Separation of drug stereoisomers by capillary electrophoresis with cyclodextrins

Using capillary electrophoresis, the enantiomers and isomers of several chiral drug molecules were resolved with cyclodextrins. Parameters affecting the resolution between (+)- and (−)-epinephrine, such as pH, cyclodextrin concentration, buffer concentration, and capillary dimensions were investigated. In addition to this, the effect of cyclodextrin type (β and several derivatized β-cyclodextrins) on resolution between stereoisomers of several chiral drug was also investigated. This study showed that the structural features of the molecule, the derivative groups on the cyclodextrin, the buffer composition and the capillary dimensions influence resolution. The chiral drugs used in this study were propranolol, atenolol, betaxolol, dipivefrin, AL03152 (an aldose reductase inhibitor), AL03363 (an oxidation product of AL03152) and the cis/trans isomers of pilocarpine.     

Separation of aminoalkanephosphonic acid enantiomers by indirect UV detection capillary electrophoresis with application of cyclodextrins

Indirect UV detection capillary electrophoresis (CE) was used for the separation of aminoalkanephosphonic acid (AP) enantiomers by applying commercially available cyclodextrins as chiral discriminators. The results show that the separation of the enantiomers depends on pH of the background electrolyte, the molar ratio of cyclodextrin to aminophosphonic acid, and on the type of the applied chiral selector. Optimization of process conditions allowed enantiomeric baseline separation or partial separation of 12 out of 14 alpha-aminophosphonic acids studied. This type of CE might therefore be successfully used for routine determination of enantiomeric purity of aminophosphonic acids.     

Derivatized cyclodextrins for the separation of enantiomers in capillary electrophoresis

The use of cyclodextrin derivatives for the efficient separation of enantiomeric drugs is described. Hydroxypropylation, methylation or carboxymethylation of the cyclodextrin not only result in a better solubility of the cyclodextrin in aqueous solutions, but also favor, via additional hydrogen bonding, the stabilization of one of the cyclodextrin-analyte complexes. The influence of the background electrolyte on peak shape is also described here. Carboxymethylated cyclodextrin can be used in similar manner to uncharged cyclodextrins at low pH values (below 4). At pH values above 5, however, its charge also allows the separation of uncharged enantiomers as in a micellar-like system.     

Separation of neutral carbohydrates by capillary electrophoresis

The basic strategies for analysis of neutral carbohydrates by capillary electrophoresis are summarized. Neutral carbohydrates are dissociated in strong alkali to give anions, hence they can be separated directly by zone electrophoresis based on the difference between their dissociation constants. However, neutral carbohydrates are not electrically charged under normal conditions. Therefore, they should be converted to ions prior to or during analysis. Precapillary introduction of a basic or an acidic group to a neutral carbohydrate gives the derivative positive (in acidic media) or negative (in alkaline media) charge, respectively. The derivatives thus obtained can be separated by zone electrophoresis. Analysis of carbohydrates in a carrier containing an oxyacid salt (such as sodium borate) or an alkaline metal salt (such as calcium acetate) causes in situ conversion to anionic or cationic complexes, respectively, which are separated by zone electrophoresis. The effective uses of electrokinetic chromatography in sodium dodecyl sulfate micelles for hydrophobic derivatives (such as 1-phenyl-3-methyl-5-pyrazolone derivatives) and size-exclusion electrophoresis in gel-packed capillaris for size different oligosaccharides are also discussed. Each separation mode has its inherent method(s) for detection, which are also described here.     

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.     

