Capillary electrophoretic separation and computational modeling of inclusion complexes of β‐cyclodextrin and 18‐crown‐6 ether with primaquine and quinocide

The antimalarial drug primaquine (PQ) and its contaminant, the positional isomer quinocide (QC) have been successfully separated using capillary electrophoresis with either β‐cyclodextrin (β‐CD) or 18‐crown‐6 ether (18C6) as chiral mobile phase additive. The interactions of the drugs with cyclodextrins and 18C6 were studied by the semiempirical method (Parametric Model 3) PM3. Theoretical calculations for the inclusion complexes of PQ and QC with α‐CD, β‐CD and 18C6 were performed. Data from the theoretical calculations are correlated and discussed with respect to the electrophoretic migration behavior. More stable complexes are predicted for the PQ–β‐CD and PQ–18C6 complexes. The coelution of PQ and QC when α‐CD was used as buffer additive can be explained by their comparable stabilities of the inclusion complex formed, while significant differences in the complexation stabilities of the drugs with β‐CD is responsible for their separation. The stronger hydrogen bonding in PQ–18C6 system is responsible for the separation between PQ and QC when 18C6 was used as chiral mobile phase additive. Copyright © 2009 John Wiley & Sons, Ltd.     

Enantiomeric separation of 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs by HPLC with hydroxypropyl‐β‐cyclodextrin as chiral mobile phase additive

The enantio‐separations of eight 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs (2‐APA NSAIDs) were established using reversed‐phase high‐performance liquid chromatography with hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as chiral mobile phase additive for studying the stereoselective skin permeation of suprofen, ketoprofen, naproxen, indoprofen, fenoprofen, furbiprofen, ibuprofen and carprofen. The effects of the mobile phase composition, concentration of HP‐β‐CD and column temperature on retention and enantioselective separation were investigated. With 2‐APA NSAIDs as acidic analytes, the retention times and resolutions of the enantiomers were strongly related to the pH of the mobile phase. In addition, both the concentration of HP‐β‐CD and temperature had a great effect on retention time, but only a slight or almost no effect on resolutions of the analytes. Enantioseparations were achieved on a Shimpack CLC‐ODS (150 × 4.6 mm i.d., 5 μm) column. The mobile phase was a mixture of methanol and phosphate buffer (pH 4.0–5.5, 20 mM) containing 25 mM HP‐β‐CD. This method was flexible, simple and economically advantageous over the use of chiral stationary phase, and was successfully applied to the enantioselective determination of the racemic 2‐APA NSAIDs in an enantioselective skin permeation study. Copyright © 2009 John Wiley & Sons, Ltd.     

Selective adsorption of metoprolol enantiomers using 2‐hydroxypropyl‐β‐cyclodextrin cross‐linked multiwalled carbon nanotube

This study investigates the ability of functionalized multiwalled carbon nanotubes (MWCNTs) for enantio‐separation of metoprolol chiral forms. 2Hydroxypropyl‐β‐cyclodextrin (2HP‐β‐CD) was applied as a chiral selector to functionalize carbon nanotubes (CNTs). The modified multiwalled CNT samples were characterized using scanning electron microscopy and Fourier transform infrared spectroscopy. The results of analyses showed that CNTs were successfully cross‐linked with 2HP‐β‐CD. To evaluate the enantio‐separation property of the products, the separation of metoprolol chiral forms on the initial and final products was examined. Further, UV–visible spectroscopy and polarimeter analyses were used for characterization. The results indicate that MWCNT does not have any intrinsic enantio‐separation ability, although its selectivity for enantio‐separation can be enhanced by cross‐linking it to 2HP‐β‐CD. Moreover, the optimal mass of adsorbent as well as optimal mass of functional groups is estimated to achieve maximum enantio‐separation efficiency. The results indicate that applying large amounts of 2HP‐β‐CD to CNTs functionalization decreases the cross‐linking efficiency, which consequently reduces enantio‐separation efficiency. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis and application of a chiral ionic liquid functionalized β‐cyclodextrin as a chiral selector in capillary electrophoresis

