Infinite enantiomeric resolution of basic compounds using highly sulfated cyclodextrin as chiral selector in capillary electrophoresis

Four chiral basic analytes, namely methadone, fluoxetine, venlafaxine, and tramadol, were selected as model compounds for investigating their stereoselective separation with highly sulfated gamma-cyclodextrin (HS gamma-CD) by capillary electrophoresis (CE)-UV and CE-mass spectrometry (MS). At high concentration of chiral selector, the preferentially bonded enantiomer migrated faster in the anodic mode to the detector and high resolutions were obtained for all analytes. In the cathodic mode, at lower highly sulphated cyclodextrin (HS-CD) concentration, basic compounds could be detected, with the weakly bonded enantiomer migrating first (enantiomeric migration order inversion). It was also then possible, at intermediate HS-CD concentration, that only one enantiomer migrated to the detector as cation while the other enantiomer complexed with the CD was negatively charged and presented an opposite mobility. The latter never reached the detector achieving a perfect enantiomeric selectivity. Infinite chiral resolutions were thus achieved by CE-UV as well as by CE-electrospray ionisation (ESI)-MS where concentrations of HS-CD were adapted according to the negative contribution of the nebulization gas pressure of the interface.     

High-performance chiral separation of fourteen triazole fungicides by sulfated beta-cyclodextrin-mediated capillary electrophoresis

In this paper, sulfated beta-cyclodextrin-mediated capillary electrophoresis (CE) is evaluated as a new approach for the chiral separation of triazole-type fungicides. The 14 fungicides investigated were bitertanol, cyproconazole, difenoconazole, diniconazole, flutriafol, hexaconazole, myclobutanil, paclobutrazol, penconazole, propiconazole, tebuconazole, tetraconazole, triadimefon and triadimenol. Under the optimal conditions, excellent enantioseparation was achieved for all the 14 fungicides, including those fungicides containing two chiral centers. To our knowledge, this is the only system to date that offers outstanding enantiodiscrimination towards all triazole-type fungicides. The impact of the molecular structures of the triazole compounds on their migration behavior was studied. Similar to other chemical systems involving host-guest complexation, the interaction between sulfated beta-cyclodextrin and the triazole compounds was found to be affected by a variety of factors, including electrostatic force, hydrogen bonding, steric effect and hydrophobicity. These factors, coupled with the countercurrent electroosmotic flow (EOF), were believed to be the major forces behind the exceptional chiral selectivity.     

Simultaneous chiral separation of triadimefon and triadimenol by sulfated beta-cyclodextrin-mediated capillary electrophoresis

Enantiomeric separation of two triazole fungicides, triadimefon and triadimenol, was investigated in sulfated beta-cyclodextrin (sulfated beta-CD)-mediated capillary electrophoresis (CE) systems. It was found that, at pH 2-4, sulfated beta-CD exhibited strong chiral recognition towards both triadimefon and triadimenol. The enantiorecognition was believed to result from the multiple interactions between sulfated beta-CD and the analytes, which included inclusion effect, electrostatic interaction, and hydrogen bonding. Under optimal conditions (phosphate buffer with 2% sulfated beta-CD, pH 2.5), simultaneous resolution of all chiral isomers of triadimefon and triadimenol was achieved in less than half an hour. In conjunction with solvent extraction and subsequent enrichment by solid-phase extraction (SPE), this new enantioseparation method was applied successfully in the study of stereoselectivity associated with the biotransformation of triadimefon to triadimenol by soil microorganisms. The present methodology was superior to the commonly adopted chiral gas chromatography (GC) approach in that a very mild procedure was involved from sample extraction to the ultimate chiral separation. Thus, the disturbance of the enantiomeric distribution patterns of the original soil samples by heat stress was an unlikely scenario. Furthermore, it was discovered that, owing to the unique selectivity of the present separation strategy, there was virtually no interference from the soil matrix, which led to improvements in both sensitivity and selectivity in real sample determination.     

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.     

Enantioselective separation of epoxides by capillary electrophoresis employing sulfated beta-cyclodextrin as chiral selector

A capillary electrophoresis method was developed for the enantioseparation of epoxide compounds. Sulfated beta-cyclodextrin was employed as a chiral selector. Phosphate-triethanolamine buffer showed a chiral separation effect when employing charged sulfated beta-cyclodextrin. The effect of pH, triethanolamine concentration and sulfated beta-cyclodextrin concentration on the resolution was studied. Methanol was tested as an organic modifier. Several other epoxides were successfully separated by the proposed method.     

