Enantiomeric separation of alanyl and leucyl dipeptides by capillary electrophoresis with cyclodextrins as chiral selectors

Eight neutral cyclodextrins were tested for the enantiomeric separation of alanyl and leucyl dipeptides by capillary electrophoresis at pH 3, and seven out of the eight cyclodextrins proved suitable for the separation of one or more of the dipeptide enantiomer pairs. The best results were obtained with heptakis(2,6-di-O-methyl)-beta-cyclodextrin. The dipeptides that were separated were mainly the aromatic and the more lipophilic aliphatic dipeptides. Mobility difference plots at pH 3.0 with malonic acid-triethanolamine as background electrolyte showed that the aromatic dipeptides had higher affinities for the cyclodextrin than the nonpolar, aliphatic dipeptides. The results suggested that, under the conditions applied, the C-terminal amino acid rather than the N-terminal one is involved in the chiral discrimination.     

Enantioselective determination by capillary electrophoresis with cyclodextrins as chiral selectors

This review surveys the separation of enantiomers by capillary electrophoresis using cyclodextrins as chiral selector. Cyclodextrins or their derivatives have been widely employed for the direct chiral resolution of a wide number of enantiomers, mainly of pharmaceutical interest, selected examples are reported in the tables. For method optimisation, several parameters influencing the enantioresolution, e.g., cyclodextrin type and concentration, buffer pH and composition, presence of organic solvents or complexing additives in the buffer were considered and discussed. Finally, selected applications to real samples such as pharmaceutical formulations, biological and medical samples are also discussed.     

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.     

Chiral separation of hydroxyflavanones in cyclodextrin-modified capillary zone electrophoresis using sulfated cyclodextrins as chiral selectors

Chiral separations of three hydroxyflavanone aglycones, including 2'-, 3'-, and 4'-hydroxyflavanone, in capillary zone electrophoresis (CZE) using randomly sulfate-substituted beta-cyclodextrin (S-beta-CD) or dual cyclodextrin (CD) systems consisting of S-beta-CD and a neutral CD at low pH were investigated. The results indicate that S-beta-CD is an excellent chiral selector for enantioseparation of 2'-hydroxyflavanone and is a good chiral selector for 3'-hydroxyflavanone. Depending on the concentration of S-beta-CD ranging from 2.0 to 0.75% (w/v), the enantioresolution values were 10.5-19.5 and 1.8-3.4 for 2'- and 3'-hydroxyflavanone, respectively. The enantiomers of 4'-hydroxyflavanone could be effectively separated with S-beta-CD at a concentration of 2.0% (w/v) within 20 min. The enantioselectivity and enantioresolution follow the order 2'-hydroxyflavanone>3'-hydroxyflavanone>4'-hydroxyflavanone. Alternatively, with the addition of sodium dodecyl sulfate (SDS) monomers at low concentrations in the electrophoretic system, enantioselectivity of these hydroxyflavanone aglycones could be enhanced with dual CD systems. In this case, SDS monomer acted as a complexing agent probably first with S-beta-CD and then subsequently with the analytes for increasing the effective electrophoretic mobility of the analytes towards the anode and as a selectivity controller for affecting the selectivity of hydroxyflavanones. Better enantioseparation between 2'-hydroxyflavanone and 3'-hydroxyflavanone could be achieved with a dual CD system consisting of S-beta-CD and gamma-CD than that with S-beta-CD and beta-CD. In addition, possible chiral recognition mechanisms of hydroxyflavanones are discussed.     

The separation of the enantiomers of diquats by capillary electrophoresis using randomly sulfated cyclodextrins as chiral selectors

Diquats, derivatives of the widely used herbicide diquat, represent a new class of functional organic molecules. A combination of their special electrochemical properties and axial chirality could potentially result in their important applications in supramolecular chemistry, chiral catalysis, and chiral analysis. However, prior to their practical applications, the diquats have to be prepared in enantiomerically pure forms and the enantiomeric purity of their P- and M-isomers has to be checked. Hence, a chiral capillary electrophoresis (CE) method has been developed and applied for separation of P- and M-enantiomers of 11 new diquats. Fast and better than baseline CE separations of enantiomers of all 11 diquats within a short time 5–7 min were achieved using acidic buffer, 22 mM NaOH, 35 mM H₃PO₄, pH 2.5, as a background electrolyte, and 6 mM randomly sulfated α-, β-, and γ-cyclodextrins as chiral selectors. The most successful selector was sulfated γ-cyclodextrin, which baseline separated the enantiomers of all 11 diquats, followed by sulfated β-cyclodextrin and sulfated α-cyclodextrin, which baseline separated enantiomers of 10 and nine diquats, respectively. Using this method, a high enantiopurity degree of the isolated P- and M-enantiomers of three diquats with a defined absolute configuration was confirmed and their migration order was identified.     

