Enantioselective separation of chiral vicinal diols in capillary electrophoresis using a mono‐6A‐aminoethylamino‐β‐cyclodextrin as a chiral selector

This paper describes an improved access to mono‐6^A‐aminoethylamino‐β‐CD (β‐CDen), a very efficient cationic chiral selector for CZE in the separation of eight chiral aromatic vicinal diols. The β‐CDen concentration has a strong influence on the efficiency of enantioseparation. The effects of the pH and concentration of the BGE, the capillary temperature, and the applied voltage on the resolution and separation selectivity have been studied. Excellent chiral resolution was achieved under the optimal conditions of β‐CDen 10 mM, pH 10, 200 mM borate buffer at 15 kV and 20°C within 20 min. Moreover, the developed method was successfully applied to the determination of the enantiomeric purity of the catalytic asymmetric dihydroxylation (AD) reaction products.     

Enantioseparation of hydroxyeicosatetraenoic acids by hydroxypropyl‐γ‐cyclodextrin‐modified micellar electrokinetic chromatography

Complete resolution of hydroxyeicosatetraenoic acid (HETE) enantiomers was achieved using hydroxypropyl‐γ‐cyclodextrin (HP‐γ‐CD)‐modified MEKC. The optimum running conditions were determined to be utilizing a 30 mM phosphate–15 mM borate buffer (pH 9.0) containing 30 mM HP‐γ‐CD and 75 mM SDS as the BGE, application of +30 kV as the effective voltage, and carrying out the experiment at 15°C. The eluents were detected at 235 nm. The method was used successfully for the simultaneous separations of (S)‐ and (R)‐enantiomers of regioisomeric 8‐, 11‐, 12‐, and 15‐HETEs. Subsequently, the optimized method was applied to evaluate the stereochemistry of 8‐ and 12‐HETEs from the marine red algae, Gracilaria vermiculophylla and Gracilaria arcuata, respectively. The 8‐HETE was found to be a mixture of 98% (R)‐enantiomer and 2% (S)‐enantiomer, while the 12‐HETE was a mixture of 98% (S)‐enantiomer and 2% (R)‐enantiomer. The present study demonstrates that the HP‐γ‐CD‐modified MEKC method is simple and sensitive and provides unambiguous information on the configuration of natural and synthetic HETEs.     

Separation and Migration Behavior of Dichlorophenols in β‐Cyclodextrin‐Modified Capillary Zone Electrophoresis

The separation and migration behavior of six isomeric dichlorophenols (DCPs) in cyclodextrin‐modified capillary zone electrophoresis (CD‐CZE) using a phosphate‐borate buffer at alkaline pH with β‐CD and hydroxypropyl‐β‐CD (HP‐β‐CD) as electrolyte modifiers were investigated. The influence of buffer pH and the concentration of β‐cyclodextrins were examined. The results indicate that baseline separation of six isomeric DCPs can be achieved with addition of β‐CD concentration in the range of 2.0‐10 mM or HP‐β‐CD concentration in the range of 4.0‐10 mM at pH 10.0. Binding constants of DCPs to β‐CDs were evaluated for a better understanding of the interaction of DCPs with β‐CDs.     

Capillary Electrophoretic Separation of Tricyclic Antidepressants Using 1‐Alkyl‐3‐Methylimidazolium‐Based Ionic Liquids as Background Electrolyte

A capillary electrophoretic (CE) method coupled with the use of 1‐ethyl‐3‐methylimidazolium tetrafluoroborate (1E‐3MI‐TFB) ionic liquid as background electrolyte (BGE) has been developed for the simultaneous separation of nine tricyclic antidepressants, viz. amitriptyline (Ami), clomipramine (Clo), desipramine (Des), fluphenazine (Flu), imipramine (Imi), nortriptyline (Nor), promazine (Pro), thioridazine (Thi) and trimipramine (Tri). Resolution of TCAs with similar molecular structures and pK_a values was accomplished by minute manipulation of the electrophoretic velocities of TCAs via reversed electroosmotic flow (EOF) generated by adsorption of 1E‐3MI cations onto the capillary wall. The optimal separation was obtained with a 50 mM 1E‐3MI‐TFB as the sole BGE at pH 3. Symmetric peaks with efficiencies up to 2.4 × 10⁵ plates/m were achieved. RSD values on migration times and peak areas were in the ranges of 0.63–0.95% and 3.41–6.34% (n = 4), respectively. The role of different alkyl groups on the imidazolium cations was also investigated.     

