electrolytic conditions.     

Evaluation of the liquid-chromatographic resolution of indenoindolic racemic compounds on three protein-based chiral stationary phases

Three liquid-chromatography columns, containing immobilized proteins as chiral stationary phases (CSPs) were investigated for the direct separation of enantiomers of racemic indenoindolic compounds. By using an experimental design the effects of column temperature, pH, ionic strength, and type and concentration of organic solvent in the mobile phase on retention and resolution of racemic substances were systematically studied. The three CSPs investigated consisted of alpha(1)-acid glycoprotein (Chiral-AGP), bovine serum albumin (BSA-DSC) and ovomucoid (Ultron ES-OVM). The two enantiomers of all studied compounds could be separated on at least one of the three CSPs, which have different enantioselective properties.     

Relationship of adverse events to serum drug levels in patients receiving high-dose azithromycin for mycobacterial lung disease

The polymerized surfactant poly(sodium N-undecylenyl amino L-valinate) [poly(L-SUV)] has been used in micellar electrokinetic capillary chromatography for the chiral separation of various acidic and basic drugs, as well as neutral compounds. Under the conditions studied, poly(L-SUV) was shown to be a very versatile anionic chiral selector in the pH range of 5.6-11. The micelle was used for the enantioseparation of coumarinic anticoagulant drugs with various buffers under moderately acidic conditions. Neutral and alkaline buffer conditions were used to successfully separate the neutral atropisomers (+/-)-1,1'-bi-2-naphthol, (+/-)-1,1'-binaphthyl-2,2'-diamine, and Tr?ger's base. Chiral separation of the cationic paveroline drugs, laudanosine, norlaudanosoline, and laudanosoline, was influenced by pH and the use of coated capillaries. The acquired data focused on optimizing the migration times, capacity and separation factors, and electrophoretic mobilities of the various racemic mixtures.     

Stereoselective determination of S-naproxen in tablets by capillary electrophoresis

A capillary electrophoresis (CE) method was developed for the stereoselective determination of the non-steroidal anti-inflammatory drug (NSAID), S-naproxen, in tablets. Several beta-cyclodextrin derivatives (CDs) were tested as chiral selectors, including sulfobutyl-beta-CD (SBCD), carboxymethyl-beta-CD (CMCD), dimethyl-beta-CD (DMCD) and trimethyl-beta-CD (TMCD), in a phosphoric acid/triethanolamine pH 3 buffer. Under these conditions, the analyte was mainly present in an uncharged form and therefore, the use a neutral CD (DMCD or TMCD) alone could not lead to enantiomeric separation. On the contrary, by addition of a charged CD (SBCD or CMCD) to the running buffer, giving the analyte enantiomers an adequate mobility, chiral resolution could be achieved, although the resolution values obtained in this case were not quite satisfactory (Rs < 1.5). Dual systems, based on the use of mixtures of charged and neutral CDs, were then investigated. The SBCD/TMCD system was found to be particularly well suited to the enantioseparation of naproxen and after optimisation of the concentrations of both CDs, a resolution value of 5.4 could be obtained. The method was validated for the determination of R-naproxen (enantiomeric impurity) in the 0.1-2% range, using the racemic mixture of the analyte. A second validation was performed in the 50-150% range for the quantitation of S-naproxen. In both cases, good results with respect to linearity, precision and accuracy were obtained.     

Separations of derivatized amino acid enantiomers by cyclodextrin-modified capillary electrophoresis: mechanistic and molecular modeling studies