A novel chiral ionic liquid functionalized β‐cyclodextrin, 6‐O‐2‐hydroxpropyltrimethylammonium‐β‐cyclodextrin tetrafluoroborate ([HPTMA‐β‐CD][BF₄]), was synthesized and used as a chiral selector in capillary electrophoresis. [HPTMA‐β‐CD][BF₄] not only increased the solubility in aqueous buffer in comparison with the parent compound, but also provided a stable reversal electroosmotic flow, and the enantioseparation of eight chiral drugs was examined in phosphate buffer containing [HPTMA‐β‐CD][BF₄] as the chiral selector. The effects of the [HPTMA‐β‐CD][BF₄] concentration and the background electrolyte pH were studied. Moreover, the chiral separation abilities of β‐CD and [HPTMA‐β‐CD][BF₄] were compared and possible mechanisms for the chiral recognition of [HPTMA‐β‐CD][BF₄] are discussed. Copyright © 2013 John Wiley & Sons, Ltd.     

Combined use of l‐alanine tert butyl ester lactate and trimethyl‐β‐cyclodextrin for the enantiomeric separations of 2‐arylpropionic acids nonsteroidal anti‐inflammatory drugs

In this study, a new CE method, employing a binary system of trimethyl‐β‐CD (TM‐β‐CD) and a chiral amino acid ester‐based ionic liquid (AAIL), was developed for the chiral separation of seven 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs (NSAIDs). In particular, the enantioseparation of ibuprofen, ketoprofen, carprofen, indoprofen, flurbiprofen, naproxen, and fenoprofen was improved significantly by supporting the BGE with the chiral AAIL l ‐alanine tert butyl ester lactate (l ‐AlaC₄Lac). Parameters, such as concentrations of TM‐β‐CD and l ‐AlaC₄Lac, and buffer pH, were systematically examined in order to optimize the chiral separation of each NSAID. It was observed that the addition of the AAIL into the BGE improved both resolution and efficiency significantly. After optimization of separation conditions, baseline separation (R_s>1.5) of five of the analytes was achieved in less than 11 min, while the resolution of ibuprofen and flurbiprofen was approximately 1.2. The optimized enantioseparation conditions for all analytes involve a BGE of 5 mM sodium acetate/acetic acid (pH 5.0), an applied voltage of 30 kV, and a temperature of 20°C. In addition, the results obtained by computing the %‐RSD values of the EOF and the two enantiomer peaks, demonstrated excellent run‐to‐run, batch‐to‐batch, and day‐to‐day reproducibilities.     

The study of enantioselectivity of all regioisomers of mono‐carboxymethyl‐β‐cyclodextrin used as chiral selectors in CE

This work documents the influence of the position of single carboxymethyl group on the β‐cyclodextrin skeleton on the enantioselectivity. These synthesized monosubstituted carboxymethyl cyclodextrin (CD) derivatives, native β‐cyclodextrin, and commercially available carboxymethyl‐β‐cyclodextrin with degree of substitution approximately 3 were used as additives into the BGE consisting of phosphate buffer at 20 mmol/L concentration, pH 2.5, and several biologically significant low‐molecular‐mass chiral compounds were enantioseparated by CE. The results indicate that different substituent location on β‐cyclodextrin skeleton has a significant influence on the enantioseparation of the investigated enantiomers. The enantioselectivity of 2^I‐O‐regioisomer was better than with native β‐cyclodextrin. Comparable results to native β‐cyclodextrin were obtained for 6^I‐O‐ regioisomer and the enantioselectivity of 3^I‐O‐regioisomer was even worse than with native β‐cyclodextrin. Commercially available derivative of CD provides better resolutions than the monosubstituted carboxymethyl CD derivatives for most of the investigated analytes.     