Chiral separation of 12 pairs of enantiomers by capillary electrophoresis using heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector and the elucidation of the chiral recognition mechanism by computational methods

Chiral separation of 12 pairs of basic analyte enantiomers including oxybutynin, bambuterol, tradinterol, clenbuterol, clorprenaline, terbutaline, tulobuterol, citalopram, phencynonate, fexofenadine, salbutamol, and penehyclidine was conducted by capillary electrophoresis using a single‐isomer anionic β‐cyclodextrin derivative, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector. Parameters influencing separation were studied, including background electrolyte pH, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin concentration, buffer concentration, and separation voltage. A background electrolyte consisting of 50 mM Tris‐H₃PO₄ and 6 mM heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin at pH 2.5 was found to be highly efficient for the separation of most enantiomers, with other conditions of normal polarity mode at 10 kV, detection wavelength of 210 nm using hydrodynamic injection for 3 s. Under the optimal conditions, baseline resolution (>1.50) for 11 pairs of enantiomers and somewhat lower resolution for penehyclidine enantiomers (1.17) were generated. Moreover, the possible mechanism of separation of clenbuterol, oxybutynin, salbutamol, and penehyclidine was investigated using a computational modeling method.     

Characterization of complexes between phenethylamine enantiomers and β‐cyclodextrin derivatives by capillary electrophoresis—Determination of binding constants and complex mobilities

To optimize chiral separation conditions and to improve the knowledge of enantioseparation, it is important to know the binding constants K between analytes and cyclodextrins and the electrophoretic mobilities of the temporarily formed analyte‐cyclodextrin‐complexes. K values for complexes between eight phenethylamine enantiomers, namely ephedrine, pseudoephedrine, methylephedrine and norephedrine, and four different β‐cyclodextrin derivatives were determined by affinity capillary electrophoresis. The binding constants were calculated from the electrophoretic mobility values of the phenethylamine enantiomers at increasing concentrations of cyclodextrins in running buffer. Three different linear plotting methods (x ‐reciprocal, y ‐reciprocal, double reciprocal) and nonlinear regression were used for the determination of binding constants with β‐cyclodextrin, (2‐hydroxypropyl)‐β‐cyclodextrin, methyl‐β‐cyclodextrin and 6‐O‐α‐maltosyl‐β‐cyclodextrin. The cyclodextrin concentration in a 50 mM phosphate buffer pH 3.0 was varied from 0 to 12 mM. To investigate the influence of the binding constant values on the enantioseparation the observed electrophoretic selectivities were compared with the obtained K values and the calculated enantiomer‐cyclodextrin‐complex mobilities. The different electrophoretic mobilities of the temporarily formed complexes were crucial factors for the migration order and enantioseparation of ephedrine derivatives. To verify the apparent binding constants determined by capillary electrophoresis, a titration process using ephedrine enantiomers and β‐cyclodextrin was carried out. Furthermore, the isothermal titration calorimetry measurements gave information about the thermal properties of the complexes.     

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.     

Estimation of apparent binding constant of complexes of selected acyclic nucleoside phosphonates with β‐cyclodextrin by affinity capillary electrophoresis

Affinity capillary electrophoresis (ACE) has been applied to estimation of apparent binding constant of complexes of (R,S)‐enantiomers of selected acyclic nucleoside phosphonates (ANPs) with chiral selector β‐cyclodextrin (βCD) in aqueous alkaline medium. The noncovalent interactions of five pairs of (R,S)‐enantiomers of ANPs‐based antiviral drugs and their derivatives with βCD were investigated in the background electrolyte (BGE) composed of 35 or 50 mM sodium tetraborate, pH 10.0, and containing variable concentration (0–25 mM) of βCD. The apparent binding constants of the complexes of (R,S)‐enantiomers of ANPs with βCD were estimated from the dependence of effective electrophoretic mobilities of (R,S)‐enantiomers of ANPs (measured simultaneously by ACE at constant reference temperature 25°C inside the capillary) on the concentration of βCD in the BGE using different nonlinear and linear calculation methodologies. Nonlinear regression analysis provided more precise and accurate values of the binding constants and a higher correlation coefficient as compared to the regression analysis of the three linearized plots of the effective mobility dependence on βCD concentration in the BGE. The complexes of (R,S)‐enantiomers of ANPs with βCD have been found to be relatively weak – their apparent binding constants determined by the nonlinear regression analysis were in the range 13.3–46.4 L/mol whereas the values from the linearized plots spanned the interval 12.3–55.2 L/mol.     

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.     