The enantiomers of 5-dimethylamino-1-naphthalene sulfonyl (DNS)-derivatives of selected amino acids were successfully separated using capillary electrophoresis (CE) employing cyclodextrins (CD) as enantio-selective running buffer additives. A previously described model for retention and chiral recognition in CD-modified CE is shown to adapt well in this application. Resolution of the isomers is strongly influenced by the type and concentration of cyclodextrin employed, as predicted by the model. Although data indicates differences in the electrophoretic mobilities for some of the completely complexed enantiomer pairs, selectivity generally requires exploiting differences in the amino acid-CD complexation constants for enantiomer pairs. In this work, the D-enantiomers exhibit larger formation constants and are complexed to a greater degree (elute first) at moderate CD concentration. When mixtures of amino acids are analyzed, the effects of separation conditions on general elution behavior must be considered or separated enantiomer pairs will co-elute with other enantiomers. Preliminary results aimed at predicting the strength of DNS-amino acid enantiomer-CD interactions based on molecular modeling studies are presented. A statistical mechanical approach to treating computationally derived enantiomer-CD interaction energies is shown to provide reasonable correlation with separation performance.     

Pharmaceutical applications of micelles in chromatography and electrophoresis

This review surveys enantiomer separation by capillary electrophoresis (CE) using polysaccharides as chiral selectors. Many ionic or electrically neutral polysaccharides, such as heparin, chondroitin sulfate, dextrin, etc., have been employed successfully for the CE separation of enantiomers. The operational conditions that affect the enantioselectivity of the chiral separation system will be described. The mechanism of enantioseparation will also be discussed briefly.     

Chiral separation of pharmaceuticals possessing a carboxy moiety

The separation of carboxylic enantiomers in the pharmaceutical field using high-performance liquid chromatographic and capillary electrophoretic techniques is reviewed. The techniques used for chiral separation include diastereomer derivatization, a chiral mobile phase, a chiral stationary phase (high-performance liquid chromatography) and chiral additives (capillary electrophoresis). Practical and conventional separation systems for pharmaceutical applications, such as pharmacokinetics, optical purity testing and stability studies, are described. A comprehensive collection of applications to carboxylic drugs and other carboxylic compounds of pharmaceutical interest is listed in the tables. The characteristics of each enantioseparation method are also discussed briefly.     

Facile liquid chromatographic enantioresolution of native amino acids and peptides using a teicoplanin chiral stationary phase

The glycopeptide antibiotic teicoplanin is shown to be a highly effective stationary phase chiral selector for the resolution of underivatized amino-acid and imino-acid enantiomers. Fifty four of these compounds (including all chiral protein amino acids) as well as a number of dipeptides were resolved. Hydro-organic mobile phases are used and no buffers or added salts are needed in most cases. Hence the purified analytes are easily isolated in pure form, if needed, by evaporating of the solvent. The effect of pH, organic modifier type and amount are discussed. The enantioselective separation mechanism is examined using both molecular modeling and retention data. The strongest stereoselective interaction is for carboxy-terminated D-amino-acids. In case of peptides, it is not necessary for these to be a D-, D-, terminal sequence for strong interactions. In some cases, including Ala-Ala, the L-, D-, terminal sequence showed greater interaction with the teicoplanin chiral stationary phase.     

Enantiomeric separation of tramadol hydrochloride and its metabolites by cyclodextrin-mediated capillary zone electrophoresis

The enantiomeric separation of tramadol hydrochloride and its major metabolites, O-demethyltramadol (M1) and N-demethyltramadol (M2) was studied using cyclodextrin (CD)-mediated capillary zone electrophoresis (CZE). Influence of the choice of type and concentration of CD, capillary temperature, length of capillaries, buffer pH and the addition of polymer modifier on the chiral separation of tramadol and its metabolites was evaluated. It was found that the drug and the metabolites can be baseline-separated simultaneously by using 50 mM phosphate buffer (pH 2.5) containing 75 mM methyl-beta-CD, 220 mM urea and 0.05% (w/v) hydroxypropylmethyl cellulose.     