Enantiomeric Separations in Capillary Electrochromatography with Crown Ether‐Capped β‐Cyclodextrin‐Bonded Silica Particles as Chiral Stationary Phase

Two novel types of crown ether capped β‐cyclodextrin (β‐CD) bonded silica, namely, 4′‐aminobenzo‐X‐crown‐Y (X=15, 18 and Y=5, 6, resp.) capped [3‐(2‐O‐β‐cyclodextrin)‐2‐hydroxypropoxy] propylsilyl‐appended silica, have been prepared and used as stationary phases in capillary electrochromatography (CEC) to separate chiral compounds. The two stationary phases have a chiral selector with two recognition sites: crown ether and β‐CD. They exhibit excellent enantioselectivity in CEC for a wide range of compounds. After inclusion of metal ions (Na⁺ or K⁺) from the running buffer into the crown ether units, the stationary phases become positively charged and can provide extra electrostatic interaction with ionizable solutes and enhance the dipolar interaction with polar neutral solutes. This enhances the host‐guest interaction with the solute and improves chiral recognition and enantioselectivity. Due to the cooperation of the anchored β‐CD and the crown ether, this kind of crown ether capped β‐CD bonded phase shows better enantioselectivity than either β‐CD‐ or crown ether bonded phases only. These new types of stationary phases have good potential for fast chiral separation with CEC.     

Enantiomeric separation of R,S‐tolterodine and R,S‐methoxytolterodine with negatively charged cyclodextrins by capillary electrophoresis

The methods for separation of R,S‐tolterodine and R,S‐methoxytolterodine enantiomers using sulfated α‐, β‐CD and phosphated‐γ‐CD by CE in acidic BGE based on Tris/phosphate pH 2.5 buffer were developed. Sulfated α‐ and β‐CD allow anodic detection while phosphated‐γ‐CD allows only cathodic detection of the separated enantiomers. The influence of chiral selector (CS)'s concentration as well as the influence of composition and concentration of BGE on resolutions were studied. Reversal migration order of tolterodine and methoxytolterodine enantiomers was observed, when sulfated‐α‐ and sulfated‐β‐CD were used. The developed methods with all three studied CSs, were validated and compared. All proposed methods enable determination of 0.2% of S‐tolterodine as an optical impurity in pills, however the method with phosphated‐γ‐CD provided lower detection limit, better repeatability of peak areas and migration times, and also lower consumption of CS. Developed method employing phosphated‐γ‐CD that was applied for the determination of optical purity of R‐tolterodine in commercial pills.     

New single isomer negatively charged β‐cyclodextrin derivatives as chiral selectors in capillary electrophoresis

To improve resolution power of chiral selector and enantiomeric peak efficiency in CE, single isomer negatively charged β‐CD derivatives, mono(6‐deoxy‐6‐sulfoethylthio)‐β‐CD (SET‐β‐CD) bearing one negative charge and mono[6‐deoxy‐6‐(6‐sulfooxy‐5,5‐bis‐sulfooxymethyl)hexylthio]‐β‐CD (SMHT‐β‐CD) carrying three negative charges, were synthesized. The structure of these two β‐CD derivatives was confirmed by ¹H NMR and MS. SET‐β‐CD and SMHT‐β‐CD successfully resolved the enantiomers of several basic model compounds. SMHT‐β‐CD provided for a significantly greater enantioseparation than SET‐β‐CD at lower concentrations. This appears to be due to the higher binding affinity of SMHT‐β‐CD to the model compounds and the wider separation window resulting from an increased countercurrent mobility of the selector. Overall, the new chiral selectors provided enantioseparations with high peak efficiency while avoiding peak distortion due to polydispersive and electrodispersive effects. The information obtained from an apparent binding constant study suggested that the enantioseparation of the model compounds followed the predictions of charged resolving agent migration model and that the observed degree of enantioseparation difference were due to the magnitude of differences in both enantiomer‐chiral selector binding affinities (ΔK) and the mobilities of the complexed enantiomers (Δμ_c).     

Enantioselective Partitioning of Racemic Ibuprofen in a Biphasic Recognition Chiral Extraction System

Enantioselective partitioning of ibuprofen enantiomers in a biphasic recognition chiral extraction system was studied. A combination of hydrophobic L‐isobutyl tartrate in organic phase and hydrophilic β‐cyclodextrin derivative in aqueous phase is necessary to establish a biphasic recognition chiral extraction system. The studies performed involve an enantioselective extraction in a biphasic system, where ibuprofen enantiomers form four complexes with the β‐cyclodextrin derivative in aqueous phase and the D(L)‐isobutyl tartrate in organic phase, respectively. In these biphasic resolutions, the types and the concentrations of the extractants, pH and temperature all exert a considerable influence on the biphasic recognition process. Good enantioselectivities for ibuprofen enantiomers were obtained at pH≦2.5 and a ratio of 2:1 of [L‐isobutyl tartrate] to [HP‐β‐CD]. Biphasic recognition chiral extraction is of strong chiral separation ability, and may be very helpful to optimize the extraction systems and realize the large‐scale production of enantiomers.     

