Direct enantioseparation of catechin and epicatechin in tea drinks by 6-O-alpha-D-glucosyl-beta-cyclodextrin-modified micellar electrokinetic chromatography

Cyclodextrin-modified micellar electrokinetic chromatography was applied to the enantioseparation of catechin and epicatechin using 6-O-alpha-D-glucosyl-beta-cyclodextrin together with sodium dodecyl sulfate and borate-phosphate buffer. Factors affecting chiral resolution and migration time of catechin and epicatechin were studied. The optimum running conditions were found to be 200 mM borate-20 mM phosphate buffer (pH 6.4) containing 25 mM 6-O-alpha-D-glucosyl-beta-cyclodextrin and 240 mM sodium dodecyl sulfate with an effective voltage of +25 kV at 20 degrees C using direct detection at 210 nm. Under these conditions, the resolution (Rs) of racemic catechin and epicatechin were 4.15 and 1.92, respectively. With this system, catechin and epicatechin enantiomers along with other four catechins ((-)-catechin gallate, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-epigallocatechin gallate) and caffeine in tea samples were analyzed successfully. The difference of migration time between catechin and epicatechin is discussed.     

Separation and quantitation of the four stereoisomers of itraconazole in pharmaceutical formulations by electrokinetic chromatography

The four stereoisomers of itraconazole were resolved for the first time by EKC using a CD as chiral selector. A study on the enantiomeric separation ability of different neutral CDs was carried out. Heptakis-2,3,6-tri-O-methyl-beta-CD was shown to provide the highest values for the enantiomeric resolution. The influence of some experimental conditions, such as pH, chiral selector concentration, and temperature, on the enantiomeric separation was also studied. The use of a 100 mM phosphate buffer (pH 2.5), 30 mM in heptakis-2,3,6-tri-O-methyl-beta-CD together with an applied voltage of 30 kV and a temperature of 20 degrees C enabled the separation of the enantiomers of itraconazole with high resolutions (Rs > 3.0). Finally, the method was validated and successfully applied to the quantitation of itraconazole in three pharmaceutical formulations.     

Enantioseparation of dopa and related compounds by cyclodextrin-modified microemulsion electrokinetic chromatography

A chiral microemulsion electrokinetic chromatography method has been developed for the enantiomeric separation of 3,4-dihydroxyphenylalanine (dopa), its precursors phenylalanine and tyrosine, and the structurally related substance methyldopa. The separations were achieved using an oil-in-water microemulsion, which consisted of the oil-compound ethyl acetate, the surfactant sodium dodecylsulfate (SDS), the co-surfactant 1-butanol, the organic modifier propan-2-ol and 20mM phosphate buffer pH 2.5 or 2.0 as aqueous phase. For enantioseparation sulfated beta-cyclodextrin was added. The resolution of each racemate was optimized by varying the concentration of the buffer and all components of the microemulsion. Enantioseparation could be achieved for dl-dopa, dl-phenylalanine and dl-tyrosine within 13min with a resolution of 4.3, 3.1 and 3.3, respectively, and for methyldopa in 17min (Rs: 1.4). The established methods allowed the detection of dopa, phenylalanine, tyrosine and methyldopa with a limit at 0.5, 1.0, 0.2 and 2.0mug/ml.     

Characterization of amino acids in silk sericin protein from Gonometa rufobrunnae by MEKC with phenyl isothiocyanate derivatization

An MEKC method was developed for the separation and characterization of phenyl-isothiocyanate (PITC)-labeled amino acids derived from Gonometa rufobrunnae silkworm after microdialysis sample cleanup. The influence of the buffer and SDS concentration on the resolution of the amino acids was investigated. A buffer system consisting of 25 mM phosphate, 10 mM borate buffer at pH 9.00, and 70 mM SDS showed the best results, with 13 PITC-amino acid derivatives being resolved out of 15 possible amino acids that were under study. Microdialysis sampling demonstrated its efficiency as a sample cleanup technique. Sericin protein from G. rufobrunnae was found to be characterized by at least 11 positively identified amino acids. These included His, Tyr, Ser, Ala, Phe, Lys, Gly, Arg, Cys, Glu, and Asp. Leu/Met and Val/Thr were coeluting pairs and hence could not be positively confirmed.     