Separation of enantiomers of norephedrine by capillary electrophoresis using cyclodextrins as chiral selectors: comparative CE and NMR studies

In this study, the enantiomer migration order (EMO) of norephedrine (NEP) in the presence of various CDs was investigated by CE. NMR and CE techniques were used to analyze the mechanism of the chiral recognition between NEP enantiomers and four CDs, i.e., native α-CD, β-CD, heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD), and heptakis(2,3-di-O-methyl-6-O-sulfo)-β-CD (HDMS-β-CD). EMO was reversed in the presence of α-CD and β-CD, although only minor differences in the structures of the complexes formed between NEP and these CDs could be derived from rotating frame nuclear Overhauser experiments (ROESY). The complexes between the enantiomers of NEP and the sulfated CDs, HDMS-β-CD, and HDAS-β-CD, were substantially different. However, EMO of NEP was identical in the presence of these CDs. HDAS-β-CD proved to be the most suitable chiral selector for the CE enantioseparation of NEP.     

Separation of enantiomers of butorphanol and cycloamine by capillary zone electrophoresis

Butorphanol tartrate is a synthetic opioid agonist-antagonist used as analgesic, possessing three chiral centres in the basic part of the molecule. Its chiral purity is routinely controlled only by optical rotation. A new capillary zone electrophoresis method, capable to separate the enantiomers of butorphanol and intermediate of its synthesis, cycloamine, was developed. Different electrolyte composition (type and concentration of carrier ion, pH, and organic solvent addition), and type and concentration of several chiral selectors (natural and modified cyclodextrins) were tested. Using the optimized conditions (acidic electrolyte with the addition of highly sulphated gamma-cyclodextrin) as low as 0.05% of undesirable enantiomers can be detected. Selected method characteristics, i.e., linearity (0-50 mg/l), precision (2.5% at 20 mg/l), and accuracy (101 +/- 2% at 20 mg/l) were evaluated. The optimized method was applied for the analysis of real batches of butorphanol and cycloamine. It was found that butorphanol tartrate manufactured by IVAX Pharmaceuticals contains less than 0.05% of undesirable enantiomer.     

Separation of binaphthyl enantiomers by capillary zone electrophoresis and electrokinetic chromatography

The capillary electrophoretic (CE) separation of the enantiomers of three binaphthyl compounds is investigated. Several CE modes such as cyclodextrin (CD) modified capillary zone electrophoresis (CZE) (CD-CZE), micellar electrokinetic chromatography (MEKC), cyclodextrin electrokinetic chromatography (CD-EKC), etc. are employed for the simultaneous enantiomer separation of the three solutes. The successful separation was achieved by combining two modes, in other words by using more than two chiral selectors. A development of the CE enantiomer separation is demonstrated for the binaphthyl compounds. The enantioselectivity of binaphthyl compounds is alo briefly discussed.     

Separation of enantiomers in capillary electrophoresis with contactless conductivity detection

Contactless conductivity detection is successfully demonstrated for the enantiomeric separation of basic drugs and amino acids in capillary electrophoresis (CE). Derivatization of the compounds or the addition of a visualization agent as for indirect optical detection schemes were not needed. Non-charged chiral selectors were employed, hydroxypropylated cyclodextrin (CD) for the more lipophilic basic drugs and 18-crown-6-tetracarboxylic acid (18C6H4) for the amino acids. Acidic buffer solutions based on lactic or citric acid were used. The detection limits were determined as 0.3 microM for pseudoephedrine as an example of a basic drug and were in the range from 2.5 to 20 microM for the amino acids.     

Chiral separation of anticholinergic drug enantiomers in nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoretic (NACE) method for the separation of nine structurally similar chiral anticholinergic drugs was developed. The eight drug enantiomers were separated on baseline within 18 min using 20mM phosphoric acid and 10 mM NaOH, containing 10 mM heptakis(2,3-dimethyl-6-sulfato)-4beta-cyclodextrin (HDMS-beta-CD) in methanol. The results were compared with those obtained in the high performance liquid chromatography system.     