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.     

Comparison of charged cyclodextrin derivatives for the chiral separation of atropisomeric polychlorinated biphenyls by capillary electrophoresis

Charged cyclodextrin (CD) derivatives were used as chiral selectors in electrokinetic chromatography (EKC) for the chiral separation of highly hydrophobic neutral racemates such as atropisomeric polychlorinated biphenyls (PCBs). beta-CD-phosphated, beta-CD sulfated, succinylated-gamma-CD (Succ-gamma-CD) and succinylated-beta-CD (Succ-beta-CD) were used as anionic CDs. As cationic CD, 6-monodeoxy-6-monoamino-beta-CD (beta-CD-NH(2)) was tested for the first time in order to separate PCBs. From the different CD derivatives employed, the best separations were obtained with the cationic CD derivative. Thus, the use of beta-CD-NH(2 )in phosphate buffer at pH 2.0 containing urea allowed the chiral recognition of eleven PCBs (45, 84, 88, 91, 95, 131, 136, 144, 149, 176, and 197). In this case, the addition of 2 M urea to the buffer solution was crucial to achieve the chiral separation of PCBs. The addition of acetonitrile to 10 mM phosphate buffer (pH 2.0) with 30 mM beta-CD-NH(2) and 2 M urea improved considerably the chiral resolution obtained for PCBs 91, 95, 136, 144, 149, and 197 although an increase in the analysis time was also observed. All the results obtained were compared with those previously obtained with the dual CD system carboxymethyl-gamma-CD/beta-CD.     

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.     

Hydroxyethylammonium monosubstituted cyclodextrin as chiral selector for capillary electrophoresis

A novel cationic cyclodextrin, mono-6^A-(2-hydroxyethyl-1-ammonium)-6^A-β-cyclodextrin chloride (HEtAMCD) has been successfully synthesized and applied as chiral selector in capillary electrophoresis. The NMR study revealed this chiral selector has three recognition sites: β-CD, ammonium cation and hydroxy group in the sidearm to contribute three corresponding driving forces including inclusion complexation, electrostatic interaction and hydrogen bonding. The effect of buffer pH and HEtAMCD concentration (2.5–10 mM) on enantioselectivity, chiral resolution as well as effective mobility of analytes was investigated. This elegantly designed CD exhibits outstanding enantioselectivities toward the studied hydroxyl acids and ampholytic racemates in CE with the aid of extra hydrogen bonding. Under optimum pH 6.0, chiral resolutions over 5 can be readily obtained for hydroxy acids with CD concentration below 5 mM. The comparison study between HEtAMCD and our earlier reported ammonium CDs indicates the hydroxyethylammonium group of HEtAMCD significantly increased the enantioselective capability.     

Ligand exchange chiral separations by cyclodextrin derivatives in capillary electrophoresis

A beta-cyclodextrin substituted by an imidazole-bound histamine (CDmh) was successfully used to separate underivatised tryptophan racemate in capillary electrophoresis in the presence of copper(II) ion by the ligand exchange mechanism. To the best of our knowledge, this is the first example of a cyclodextrin derivative behaving as the first coordination sphere ligand in the complex added to the background electrolyte (BGE).     

毛细管电泳在手性化合物分离中的应用进展

由于手性化合物尤其是手性药物的两个对映体具有不同的化学性质和生理活性,对手性化合物进行分离在医药、生物、食品和环境等领域都具有十分重要的意义。毛细管电泳由于其独特的优势已广泛应用于手性物质的分离。本文对2013~2015年毛细管电泳用于手性分离的最新进展进行了综述,并对其发展前景进行了展望。     