Chiral separation of phenylalanine and tryptophan by capillary electrophoresis using a mixture of β‐CD and chiral ionic liquid ([TBA] [l‐ASP]) as selectors

In this paper, a simple, effective and green capillary electrophoresis separation and detection method was developed for the quantification of underivatized amino acids (dl ‐phenylalanine; dl ‐tryptophan) using β‐Cyclodextrin and chiral ionic liquid ([TBA] [l ‐ASP]) as selectors. Separation parameters such as buffer concentrations, pH, β‐CD and chiral ionic liquid concentrations and separation voltage were investigated for the enantioseparation in order to achieve the maximum possible resolution. A good separation was achieved in a background electrolyte composed of 15 mm sodium tetraborate, 5 mm β‐CD and 4 mm chiral ionic liquid at pH 9.5, and an applied voltage of 10 kV. Under optimum conditions, linearity was achieved within concentration ranges from 0.08 to 10 µg/mL for the analytes with correlation coefficients from 0.9956 to 0.9998, and the analytes were separated in less than 6 min with efficiencies up to 970,000 plates/m. The proposed method was successfully applied to the determination of amino acid enantiomers in compound amino acids injections, such as 18AA‐I, 18AA‐II and 3AA. Copyright © 2013 John Wiley & Sons, Ltd.     

Investigation of Mixed Chiral Selectors of Different Metal Ion-L-Alanine Complex and β-Cyclodextrin on the Chiral Separation of Dansyl Amino Acids with Capillary Electrophoresis

Chiral separation of dausyl amino acids by capillary electrophoresis using mixed selectors of Mn(ll)-L-alanine complex and β-cyclodextrin (β-CD) was studied. Resolution was considerably superior to that obtained by using either Mn (Ⅱ)-L-alanine complex or β-CD alone. The effects of separation parameters, such as pH value of buffer solution, capillary temperature, the concentration of Mn (Ⅱ)-L-alanine complex, the types of CD and ligand on the migration times and resolutions were investigated. Six different transition metal complexes,Cu(Ⅱ), Zn(Ⅱ), Co(Ⅱ), Ni(Ⅱ), Hg(Ⅱ) and Cd(Ⅱ)-L-alanine complexes have been employed and compared with Mn(Ⅱ)complex. Differences in retention and selectivity were found.The substitution of Cu(Ⅱ), Zn(Ⅱ), Co(Ⅱ) and Ni(Ⅱ) for Mn(Ⅱ) resulted in a better chiral resolution while Hg(Ⅱ) and Cd(Ⅱ) showed poorer resolution abilities. The chiral separation mechanism was also discussed briefly.     

Mono‐6A‐(4‐methoxybutylamino)‐6A‐β‐cyclodextrin as a chiral selector for enantiomeric separation

A new member of the family of methoxylalkylamino monosubstituted β‐cyclodextrins, mono‐6^A‐(4‐methoxybutylamino)‐6^A‐β‐cyclodextrin, has been developed as a chiral selector for enantioseparation in capillary electrophoresis. This amino cyclodextrin exhibited good enantioselectivities for 16 model acidic racemates including three dansyl amino acids at an optimum pH of 6.0. Excellent chiral resolutions over six were obtained for α‐hydroxy acids and 2‐phenoxypropionic acids with 3.0 mM chiral selector. The good chiral recognition for α‐hydroxyl acids was attributed to inclusion complexation, electrostatic interactions, and hydrogen bonding. The hydrogen‐bonding‐enhanced chiral recognition was revealed by NMR spectroscopy. The chiral separation of acidic racemates was further improved with the addition of methanol (≤10 vol%) as an organic additive.     