Spectrofluorimetric and Spectrophotometric Determination of Oxamniquine in Pharmaceuticals and Biological Fluids via Derivatization with 4‐Chloro‐7‐Nitrobenzo‐2‐oxa‐1,3‐Diazole (NBD‐Cl)

Spectrophotometric and spectrofluorimetric methods were developed for the determination of oxamniquine (OXM). Both methods are based on coupling with 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl) in borate buffer of pH 7.6, and the reaction product was measured at 400 nm (Method I). The same product was measured by spectrofluorimetry at 480 nm upon excitation at 400 nm (Method II). The absorbance and the fluorescence intensity were enhanced by addition of sodium dodecyl sulphate (SDS). The absorbance‐concentration plot is rectilinear over the range of 5–25 μg/mL with an LOD of 0.31 μg/mL. The fluorescence‐concentration plot is linear over the range of 0.2–1.2 μg/mL with an LOD of 0.03 μg/mL. Both methods were applied to the analysis of capsules, and the results were in good agreement with those obtained using the official method. The method was applied to spiked human plasma; the mean % recovery (n = 5) is 101.05 ± 1.65. A proposal of the reaction pathway is presented.     

Chiral capillary electrophoresis with cationic pyrrolidinium‐β‐cyclodextrin derivatives as chiral selectors

New single‐isomer, cationic β‐cyclodextrins, including mono‐6‐deoxy‐6‐pyrrolidine‐β‐cyclodextrin chloride (pyCDCl), mono‐6‐deoxy‐6‐(N‐methyl‐pyrrolidine)‐β‐cyclodextrin chloride (N‐CH₃‐pyCDCl), mono‐6‐deoxy‐6‐(N‐(2‐hydroxyethyl)‐pyrrolidine)‐β‐cyclodextrin chloride (N‐EtOH‐pyCDCl), mono‐6‐deoxy‐6‐(2‐hydroxymethyl‐pyrrolidine)‐β‐cyclodextrin chloride (2‐MeOH‐pyCDCl) were synthesized and used as chiral selectors in capillary electrophoresis for the enantioseparation of carboxylic and hydroxycarboxylic acids and dansyl amino acids. The unsubstituted pyCDCl exhibited the greatest resolving ability. Most analytes were resolved over a wide range of pH from 6.0 to 9.0 with this chiral selector. In general, increasing pH led to a decrease in resolution. The effective mobilities of all the analytes were found to decrease with increasing CD concentration. The optimal concentration for most carboxylic acids and dansyl amino acid was in the range 5–7.5 mM and >15 mM for hydroxycarboxylic acids. ¹H NMR experiments provided direct evidence of inclusion in the CD cavity.     

Combined use of ionic liquid and sulfobutylether‐β‐cyclodextrin as electrolyte additives for separation and determination of camptothecin alkaloids by CZE

This work reported that ionic liquid (IL) ([Bmim] [PF₆]) and sulfobutylether‐β‐CD (SBE‐β‐CD) were used as electrolyte additives for the separation and determination of camptothecin (CPT) alkaloids by CZE. Separation parameters such as the buffer type, pH, and concentration of the running buffer, the concentration of SBE‐β‐CD and IL, temperature, and separation voltage were all investigated in order to achieve the maximum possible resolution. The four analytes were baseline separated within 10 min in capillary at the separation voltage of 15 kV with a running buffer consisting of 20 mM borate buffer, 20 mM IL, and 100 mM SBE‐β‐CD at pH 9.0. Under such conditions, good linearity about two orders of magnitudes of peak areas was achieved for the investigated CPT alkaloids with the correlation coefficients ranging from 0.9946 to 0.9985. For all analytes, detection limits (S/N = 3) and quantitation limits (S/N = 10) range from 0.05 to 0.92 μg/mL and 0.17 to 3.06 μg/mL, respectively. The proposed method has not only been successfully applied to the separation and determination of CPT alkaloids but also showed that IL seemed to be a promising additive in CZE separation.     

Therapeutic deferoxamine and deferiprone monitoring in β‐thalassemia patients’ plasma by field‐amplified sample injection and sweeping in capillary electrophoresis

One CE method was established for detecting deferoxamine (DFO) and deferiprone (DFR) in plasma. For β‐thalassemia patients, DFO and DFR are major medicines to treat the iron overload caused by blood transfusion. Field‐amplified sample injection combined with sweeping was used for sensitivity enhancement in CE. This method was performed on an uncoated fused‐silica capillary. After liquid–liquid extraction, the plasma samples were electrokinetically injected into capillary at +10 kV for 180 s. The phosphate buffer (100 mM) containing 50 mM triethanolamine was used as the BGE (pH 6.6). Separation buffer was phosphate buffer (100 mM, pH 3.0) containing 150 mM SDS. This method showed good linearity (r ≥ 0.9960). Precision and accuracy were evaluated by the results of RSD and relative error of intrabatch and interbatch analyses, and all of the absolute values were less than 6.12%. The LODs (S/N = 3) were 200 ng/mL for DFO, and 25 ng/mL for DFR. The LOQ (S/N = 10) of DFO and DFR were 600 and 75 ng/mL, respectively. This method was applied for clinical applications of five β‐thalassemia patients.     