Development of a capillary zone electrophoresis method for the separation of a furan combinatorial library

Nine component mixtures of a furan library were simultaneously separated by capillary zone electrophoresis (CZE) using a phosphate buffer as a background electrolyte at low pH. The effects of buffer concentration, buffer pH, type and concentration of organic solvents on the electrophoretic mobility, resolution, and analysis time were systematically investigated. Resolution and efficiency of furan library components were further improved using cyclodextrin (CD)-modified CZE. Under optimum conditions, eight of the nine furans were baseline-resolved in less than 10 min at 30 kV using 50 mM phosphate buffer, 10% v/v acetonitrile (ACN), pH 2.0, with 5 mM gamma-CD.     

Chiral separation of N-imidazole derivatives, aromatase inhibitors, by cyclodextrin-capillary zone electrophoresis. Mechanism of enantioselective recognition

Baseline separation of ten new, substituted [1-(imidazo-1-yl)-1-phenylmethyl)] benzothiazolinone and benzoxazolinone derivatives with one chiral center was achieved using cyclodextrin-capillary zone electrophoresis (CD-CZE). A method for the enantiomeric resolution of these compounds was developed using neutral CDs (native alpha-, beta-, gamma-CDs or alpha-, beta-, gamma-hydroxypropyl (HP)-CDs) as chiral selectors. Operational parameters including the nature and concentration of the chiral selectors, pH, ionic strength, organic modifiers, temperature, and applied voltage were investigated. The use of neutral CDs provides enantiomeric resolution by inclusion of compounds in the CD cavity. The HP-alpha-CD and HP-beta-CD were found to be the most effective complexing agents and allowed efficient enantiomeric resolutions. Optimal separation of N-imidazole derivatives was obtained using 50 mM phosphate buffer at pH 2.5 containing either HP-alpha-CD or HP-beta-CD (7.5-12.5 mM) at 25 degrees C, with an applied field of 0.50 kV.cm(-1) giving resolution factors Rs superior to 1.70 with migration times of the second enantiomer less than 13 min. The same enantiomer migration order observed for all molecules can be related to a close interaction mechanism with CDs. The influence of structural features of the solutes on Rs and tm was studied. The lipophilic character (log kw) of the solutes and the apparent and averaged association constants of inclusion complexes for four compounds with the six different CDs led us to rationalize the enantioseparation mechanisms. The conclusions were corroborated with reversed-phase high-performance liquid chromatography (HPLC) on chiral stationary phases (CSPs) based on CDs.     

Micellar electrokinetic capillary chromatography of macrolide antibiotics. Separation of tylosin, erythromycin and their related substances.

The separation of tylosin by micellar electrokinetic capillary chromatography with a mixed micelle system is described. Good selectivity was obtained with sodium phosphate buffer (80 mM, pH 7.5) containing 20 mM sodium cholate and 7 mM cetyltrimethylammonium bromide (CTAB). This method permits tylosin to be separated from its closely related substances within 15 min. The influences of type of buffer, buffer pH, the concentrations of sodium cholate and CTAB were investigated. The robustness of the method was examined for tylosin by means of a full-fraction factorial design. Quantitative results are presented. Using a similar buffer system (80 mM sodium phosphate, pH 6.0, 20 mM sodium cholate and 5 mM CTAB), separation of erythromycin and its main related substances was also obtained. However, detection sensitivity and resolution are not sufficient for analysis of related substances in erythromycin commercial samples.     

Resolution of structural isomers of sialylated oligosaccharides by capillary electrophoresis

The resolution of structural isomers in mixtures of oligosaccharides is often challenging. Capillary electrophoresis was employed to separate three sets of structural isomers of sialylated oligosaccharides found in human milk and bovine colostrum. Different running buffers were necessary to achieve optimal baseline resolution. To resolve 3'- and 6'-sialyllactoses, 0.2 M aqueous sodium phosphate containing 40% methanol as an organic modifier was used as a running buffer. To resolve 3'- and 6'-sialyllactosamines, 0.4 M aqueous sodium phosphate without organic modifier was used. Baseline resolution of sialyllacto-N-tetraose-a and -b and sialyllacto-N-neotetraose-c was achieved with a 0.4 M Tris-HCl buffer containing 250 mM sodium dodecyl sulfate and 10% methanol as the organic modifier. Thus, each of these sets of structural isomers of sialylated oligosaccharides required a unique running buffer with respect to buffer type, concentration, pH, presence of organic modifiers, and surfactants. Similar electrophoresis conditions may be useful for resolving and analyzing other structural isomers of acidic oligosaccharides by capillary electrophoresis.     

Comparison of sulfobutylether- and sulfated-beta-cyclodextrins as additives for the chiral separation of basic spirobenzopyrans by capillary electrophoresis.