Chiral separation and determination of the optical purity of thiamphenicol-related chiral compound by capillary zone electrophoresis with cyclodextrins as chiral selectors

Summary One classical method for quantitation of amino acids in proteins is hydrolysis of the proteins and determination of the free amino acids. Although the drastic experimental conditions necessary for complete hydrolysis always cause degradation of some of the amino acids, if mild hydrolysis conditions are used, a mixture of amino acids and oligopeptides is obtained. If these conditions are adequately tuned, the oligopeptides are almost exclusively dipeptides. For this reason we have initiated a study to find a derivatizing agent suitable for the analysis of amino acids and dipeptides by an absolute method of quantitation already tested for amino acids. FMOC-Cl was found to be a suitable derivatizing agent for this purpose.     

Enantiomer separation of drugs by capillary electrophoresis using proteins as chiral selectors

The separation of drug enantiomers using proteins as the chiral selectors in capillary electrophoresis (CE) is considered in this review. The proteins used include albumins such as bovine serum albumin, human serum albumin and serum albumins from other species, glycoproteins such as alpha1-acid glycoprotein, crude ovomucoid, ovoglycoprotein, avidin and riboflavin binding protein, enzymes such as fungal cellulase, cellobiohydrolase I, pepsin and lysozyme and other proteins such as casein, human serum transferrin and ovotransferrin. Protein-based CE is carried out in two modes: in one proteins are immobilized or adsorbed within the capillary, or protein-immobilized silica gels are packed into the capillary (affinity capillary electrochromatography mode), and in the other proteins are dissolved in the running buffer (affinity CE mode). Furthermore, the advantages and limitations of the two modes and the factors affecting the chiral separations of various drugs by protein-based CE are discussed.     

Chiral separation of newly synthesized arylpropionic acids by capillary electrophoresis using cyclodextrins or a glycopeptide antibiotic as chiral selectors

Summary The chiral separation of two newly synthesized arylpropionic acids of pharmaceutical interest, namely 2-[(5′-benzoil-2′-hydroxy)phenyl]-propionic acid (DF-1738y) and 2-[(4′-benzoiloxy-2′-hydroxy)phenyl]-propionic acid (DF-1770y), was performed by Capillary Zone Electrophoresis (CZE) using either cyclodextrins or antibiotics as chiral selectors in coated capillary. In order to optimize the separation, the effect on the migration time and resolution of type and concentration of the chiral selector, the buffer pH and the capillary temperature were studied. Several cyclodextrins, namely the charged 6^A-monomethylamino-β-cyclodextrin (MeNH-β-CD) and the neutral methyl-β-cyclodextrins (M-β-CD) and heptakis-2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD), were tested for the enantiomeric separation of aryl propionic acids (APAs) compounds. Of these TM-β-CD provided the highest enantiomeric resolution at pH 5, however only DF-1738y optical isomers were baseline resolved. Using background electrolytes (BGEs) at higher pHs (pH=6–7) supported with the above listed CDs, an enantioresolution increase was recognized only for compound DF-1738y. In contrast DF-1770y exhibited the highest resolution at the lowest pH value studied (pH 4). The macrocyclic antibiotic vancomycin was therefore added to the BGE and tested as chiral selector using the partial filling counter current mode in order to obtain a sensitive analysis, high resolution and reduced antibiotic adsorption on the capillary wall. 5 mM vancomycin dissolved in the BGE at pH 5 and 25°C provided relatively high enantiomeric resolution (R _DF-1738y=3.4,R _DF-1770y=2.22) of both compounds.     

Enantioseparation of novel psychoactive chiral amines and their mixture by capillary electrophoresis using cyclodextrins as chiral selectors

The prevalence of new psychoactive substances (NPS) has been increasing during the last decade as well as their constant growth of availability across the whole world. Regardless of the potential health hazard, NPS (often racemic compounds) are frequently sought after and abused for their psychoactive effects that may differ for individual enantiomers. In this work, capillary electrophoresis was used for the chiral separation of a mixture of eleven psychoactive chiral amines using β-cyclodextrin and carboxymethyl-β-cyclodextrin as chiral selectors at various concentrations. Chiral separation was successful for all the analytes studied. A mixture of these analytes was subsequently analyzed under optimal conditions, i.e., when using 20 mmol/L carboxymethyl-β-cyclodextrin in 50 mmol/L sodium phosphate buffer, pH 2.5. In this case, chiral separation occurred in nine out of eleven analytes. To our best knowledge, we achieved enantioseparations of seven analyzed compounds by CE for the first time.     