Enantiomeric separation of denopamine by capillary electrophoresis and high-performance liquid chromatography using cyclodextrins

Direct separation of enantiomers of denopamine was investigated by two separation methods. One is capillary zone electrophoresis (CZE) using cyclodextrins (CDs) (CD-CZE) and the other is high-performance liquid chromatography (HPLC) using CD immobilized chiral stationary phases (CD-CSPs). Enantiomers of denopamine were successfully resolved by employing heptakis (2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD) and acetyl-beta-cyclodextrin (AC-beta-CD), and partially resolved with heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TM-beta-CD), hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and beta-CD polymer under acidic conditions. Separation of enantiomers of denopamine by HPLC was also successful with one of the CD-CSPs, where perphenylated beta-CD (Ph beta-CD) was immobilized. In CD-CZE, some polymeric additives, such as hydroxy-propylmethylcellulose (HPMC) and polyvinylalcohol (PVA), and a coated capillary were used to improve the enantioseparation of denopamine. Method validations such as linearity, recovery and repeatability, etc., were investigated for HPLC, and the method was applied to the optical purity testing of the drug substances and in tablet form.     

Vancomycin as a chiral selector in capillary electrophoresis: an appraisal of advantages and limitations

The properties of the macrocyclic antibiotic vancomycin, used as a chiral selector, were studied with aminoquinolycarbamate derivatives of amino acids, containing sulfur and selenium, as well as with other organic ions. Vancomycin combines the ability to resolve fully ionized anionic enantiomers, typical of proteins, with excellent separation efficiency, exceeding that of cyclodextrins. It allows better than baseline chiral separations of several anionic analytes within 3-5 min. The resolving power of vancomycin results from its great skill in discriminating enantiomers rather than from high affinities to the separated enantiomers. The association constants of vancomycin are of the same order of magnitude, 10(2) L/mol, as that found for beta-cyclodextrin (beta-CD). The difference in association constants of separated cystine enantiomers with vancomycin, 2 x 10(2) L/mol, is one order of magnitude higher than that of enantiomers separated with beta-CD. Analytically convenient mobility differences up to 1-2 x 10(9) m2V-1s-1, with only one of the enantiomers appreciably decelerated, are obtained at submillimolar vancomycin concentrations. Typical separation efficiencies are close to 250,000 theoretical plates per meter of capillary. Deceleration of various organic ions by millimolar vancomycin implies that chiral separations with vancomycin need not be restricted to carboxylic acids. The vancomycin-analyte interactions are strongly affected by the chemical composition and concentration of the buffer. An additional experimental variable, highly effective in manipulating the separation selectivity of analytes, is the buffer pH.     

Effects of various anionic chiral selectors on the capillary electrophoresis separation of chiral phenethylamines and achiral neutral impurities present in illicit methamphetamine

In this study, various anionic chiral selectors were investigated for the capillary electrophoresis (CE) separation of six chiral phenethylamines and three achiral neutral impurities which are commonly identified in illicit methamphetamine. Analyses were carried out at pH 8 (high osmotic flow) with untreated capillaries using 25 mM chiral surfactant or 10 mM charged cyclodextrin. The chiral selectors included the micelle (R)-N-dodecoxycarbonylvaline (EnantioSelect (R)-Val-1) (ES) and the cyclodextrins sulfobutyl(IV)-ether-beta-cyclodextrin (SBE(IV)-beta-CD) (BSB4), SBE(VII)-beta-CD (BSB7), SBE(XII)-beta-CD (BSB 12), SBE(IV)-gamma-CD (GSB-4), SBE(VII)-gamma-CD (GSB-7), sulfated(XI)-alpha-cyclodextrin (SU(XI)-alpha-CD (AS11), SU(VII)-beta-CD (BS7), SU(XII)-beta-CD (BS12) and SU(XIII)-beta-CD (GS13). Enantiomeric and achiral selectivity strongly depends on the size of the CD, the average degree of substitution, and the type of substitution. ES exhibits good performance for the neutral solutes, but exhibits enantiomeric selectivity only for the alpha-hydroxyphenethylamines. GS13 provides the best overall enantiomeric selectivity. All fifteen solutes related to methamphetamine are simultaneously separated using BSB7.     