Stereoisomer separation of flavanones and flavanone‐7‐O‐glycosides by means of nanoliquid chromatography employing derivatized β‐cyclodextrins as mobile‐phase additive

A nanoliquid chromatographic method for the stereoisomer separation of some flavanone aglycones and 7‐O‐glycosides has been proposed employing a C18 capillary column and a chiral mobile‐phase additive such as cyclodextrin. The chiral separation of eriodictyol, naringenin, and hesperitin was obtained by addition of carboxymethyl‐β‐cyclodextrin to the mobile phase, whereas eriocitrin, naringin, narirutin, and hesperidin diastereoisomers were resolved by using sulfobutyl ether‐β‐cyclodextrin. The influence of the composition of the mobile phase, the length of the capillary column, and the flow rate on the chiral recognition were investigated. At optimum conditions, baseline separation for the selected aglycones and glycosylated forms were achieved with a mobile phase consisting of 50 mM sodium acetate buffer pH 3 and 30% methanol containing 20 mM of carboxymethyl‐β‐cyclodextrin and 10 mM of sulfobutyl ether‐β‐cyclodextrin, respectively. Precision, linearity, and sensitivity of the method were tested. Limits of detection and quantification for the studied flavanone glycosides were in the range 1.3‐2.5 and 7.5‐12.5 µg/mL, respectively. The method was used for the determination of the diastereomeric composition of the flavanone‐7‐O‐glycosides in Citrus juices after solid‐phase extraction procedure.     

Efficient capillary electrophoresis separation and determination of free amino acids in beer samples

Simultaneous detection of various o‐phthalaldehyde (OPA)‐labeled amino acids (AAs) in food samples was reported based on CE separation. Ionic liquid was used for the first time for CE analysis of AAs with in‐capillary derivatization. Several other additives, including SDS, α/β‐CD, and ACN, as well as key parameters for CE separation (buffer pH value, separation voltage), were also investigated. Our results show that the multiple additive strategy exhibits good stable and repeatable character for CE analysis of OPA‐labeled AAs, for either in‐capillary derivatization or CE separation, and allows simultaneous quantification of different OPA‐labeled AAs in a large concentration range of 50 μM to 3.0 mM with LOD down to 10 μM. Seventeen OPA‐labeled AAs, except for two pairs of AAs (His/Gln and Phe/Leu), which were separated with resolutions of 1.1 and 1.2, respectively, were baseline separated and identified within 23 min using the present multiple additive strategy. The method was successfully applied for simultaneous analysis of AAs in seven beer samples and as many as eleven trace‐amount AAs were detected and quantified, indicating the valuable potential application of the present method for food analysis.     

Determination of the enantiomeric and diastereomeric impurities of RS‐glycopyrrolate by capillary electrophoresis using sulfated‐β‐cyclodextrin as chiral selectors

A practical chiral CE method, using sulfated‐β‐CD as chiral selector, was developed for the enantioseparation of glycopyrrolate containing two chiral centers. Several parameters affecting the separation were studied, including the nature and concentration of the chiral selectors, BGE pH, buffer type and concentration, separation voltage, and temperature. The separation was carried out in an uncoated fused‐silica capillary of (effective length 40 cm) × 50 μm id with a separation voltage of 20 kV using 30 mM sodium phosphate buffer (pH 7.0, adjusted with 1 M sodium hydroxide) containing 2.0% w/v sulfated‐β‐CD at 25°C. Finally, the method for determining the enantiomeric impurities of RS‐glycopyrrolate was proposed. The method was further validated with respect to its specificity, linearity range, accuracy and precision, LODs, and quantification in the expected range of occurrence for the isomeric impurities (0.1%).     