Rapid screening for chiral separations by short-end injection capillary electrophoresis using highly sulfated cyclodextrins as chiral selectors

Two series of amino acid derivatives and phenylamines were used to evaluate the potential of highly sulfated cyclodextrins (HS-CDs) for the screening for chiral separations by capillary electrophoresis (CE). HS-CDs showed to be very versatile and to exhibit very high enantioselectivity. The use of short-end injection allowed to reduce dramatically the analysis time. From the results obtained, a scheme for the rapid screening of enantiomeric molecules was developed and applied to various chiral drugs. Results are very satisfying as almost all compounds (62 out of 67) could be baseline-resolved. Usually, less than three experiments were necessary to obtain very good separation.     

Enantiopurity analysis of new types of acyclic nucleoside phosphonates by capillary electrophoresis with cyclodextrins as chiral selectors

CE methods have been developed for the chiral analysis of new types of six acyclic nucleoside phosphonates, nucleotide analogs bearing [(3‐hydroxypropan‐2‐yl)‐1H‐1,2,3‐triazol‐4‐yl]phosphonic acid, 2‐[(diisopropoxyphosphonyl)methoxy]propanoic acid, or 2?(phosphonomethoxy)propanoic acid moieties attached to adenine, guanine, 2,6‐diaminopurine, uracil, and 5‐bromouracil nucleobases, using neutral and cationic cyclodextrins as chiral selectors. With the exception of the 5‐bromouracil‐derived acyclic nucleoside phosphonate with a 2‐(phosphonomethoxy)propanoic acid side chain, the R and S enantiomers of the other five acyclic nucleoside phosphonates were successfully separated with sufficient resolutions, 1.51–2.94, within a reasonable time, 13–28 min, by CE in alkaline BGEs (50 mM sodium tetraborate adjusted with NaOH to pH 9.60, 9.85, and 10.30, respectively) containing 20 mg/mL β‐cyclodextrin as the chiral selector. A baseline separation of the R and S enantiomers of the 5‐bromouracil‐derived acyclic nucleoside phosphonate with 2‐(phosphonomethoxy)propanoic acid side chain was achieved within a short time of 7 min by CE in an acidic BGE (20:40 mM Tris/phosphate, pH 2.20) using 60 mg/mL quaternary ammonium β‐cyclodextrin chiral selector. The developed methods were applied for the assessment of the enantiomeric purity of the above acyclic nucleoside phosphonates. The preparations of all these compounds were found to be synthesized in pure enantiomeric forms. Using UV absorption detection at 206 nm, their concentration detection limits were in the low micromolar range.     

Enantioseparation of phenothiazines in cyclodextrin-modified capillary zone electrophoresis using sulfated cyclodextrins as chiral selectors

In this study, enantioseparations of five phenothiazines, including promethazine, ethopropazine, trimeprazine, methotrimeprazine, and thioridazine, in CD-modified CZE using dual CD systems consisting of randomly sulfate-substituted CD (MI-S-beta-CD) and a neutral CD as chiral selectors in a citrate buffer (100 mM) at pH 3.0 were investigated. The results indicate that MI-S-beta-CD is an excellent chiral selector for enantioseparation of ethopropazine. The enantiomers of promethazine can also be baseline-resolved with MI-S-beta-CD at concentrations in the range of 0.5-1.0% w/v. On the other hand, thioridazine and trimeprazine interact strongly with neutral CDs. As a result, the enantioselectivity of these two phenothiazines is remarkably and synergistically enhanced with increasing the concentration of neutral CDs in the presence of MI-S-beta-CD and simultaneous enantioseparations of these phenothiazines, except for methotrimeprazine, could favorably be achieved with the use of dual CD systems. Moreover, by varying the concentration of beta-CD or gamma-CD at a fixed concentration of MI-S-beta-CD (0.75% w/v) reversal of the enantiomer migration order of promethazine occurred. This may be attributable to the opposite effects of charged and neutral CDs on the mobility of the enantiomers of promethazine.     