Three charged substituted beta-cyclodextrins (beta-CDs), sulfobutylether-beta-(SBE-beta-CD), degree of substitution (DS) 4 and 7), and sulfated-beta-(S-beta-CD) cyclodextrins, were compared as chiral additives in capillary electrophoresis for the enantiomeric separation of basic spirobenzopyran derivatives (pKa 9.9) which differ from each other by an N-alkyl group. The number of sulfobutylether groups attached to the cyclodextrin moiety significantly influences the enantioseparation of the basic drugs. SBE-beta-CD (DS 7) which is more strongly bound to cationic analyte than SBE-beta-CD (DS 4.6), requires smaller concentrations to achieve the same resolution. Besides, better enantioresolutions were obtained with S-beta-CD rather than with SBE-beta-CDs though higher concentrations are required, which led to high current values. However, both pairs of enantiomers cannot be resolved using S-beta-CD while SBE-beta-CDs make it possible to resolve simultaneous enantioseparation of such solutes slightly differing in hydrophobicity. This supports the hypothesis that hydrophobic interactions (outside of the CD cavity) between the butyl group attached to SBE-beta-CD and the N-alkyl group of spirobenzopyran play a role in the enantioseparation. On the other hand, the sulfate group of S-beta-CD was directly attached to the CD moiety which means that the S-beta-CD-drug complexation mechanism arises through the combination of electrostatic and hydrophobic (inside the CD cavity) interactions. Finally, enantiomers of spirobenzopyran drugs were satisfactorily resolved by CE using a 20 mg/mL S-beta-CD concentration (resolution 4.0), 7 mg/mL SBE-beta-CD DS 4 (resolution 1.3), or 5 mg/mL SBE-beta-CD DS 7 (resolution 3.3) added to the phosphate buffer (pH 2.6, 50 mM ionic strength).     

Hyphenated techniques in anticancer drug monitoring. II. Liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry

A micellar electrokinetic chromatographic (MEKC) method was developed for the separation of ten phenolic acids including cichoric acid and caftaric acids, specific marker phytochemicals of Echinacea purpurea. The MEKC method involved the use of 70 mM sodium deoxycholate (SDC) in 40 mM borate (pH 9.2) buffer and UV detection at 300 nm. The bile acid was used as biosurfactant able to provided a micellar system with different and more selective properties than sodium dodecyl sulfate. The effects of SDC and borate concentration and buffer pH on the analyte resolution were evaluated. The validated method was applied to the determination of cichoric acid and related compounds in E. purpurea root extracts, and in commercial E. purpurea based dried extracts and tablets.     

Effect of surfactant concentration and buffer selection on chromatographic figures of merit in chiral microemulsion electrokinetic chromatography

The enantiomeric resolution of 15 different pharmaceutical compounds was explored using chiral microemulsion electrokinetic chromatography (MEEKC). The microemulsion employed was comprised of the chiral surfactant dodecoxycarbonylvaline (DDCV), 1-butanol, and ethyl acetate, at an initial composition of 1% w/v:1.2% v/v:0.5% v/v, respectively. The effect of varying the background buffer composition, voltage, and ultimately the surfactant concentration and/or aggregate phase ratio were examined. Changing from a zwitterionic buffer ((2-[2-amino-2-oxoethyl)amino]ethanesulfonic acid, ACES) to the same concentration of phosphate buffer improved the efficiency and decreased overall analysis time, but also resulted in a decrease in chiral resolution. Furthermore, using phosphate buffer while simultaneously increasing the percent DDCV from 1 to 4% increased the efficiencies from a range of 34,000 to 59,000 N/m to a range of 160,000 to 400,000 N/m. While the enantioselectivities did not change significantly, the improvement in efficiencies, elution range, and retention factors provided an increase in both resolution and the number of enantiomers that were separated. Using an optimized microemulsion comprised of phosphate buffer and 4% DDCV, chiral separation was achieved for all 11 pairs of enantiomers, with a resolution ranging from 0.90 to 4.71. Moreover, the average resolution doubled in going from nonoptimized to optimized conditions for five of the eleven compounds. Finally, a comparison was made of the effect of increasing only the surfactant concentration by a factor of 4 versus increasing the overall composition (or phase ratio) by a factor of 4. Ultimately, the microemulsion containing 4% DDCV provided a larger elution range, greater resolution, and more optimal retention than that provided by the 4x phase increase.     