Phospholipid-lysozyme coating for chiral separation in capillary electrophoresis

A phospholipid coating with lysozyme as chiral recognition reagent permeated into the phospholipid membrane was developed for the chiral capillary electrophoretic (CE) separation of D- and L-tryptophan. As a kind of carriers, coated as phospholipid membranes onto the inner wall of a fused-silica capillary, liposomes are able to interact with basic proteins such as lysozyme, which may reside on the surface of the phospholipid membrane or permeate into the middle of the membrane. The interaction results in strong immobilization of lysozyme in the capillary. Coatings prepared with liposomes alone did not allow stable immobilization of lysozyme into the phospholipid membranes, as seen from the poor repeatability of the chiral separation. When 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (M1C4) was applied as a first coating layer in the capillary, the electroosmotic flow (EOF) was effectively suppressed, the phospholipid coating was stabilized, and the lysozyme immobilization was much improved. The liposome composition, the running buffer, and the capillary inner diameter all affected the chiral separation of D- and L-tryptophan. Coating with 4 mM M1C4 and then 1 mM phosphatidylcholine (PC)/phosphatidylserine (PS) (80:20 mol%), with 20 mM (ionic strength) Tris at pH 7.4 as the running buffer, resulted in optimal chiral separation with good separation efficiency and resolution. Since lysozyme was strongly permeated into the membrane of the phospholipids on the capillary surface, the chiral separation of D- and L-tryptophan was achieved without lysozyme in the running buffer. The effects of different coating procedures and separation conditions on separation were evaluated, and the M1C4-liposome and liposome-lysozyme interactions were elucidated. The usefulness of protein immobilized into phospholipid membranes as a chiral selector in CE is demonstrated for the first time.     

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.     

Experimental design for enantioselective separation of celiprolol by capillary electrophoresis using sulfated beta-cyclodextrin

A capillary zone electrophoresis method was developed for the enantioseparation of celiprolol enantiomers, using a sulfated beta-cyclodextrin (beta-CD) as a chiral selector. The use of a coated capillary was necessary to achieve stable and reproducible enantioseparations. A central composite design was applied to optimize the method and four parameters were selected for this study: the buffer pH, the buffer concentration, the sulfated beta-CD concentration and the temperature. Resolution between celiprolol enantiomers as well as analysis time and generated current were established as responses. For each response, a model was obtained by a second-degree mathematical expression. From the models, the most favorable conditions were determined by optimizing the resolution between celiprolol enantiomers and by setting the two other responses at threshold values. Response surfaces were also used to assess the robustness of the analytical method around the optimal region. Successful results were obtained with a 52 mM acetate buffer at pH 4.0 in the presence of 3.0 mM sulfated beta-CD at a temperature of 19.5 degrees C. Under these optimized conditions, baseline separation of the celiprolol enantiomers was achieved in less than 10 min. The method showed good validation data in terms of precision, accuracy and linearity, and was found to be suitable in determining celiprolol enantiomers in pharmaceutical preparations and in biological fluids.     

Evaluation of sulfated maltodextrin as a novel anionic chiral selector for the enantioseparation of basic chiral drugs by capillary electrophoresis

Introducing a new class of chiral selectors is an interesting work and this issue is still one of the hot topics in separation science and chirality. In this study, for the first time, sulfated maltodextrin (MD) was synthesized as a new anionic chiral selector and then it was successfully applied for the enantioseparation of five basic drugs (amlodipine, hydroxyzine, fluoxetine, tolterodine, and tramadol) as model chiral compounds using CE. This chiral selector has two recognition sites: a helical structure and a sulfated group which contribute to three corresponding driving forces; inclusion complexation, electrostatic interaction, and hydrogen binding. Under the optimized condition (buffer solution: 50 mM phosphate (pH 3.0) and 2% w/v sulfated MD; applied voltage: 18 kV; temperature: 20°C), baseline enantioseparation was observed for all mentioned chiral drugs. When instead of sulfated MD neutral MD was used under the same condition, no enantioseparation was observed which means the resolution power of sulfated MD is higher than neutral MD due to the electrostatic interaction between sulfated groups and protonated chiral drugs. Also, the countercurrent mobility of negatively charged MD (sulfated MD) allows more interactions between the chiral selector and chiral drugs and this in turn results in a successful resolution for the enantiomers. Furthermore, a higher concentration of neutral MD (approximately five times) is necessary to achieve the equivalent resolution compared with the negatively charged MD.     

Enantioseparation using sulfated cyclosophoraoses as a novel chiral additive in capillary electrophoresis

Highly sulfated cyclosophoraoses (HS-Cys) were synthesized by the chemical modification of a family of neutral cyclosophoraoses isolated from Rhizobium leguminosarum bv. trifolii. The HS-Cys were then analytically characterized using Fourier transform-infrared spectroscopy and elemental analysis. These HS-Cys were successfully used as a novel chiral additive, in low-pH aqueous background electrolytes, for capillary electrophoretic separation of five basic chiral drugs such as arterenol, atenolol, isoproterenol, propranolol, and metoprolol.     