Enantiomeric separation of N-protected amino acids by non-aqueous capillary electrophoresis with dimeric forms of quinine and quinidine derivatives serving as chiral selectors

A simple method for analysis of anatoxin-a in aqueous samples was developed using solid-phase microextraction (SPME) and high-performance liquid chromatography (HPLC) with fluorescence detection. Anatoxin-a was derivatized to a fluorogenic agent on the surface of the SPME fiber. In the method an SPME fiber was immersed for 30 min in the aqueous sample. The fluorogenic derivatizing reagent (4-fluoro-7-nitro-2,1,3-benzoxadiazole, 1.0 mg/ml in methanol) was dropped or sprayed onto the fiber containing extracted analytes. The fiber was then heated for 10 min in an empty vial at 70 degrees C in a waterbath to promote derivatization. The derivatives formed on the fiber were desorbed in a SPME-HPLC interface. The interface was filled with methanol-1 mM hydrochloric acid (7:3, v/v) before inserting of the fiber into the interface. For desorption, the fiber was inserted in the interface for 5 min. For anatoxin-a in an aqueous sample, the calibration curve showed linearity in the range of 50-1500 ng/ml and the limit of detection of anatoxin-a was 20 ng/ml. No interferences were found, and the time for analysis was 55 min for one sample.     

Enantiomeric separation by capillary electrophoresis using a crown ether as chiral selector.

This paper reviews chiral separations of primary amines by capillary electrophoresis and crown ether as chiral selector. Two possible mechanisms of chiral recognition by host-guest complexation are discussed: (i) The substituents of the crown ether act as barriers for the guest compounds, and (ii) lateral electrostatic interactions between host and guest occur. Experimental conditions affecting the separation are discussed in detail. A literature overview of practical applications is presented as well. More than 80 different primary amines were analyzed, whereupon the majority could be resolved using a screening method. It is shown that a synergistic effect on the resolution of chiral amines is observed when the chiral crown ether and cyclodextrins are simultaneously used in the same buffer system. This approach opens interesting perspectives for further method optimization.     

Comparing cyclodextrin derivatives as chiral selectors for enantiomeric separation in capillary electrophoresis

A total of 26 different cyclodextrin (CD) derivatives with different functional groups and degrees of substitution were tested against 35 basic pharmaceutical compounds in an effort to investigate their effectiveness as chiral selectors for enantiomeric separation in capillary electrophoresis (CE). Testing was performed under the same conditions using a low pH buffer (25 mM phosphate buffer at pH approximately 2.5). Five CD derivatives, namely, highly sulfated-beta-CD, highly sulfated-beta-CD, hydroxypropyl-beta-CD (degree of substitution approximately 1), heptakis-(2,6-O-dimethyl)-beta-CD, and heptakis(2,3,6-O-trimethyl)-beta-CD were identified to be most effective for enantiomeric separations and have a wide range of enantiomeric selectivity towards the model compounds. Over 90% of the model compounds were enantiomerically resolved with the five identified CD derivatives, at a minimum resolution of 0.5. An additional 20 compounds were also tested to demonstrate the validity of the identified CD derivatives. The five CD derivatives were recommended as the starting chiral selectors in developing enantiomeric separation methods by CE.     

Enantiomeric separation of dansylamino acids by capillary zone electrophoresis based on complexation with cyclodextrins

Summary The enantiomeric separation ability of unmodified and methylated cyclodextrins (CDs) during capillary zone electrophoresis (CZE) was investigated using twelve dansylamino acids. Unmodified - and -CDs exhibited high enantioselectivities. -CD could scarcely separate the enantiomers before and after dimethylation, but obtained enantioselectivity after trimethylation. On the other hand, dimethylation of -CD removed much of its high enantioselectivity. Moreover, the chemical modifications produced a reverse in the migration order of the enantiomers. The inclusion of dansyl-DL-phenylalanine with CDs was evaluated using 600 MHz ¹H NMR spectroscopy.     

New derivatives of cyclodextrins as chiral selectors for the capillary electrophoretic separation of dichlorprop enantiomers

-, β- and γ-cyclodextrins (CDs), as well as some of their chemical derivatives, have been tested as chiral resolving agents for the capillary zone electrophoretic resolution of the racemic herbicide dichlorprop, (±)-2-(2,4-dichlorophenoxy)propionic acid, of which only the (+)-isomer is herbicidally active. The complexation constants of the herbicide enantiomers with the cyclodextrin host molecules have been calculated from the electrophoretic migration time data at variable cyclodextrin concentration. The experimental results showed that several of the investigated CDs allowed dichlorprop enantiomer resolution. In particular, a newly synthesised ethylcarbonate derivative of β-CD showed the best enantiomer resolution properties among the tested compounds, while the remaining ones showed inferior or no performances at all. The calculated inclusion constants allowed identification of the best conditions for enantioresolution, and an explanation of the different complexation properties of the investigated compounds has been proposed on the basis of molecular modeling.     

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.