Preparative and scaled‐up separation of high‐purity α‐linolenic acid from perilla seed oil by conventional and pH‐zone refining counter current chromatography

α‐Linolenic acid is an essential omega‐3 fatty acid needed for human health. However, the isolation of high‐purity α‐linolenic acid from plant resources is challenging. The preparative separation methods of α‐linolenic acid by both conventional and pH‐zone refining counter current chromatography were firstly established in this work. The successful separation of α‐linolenic acid by conventional counter current chromatography was achieved by the optimized solvent system n‐heptane/methanol/ water/acetic acid (10:9:1:0.04, v/v), producing 466 mg of 98.98% α‐linolenic acid from 900 mg free fatty acid sample prepared from perilla seed oil with linoleic acid and oleic acid as by‐products. The scaled‐up separation in 45× is efficient without loss of resolution and extension of separation time. The separation of α‐linolenic acid by pH‐zone refining counter current chromatography was also satisfactory by the solvent system n‐hexane/methanol/water (10:5:5, v/v) and the optimized concentration of trifluoroacetic acid 30 mM and NH₄OH 10 mM. The separation can be scaled up in 180× producing 9676.7 mg of 92.79% α‐linolenic acid from 18 000 mg free fatty acid sample. pH‐zone refining counter current chromatography exhibits a great advantage over conventional counter current chromatography with 20× sample loading capacity on the same column.     

Synthesis and Bioactivity of Quinoline‐3‐carboxamide Derivatives

Twelve novel substituted 2‐chloroquinoline‐3‐carboxamide derivatives were prepared from acetanilides using the Vilsmeier–Haack reaction, producing 2‐chloro‐3‐carbaldehyde quinolines, followed by oxidation of the 3‐carbaldehyde to the carboxylic acid and coupling this group with various anilines. The structures of the synthesized compounds were confirmed by NMR, mass spectrometry, and single crystal X‐ray diffraction. The chemical shifts of H‐5 and H‐8 were shown to be influenced by the substituent at C‐6. The substituent at C‐6 was also seen to affect the chemical shift of C‐5, C‐7, and C‐8, with C‐5 and C‐7 being more shielded in 5j (F substituted) in comparison with 5g (Cl substituted) and 5d (CH₃ substituted). The compounds showed weak activity in the mM range against Gram‐positive and Gram‐negative bacteria of which 5b , 5d , and 5f showed the best activity with minimum bactericidal concentration values for 5b being 3.79 mM against methicillin‐resistant Staphylococcus aureus and 5d and 5f having minimum bactericidal concentration values of 3.77 and 1.79 mM against S. aureus ATCC 25923, respectively.     

Determination of ζ‐potential,charge, and number of organic ligands on the surface of water soluble quantum dots by capillary electrophoresis

The number of charges and/or organic ligands covalently attached to the surface of CdTe quantum dot nanoparticles has been determined from their electrophoretic mobilities measured in capillaries filled with free electrolyte buffers. Three sizes of water soluble CdTe quantum dots with 3‐mercaptopropionic and thioglycolic acids as surface ligands were prepared. Their electrophoretic mobilities in different pH and ionic strength values of separation buffers were measured by capillary electrophoresis with laser induced fluorescence detection. The ζ‐potentials determined from electrophoretic mobilities using analytical solution of Henry function proposed by Ohshima were in the range from ?30 to ?100 mV. Charges of QDs were calculated from ζ‐potentials. As a result, numbers of organic ligands bonded to QDs surface were determined to be 13, 14, and 15 for the sizes of 3.1, 3.5, and 3.9 nm, respectively. The dissociation constants of organic ligands bonded on QDs surfaces estimated from the dependence of QDs charge on pH of the separation buffer were 7.8 and 7.9 for 3‐mercaptopropionic acid and 6.9 for thioglycolic acid.     

Stereoselective Organocatalyzed Synthesis of α‐Fluorinated β‐Amino Thioesters and Their Application in Peptide Synthesis

α‐Fluorinated β‐amino thioesters were obtained in high yields and stereoselectivities by organocatalyzed addition reactions of α‐fluorinated monothiomalonates (F‐MTMs) to N‐Cbz‐ and N‐Boc‐protected imines. The transformation requires catalyst loadings of only 1 mol % and proceeds under mild reaction conditions. The obtained addition products were readily used for coupling‐reagent‐free peptide synthesis in solution and on solid phase. The α‐fluoro‐β‐(carb)amido moiety showed distinct conformational preferences, as determined by crystal structure and NMR spectroscopic 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%).     

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.