A novel kinase activity associated with Nef derived from neurovirulent simian immunodeficiency virus

A method for resolving the enantiomers of various 2-arylpropionic acids (viz. ketoprofen, ibuprofen and fenoprofen) by capillary zone electrophoresis (CZE) using a background electrolyte (BGE) containing a cyclodextrin as chiral selector is proposed. The effects of the type of cyclodextrin used and its concentration on resolution were studied and heptakis-2,3,6-tri- O-methyl-beta-cyclodextrin was found to be the sole effective choice for the quantitative enantiomeric resolution of all the compounds tested. The influence of pH, BGE concentration, capillary temperature and addition of methanol to the BGE on resolution and other separation-related parameters was also studied. The three compounds studied can be enantiomerically resolved with a high efficiency in a short time (less than 20 min) with no capillary treatment. This makes the proposed method suitable for assessing the enantiomeric purity of commercially available pharmaceuticals.     

Chiral surfactants in micellar electrokinetic capillary chromatography

Most biologically active molecules contain one or more chiral centres, giving rise to stereoisomeric forms which can behave differently in a chiral environment. Thus, only one of the enantiomers may show the desired physiological activity, whereas the other enantiomers may either be considerably less active or even show undesireable side effects. Establishing that a chiral drug consists of one single enantiomer is nowadays essential before it can be given to patients. Analytical tools that can discriminate between enantiomers play a very important role in determining the stereoisomeric composition of chiral molecules. Until recently chromatographic techniques were the most popular for enantiomeric separations. The increased use of capillary electrophoresis (CE) has provided complementary methodology for chiral discrimination. One mode of CE that has been used for this purpose is micellar electrokinetic capillary chromatography (MECC), where natural or synthetic chiral surfactants are added to the separation buffer.     

Polymorphism in the alleles of tetranucleotide repeat SE33 in Udege and in two urban populations of Russia

Capillary electrophoresis was used to separate diastereomers and enantiomers of synthesized thiopyrans and thiinopyrroles. Separation conditions were optimized by varying the buffer parameters, i.e., kind and concentration of chiral selector, pH, main buffering component, micelle forming additives, and content of organic solvents. The interaction of thiopyrans and thiinopyrroles with beta- and gamma-cyclodextrins results in the separation of enantiomers and diastereomers. The magnitude of the resolution depends on the spatial requirements of the different substituents of the analytes and the dimensions of the cyclodextrin cavity. For well-separated enantiomers or diastereomers the identification and characterization of degradation products was possible.     

Enantiomeric Separation of Antidepressant Trimipramine by Capillary Electrophoresis Combined with Electrochemiluminescence Detection in Aqueous-organic Media

The antidepressant trimipramine(Tri) enantiomers were successfully separated by capillary electrophore-sis(CE) coupled with electrochemiluminescence(ECL) detection in aqueous-organic media. A dual cyclodextrin(CD) system combining β-CD and hydroxypropyl-β-cyclodextrin(HP-β-CD) was used as chiral selector. Acetonitrile(ACN) was added to the running buffer to improve the separation efficiency, detection sensitivity and repeatability. The me-thod was also successfully applied to the chiral separation of Tri in spiked human urine sample.     

Capillary electrophoretic separation of tricyclic antidepressants using charged carboxymethyl-beta-cyclodextrin as a buffer additive

Charged carboxymethyl-beta-cyclodextrin was successful in the capillary electrophoretic separation of a series of tricyclic antidepressants. The cyclodextrin alone was successful in the separation of carbamazepine, protriptyline, desipramine, clomipramine, and opipramol using a 3-(trimethoxysilyl)propyl methacrylate capillary coating to reduce the electroosmotic flow. The ideal buffer pH was found to be in the range of 6-7 and the ideal cyclodextrin concentration to be 10 mM. All nine antidepressants were resolved using the charged cyclodextrin in the micellar electrokinetic chromatography (MEKC) mode with sodium dodecyl sulfate as the surfactant. Neither the cyclodextrin nor the surfactant alone were successful in resolving the whole series of compounds under investigation but a combination of both produced the separation. Separations were performed on a linear polyacrylamide coated capillary. The ideal pH of the buffer was in the range of 5-7.