Chiral separation of deprenyl-N-oxide isomers by capillary electrophoresis using various cyclodextrin derivatives

Chiral separation of deprenyl-N-oxide isomers is presented using capillary electrophoresis in the presence of various cyclodextrin (CD) derivatives. This recently identified metabolite of R-(-)-deprenyl may possess desirable pharmacological activities. The effect of the cavity size and the substituents of the CD are examined on the enantiomer resolution of the compound having an asymmetric center on a heteroatom. The importance of hydrophilic or hydrogen bonding interaction, as well as the position of the interacting groups is demonstrated. Outstanding selectivity and resolution values are achieved using the chargeable carboxymethyl-beta-CD. 2-Hydroxypropyl-beta-CD is also suitable for the enantiomer separation of the analyte. Native beta-CD and carboxyethyl-beta-CD provide only poor enantioselectivity, whereas heptakis-(2,6-di-O-methyl)-beta-CD is capable of separating only the diastereomers. No chiral resolution can be observed in the presence of gamma-CD.     

Enantiomer separation of miconazole by capillary electrophoresis with dual cyclodextrin systems.

A cyclodextrin-modified micellar capillary electrophoretic method (MECC) was developed using mixtures of beta, cyclodextrins (beta-CD) and mono-3-O-phenylcarbamoyl-beta-CD as chiral additives for the chiral separation of miconazole with the dual CDs systems. The enantiomers were resolved using a running buffer of 50 mmol/L borate pH 9.5 containing 15 mmol/L beta-CD and 15 mmol/L mono-3-O-phenylcarbamoyl-beta-CD containing 50 mmol/L sodium dodecyl sulfate and 1 mol/L urea. A study of the respective influence of the beta-CD and the mono-3-O-phenylcarbamoyl-beta-CD concentration was performed to determine the optimal conditions with respect to the resolution. Good repeatability of the method was obtained.     

Enantioseparation of solriamfetol and its major impurity phenylalaninol by capillary electrophoresis using sulfated gamma cyclodextrin

R-solriamfetol is a recently approved drug used for the treatment of excessive sleepiness associated with narcolepsy and sleep apnea. Herein, a capillary electrophoretic method was developed, enabling the simultaneous analysis of the API and its S-enantiomer in addition to the enantiomers of its major impurity phenylalaninol. Twenty-nine different cyclodextrins (CDs), including native, neutral, and charged ones were screened as potential chiral selectors, and the best results were obtained with sulfated CDs. Randomly sulfated-β-CD exhibited outstanding enantioresolution, the peaks of phenylalaninol enantiomers inserted between the two peaks of solriamfetol enantiomers, while sulfated-γ-CD (S-γ-CD) showed remarkable resolution values in a much shorter analysis time with the optimal enantiomer migration order. Among the single isomer sulfated CD derivatives, substituent dependent enantiomer migration order reversal could also be observed in the case of heptakis(6-O-sulfo)-β-CD (HS-β-CD) or heptakis(2,3-O-dimethyl-6-O-sulfo)-β-CD (HDMS-β-CD) with R-,S-solriamfetol, and heptakis(2,3-O-diacetyl-6-O-sulfo)-β-CD (HDAS-β-CD) resulting S-,R-solriamfetol migration order. The sulfated-γ-CD system was chosen for method optimization applying orthogonal experimental design. The optimized method (45 mM Tris-acetate buffer, pH 4.5, 4 mM S-γ-CD, 21°C, +19.5 kV) was capable for the baseline separation of solriamfetol and phenylalaninol enantiomers within 7 min. The optimized method was validated according to the ICH guidelines and successfully applied for the analysis of pharmaceutical preparation (Sunosi^® 75 mg tablet), thus it may serve as a routine procedure for the laboratories of regulatory authorities as well as in Pharmacopoeias.     

涂层技术在毛细管电泳手性分离中的应用

手性是自然界的本质属性之一。手性分离分析技术对生命科学、环境科学、生物工程和药物工程等许多学科都具有十分重要的意义。当前,对不同种类手性化合物进行拆分已成为毛细管电泳技术最具特色的研究和应用领域之一。然而,被分析物(或拆分剂)在毛细管内壁的吸附是毛细管电泳手性分离中的常见问题。涂层技术就是采用不同的方法对毛细管内壁进行改性,是抑制非特异性吸附、提高分离效率及分离重现性最简便和最有效的方法。本文主要综述了近十几年来各种涂层技术在毛细管电泳手性分离领域的应用现状,并对毛细管涂层技术今后的发展进行了展望。