Separation of enantiomers by capillary electrochromatography

The current popularity of capillary electrochromatography (CEC) has led to an increasing number of studies on the development and evaluation of enantioselective CEC systems. These studies clearly demonstrate that the most prominent advantage of electrically driven separation methods, the vastly increased column efficiency as compared to pressure-driven chromatography, can also be experimentally achieved for the separations of enantiomers. In analogy to high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), several approaches have been used. The addition of a chiral selector to the mobile phase is the simplest method. Less erroneous and more elegant approaches are those that use open-tubular, conventional packed, and monolithic columns containing chiral stationary phases that stereoselectively interact with enantiomers. This review evaluates the new techniques and compares them to enantioselective HPLC and CE. Further, it describes the various concepts of enantioselective CEC and focuses on the current ‘state-of-the-art' column technology.     

Separation of basic drug enantiomers by capillary zone electrophoresis using glucuronyl glucosyl beta-cyclodextrin as a chiral selector.

Separations of basic drug enantiomers have been investigated using glucuronyl glucosyl beta-cyclodextrin (GUG beta-CD) as a chiral selector in the background electrolyte by capillary zone electrophoresis. The effects of GUG beta-CD concentration and running buffer pH on the migration times and resolution of 16 basic drug enantiomers were precisely examined using a linear polyacrylamide-coated capillary. High resolution of 16 basic drug enantiomers was generally attained with a running buffer pH 2.5 or 3.5 containing 10 mM GUG beta-CD. Next, we compared the chiral resolution abilities of GUG beta-CD with those of beta-CD and maltosyl beta-CD (G2 beta-CD). GUG beta-CD showed higher resolution for basic drug enantiomers tested than beta-CD and G2 beta-CD. This could be due to that hydrogen bonding or ionic interactions of uncharged and charged glucuronyl glucosyl groups of GUG beta-CD with an analyte could stabilize the inclusion complex.     

Enantiomer separation of drugs by capillary electrophoresis using mixtures of beta-cyclodextrin sulfate and neutral cyclodextrins

Direct separation of enantiomers of drugs was investigated by capillary electrophoresis employing mixtures of charged cyclodextrin derivatives (CDs) and electrically neutral CDs (i.e., dual CD system). Among various charged CDs employed, it was found that beta-CD sulfate showed relatively wide enantioselectivity for a wide variety of drugs under acidic conditions. Then separation of enantiomers was performed by employing beta-CD sulfate and the effect of the addition of electrically neutral CDs to the buffers containing beta-CD sulfate was investigated. Through the addition of electrically neutral CDs to the buffers containing the charged CD, resolution of most of the enantiomers was improved, compared with those with the charged CD alone. It was also found that the ring size (alpha, beta, gamma,), the substitution groups and the concentration of the additional electrically neutral CDs affected the enantioselectivity. For example, alpha-CD addition was effective for the separation of enantiomers of chlorpheniramine and hydroxypropyl-beta-CD was effective for the enantiomer separation of trimetoquinol isomer. The application of the method in optical purity testing is also briefly mentioned.     

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.     

Separation of enantiomers of ephedrine by capillary electrophoresis using cyclodextrins as chiral selectors: comparative CE, NMR and high resolution MS studies

The enantiomer migration order (EMO) of ephedrine was investigated in the presence of various CDs in CE. The molecular mechanisms of chiral recognition were followed for the ephedrine complexes with native α- and β-CD and heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD) by CE, NMR spectroscopy and high-resolution MS. Minor structural differences were observed between the complexes of ephedrine with α- and β-CD although the migration order of enantiomers was opposite when these two CDs were applied as chiral selectors in CE. The EMO was also opposite between β-CD and HDAS-β-CD. Significant structural differences were observed between ephedrine complexes with the native CDs and HDAS-β-CD. The latter CD was advantageous as chiral CE selector not only due to its opposite electrophoretic mobility compared with that of the cationic chiral analyte, but also primarily due to its enhanced chiral recognition ability towards the enantiomers of ephedrine.     