Synthesis of the chiral selector heptakis(6‐O‐methyl)‐β‐cyclodextrin by phase‐transfer catalysis and hydrazine‐mediated transfer‐hydrogenation

The exhaustive primary‐side alkylation of cyclodextrins has never been achieved directly. The undesired and simultaneous derivatization of the secondary hydroxyl moieties generates intricate isomeric mixtures that are challenging to purify, analyse and characterize. The aim of this study was to develop a chromatography‐free and up‐scalable strategy towards the preparation of per‐6‐O‐methylated cyclodextrin and to test the compound as potential chiral selector. The target molecule was prepared according to a five‐step synthesis by using methyltriphenylphosphonium bromide as catalyst under heterogeneous conditions. The removal of benzyl moieties, used as temporary secondary‐side protecting groups, was attained by applying hydrazine‐carbonate in the presence of Pd/C. All the intermediates were obtained in high yields, thoroughly characterized and their purity was assessed by ad‐hoc developed HPLC methods. The per‐6‐O‐methylated β‐cyclodextrin showed promising chiral recognition ability as background electrolyte additive in cyclodextrin‐modified capillary electrophoresis using the recreational drug methylene‐dioxypyrovalerone as model compound. Additionally, a model for the inclusion geometry between the single isomer host and the selected drug was developed based on the extensive 2D NMR analysis. The versatility of the proposed synthetic strategy opens the way to the industrial production of homogeneously primary‐alkylated cyclodextrins and to their wide application in chiral separation of various drugs.     

Application of ionic liquid as additive in determination of three β‐agonists by capillary electrophoresis with amperometric detection

CE coupled with amperometric detection method was developed using ionic liquid 1‐ethyl‐3‐methylimidazolium tetrafluoroborate (EMImBF₄) as additive for the simultaneous detection of clenbuterol (CLB), terbutaline (TER), and ractopamine (RAC) in feed. The effects of detection potential, concentration of EMImBF₄, pH, and concentration of the running buffer, separation voltage as well as injection time on the separation and detection of these three β‐agonists were investigated in detail. Under the optimum conditions: the detection potential at 1.05 V, 50 mmol/L Tris‐HAc at pH 8.0 with 0.6% (v/v) EMImBF₄, electrokinetic injection 6 s at 16 kV and separation voltage at 16 kV, a baseline separation for these three analytes could be achieved within 11 min. Introduction of EMImBF₄ into the running buffer resulted in significant improvement in separation selectivity and enhancement in peak currents for those β‐agonists, especially for TER and RAC, which could not be separated in the running buffer without additive. The method exhibited wide linear range with LOD (S/N = 3) of 2, 1, and 2 nmol/L for CLB, TER, and RAC, respectively. The precision was determined in both intraday (n = 5) and interday (n = 3) assays, and the RSDs for both migration time and peak current were less than 6%. The proposed method was also applied to analyze β‐agonists in feed sample.     

Capillary electrophoretic enantioseparation of m‐nisoldipine using two different β‐cyclodextrins

The methods for the enantioseparation of m‐nisoldipine, a new 1,4‐dihydropyridine calcium ion antagonist, were developed. The elaborated methods of m‐nisoldipine enantiomers separation were successfully performed using an anionic CD–sulfobutyl ether‐β‐CD (SBE‐β‐CD) or carboxymethyl‐β‐CD as chiral selector. However, the results indicated that SBE‐β‐CD was a better chiral selector for enantioseparation of the neutral m‐nisoldipine. Furthermore, comparing the two SBE‐β‐CDs, the derivative with a higher degree of substitution (DS) of 7.0 induced better enantioresolution than the one with low DS (4.0). In addition, possible chiral recognition mechanisms of dihydropyridines were discussed.     

Enantioselective separation in capillary electrophoresis using a novel mono-6-propylammonium-β-cyclodextrin as chiral selector

The chiral resolving ability of a novel single-isomer cationic β-cyclodextrin (CD), mono-6^A-propylammonium-6^A-deoxy-β-cyclodextrin chloride (PrAMCD), as a chiral selector in capillary electrophoresis (CE) is reported in this work for the enantioseparation of hydroxy, carboxylic acids and amphoteric analytes. The effect of chiral selector concentration on the resolution was studied. Good resolutions were achieved for hydroxy acids. Optimum resolutions were obtained even at 3.5 mM CD concentration for carboxylic acids. The electrophoretic method showed good linearity and reproducibility in terms of migration times and peak areas, which should make it suitable for routine analysis. In addition, baseline chiral separation of a six-acid mixture was achieved within 20 min. PrAMCD proved to be an effective chiral selector for acidic analytes.