Determination of stability constants of complexes of neutral analytes with charged cyclodextrins by affinity capillary electrophoresis

A novel procedure for the determination of stability constants in systems with neutral analytes and charged complexation agents by affinity capillary electrophoresis was established. This procedure involves all necessary corrections to achieve precise and reliable data. Temperature, ionic strength, and viscosity corrections were applied. Based on the conductivity measurements, the average temperature of the background electrolyte in the capillary was kept at the constant value of 25°C by decreasing the temperature of the cooling medium. The viscosity correction was performed using the viscosity ratio determined by an external viscosimeter. The electrophoretical measurements were performed, at first, at constant ionic strength. In this case, the increase of ionic strength caused by increasing complexation agent concentration was compensated by changing of the running buffer concentration. Subsequently the dependence of the analyte effective mobility on the complexation agent concentration was measured without the ionic strength compensation (at variable ionic strength). The new procedure for determination of the stability constants even from such data was established. These stability constants are in a very good agreement with those obtained at the constant ionic strength. The established procedure was applied for determination of the thermodynamic stability constants of (R, R)-(+)- and (S, S)-(-)-hydrobenzoin and R- and S-(3-bromo-2-methylpropan-1-ol) complexing with 6-monodeoxy-6-mono(3-hydroxy)propylamino-β-cyclodextrin hydrochloride.     

Separation of basic drug enantiomers by capillary electrophoresis using chicken alpha1-acid glycoprotein: insight into chiral recognition mechanism

Recombinant chicken alpha(1)-acid glycoprotein (alpha(1)-AGP) was prepared by the Escherichia coli expression system and completely deglycosylated alpha(1)-AGP (cd-alpha(1)-AGP) was obtained by treatments of native alpha(1)-AGP with a mixture of endoglycosidase and N-glycosidase. The average molecular masses of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP were estimated to be about 29 200, 21 700 and 20 700, respectively, by matrix-assisted laser desorption-time of flight-mass spectrometry. We compared the chiral recognition ability of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP using them as chiral selectors in capillary electrophoresis. The chicken alpha(1)-AGP showed higher resolution for eperisone, pindolol and tolperisone than cd-alpha(1)-AGP or recombinant alpha(1)-AGP. Recombinant alpha(1)-AGP still showed chiral recognition for three basic drugs tested. By addition of propranolol as a competitor in the separation solution in CE, no enantioseparations of three basic drugs were observed with chicken alpha(1)-AGP, cd-alpha(1)-AGP or recombinant alpha(1)-AGP. These results reveal that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition ability, and that the chiral recognition site(s) for basic drugs exists on the protein domain.     

Chiral capillary electrophoresis as predictor for separation of drug enantiomers in continuous flow zone electrophoresis

Separation of the enantiomers of chlorpheniramine and methadone in acidic buffers containing carboxymethyl-betacyclodextrin (CMCD) as chiral selector was investigated by capillary zone electrophoresis. For a range of pH and CMCD concentrations, the mobility difference and resolution of the enantiomers were determined. Then, conditions known to provide well resolved enantiomers and optimized chiral separation were applied to chiral continuous flow electrophoresis. In that approach, a thin film of fluid flowing between two parallel plates is employed as carrier for electrophoresis. The electrolytes and the sample are continuously admitted at one end of the electrophoresis chamber and are fractionated by an array of outlet tubes at the other. The number of pure enantiomeric fractions obtained by chiral continuous flow electrophoresis was found to be directly dependent on the enantiomeric mobility difference. For racemic chlorpheniramine separated in a betaine-acetic acid buffer at a total throughput of 5 mg/h, complete enantiomeric separation is shown to require a mobility difference of about 3 x 10(-9) m2/V s. Furthermore, compared to the previous investigations with hydroxypropyl-beta-cyclodextrin, CMCD was found to permit improved fractionation of methadone enantiomers. With a total racemic drug throughput of about 15 mg/h, continuous flow zone electrophoresis processing with CMCD as chiral selector is shown to have the potential of providing pure enantiomers on a mg/h scale. The results indicate that chiral capillary zone electrophoresis data can be employed as predictor for preparative scale chiral separations based upon continuous flow zone electrophoresis.