Chiral CE of aromatic amino acids by ligand-exchange with zinc(II)-L-lysine complex

A novel method of chiral ligand-exchange CE was developed with either L- or D-lysine (Lys) as a chiral ligand and zinc(II) as a central ion. This type of chiral complexes was explored for the first time to efficiently separate either individual pairs of or mixed aromatic amino acid enantiomers. Using a running buffer of 5 mM ammonium acetate, 100 mM boric acid, 3 mM ZnSO(4) x 7H(2)O and 6 mM L-Lys at pH 7.6, unlabeled D,L-tryptophan, D,L-phenylalanine, and D,L-tyrosine were well separated, giving a chiral resolution of up to 7.09. The best separation was obtained at a Lys-to-zinc ratio of 2:1, zinc concentration of 2-4 mM and running buffer pH 7.6. The buffer pH was determined to have a strong influence on resolution, while buffer composition and concentration impacted on both the resolution and peak shape. Boric acid with some ammonium acetate was an adoptable buffer system, and some additives like ethylene diamine tetraacetic acid capable of destroying the complex should be avoided. Fine-tuning of the chiral resolution and elution order was achieved by regulating the ratio of L-Lys to D-Lys; i.e. the resolution increased from zero to its highest value as the ratio ascended from 1:0 to 1:infinitive, and L-isomers eluted before or after D-isomers in excessive D- or L-Lys, respectively.     

Enantiomeric separation of ketoconazole and terconazole antifungals by electrokinetic chromatography: Rapid quantitative analysis of ketoconazole in pharmaceutical formulations

EKC using a neutral CD as chiral selector was applied in this work to the development of a method enabling the enantiomeric separation of ketoconazole and terconazole antifungals. The influence of different experimental conditions such as temperature, CD concentration, pH, and nature and concentration of the buffer on the enantiomeric resolution of the compounds studied was investigated. The use of 10 mM heptakis-(2,3,6-tri-O-methyl)-beta-CD in a 100 mM phosphate buffer (pH 3.5) with a temperature of 15 degrees C allowed the separation of the enantiomers of ketoconazole and terconazole with high resolution (R(s) > 2.0). The rapid separation of ketoconazole enantiomers with an analysis time less than 3 min was carried out after fitting some experimental parameters. The developed method was applied to the determination of ketoconazole in different pharmaceutical formulations.     

Chiral separation of amino acids derivatized with fluoresceine-5isothiocyanate by capillary electrophoresis and laser-induced fluorescence detection using mixed selectors of beta-cyclodextrin and sodium taurocholate

Chiral separation of 20 pairs of amino acids derivatized with fluoresceine-5-isothiocyanate (FITC) by capillary electrophoresis and laser-induced fluorescence detection was studied using the mixture of beta-cyclodextrin (beta-CD) and sodium taurocholate (STC) as selector. Resolution was considerably superior to that obtained by using either beta-CD or STC alone. The molar ratio of beta-CD to STC of about 2:3 was found to be critical to achieve maximum separation. At this beta-CD-to-STC ratio, chiral separation occurred at really low total concentration of beta-CD and STC (<0.1 mM). Other impacting factors were investigated including the total concentration of beta-CD and STC, pH, and capillary conditioning procedure between two successive runs. Using a running buffer of 80 mM borate containing 20 mM beta-CD and 30 mM STC at pH 9.3, all of the 20 pairs of FITC-amino acid enantiomers were baseline resolved. The resolutions of the most pairs of the amino acid enantiomers (17 of 20) were higher than 3.0, only three pairs gave a resolution lower than 3.0 but higher than 1.90 (beta-phenylserine, pSer). The highest resolution reached 14.58 (Glu). Two derivatives of beta-CD, 2-hydroxypropyl-beta-CD (HP-beta-CD) and heptakis(2,6-di-O-methyl)-beta-CD (DM-beta-CD) were also explored. HP-beta-CD showed similar cooperative effect with STC, while DM-beta-CD together with STC led to poorer chiral separation.     

Simultaneous separation of nitrofuran antibiotics and their metabolites by using micellar electrokinetic capillary chromatography

Mixtures comprising nitrofuran antibiotics (NFA) and nitrofuran metabolites (NFM) were resolved for the first time by using MEKC. Sodium deoxycholate (SDC) was chosen as the micelle-forming surfactant. Optimization of separation conditions was achieved by using a central composite experimental design (CCD) approach. Experimental parameters such as concentration ratio of borate to phosphate in the buffer, pH of the running electrolyte and voltage were investigated. The effect of concentration of the surfactant on resolution was significant. Under optimal conditions of 80 mM SDC, pH 9.0, (20 mM borate + 20 mM phosphate) and 16 kV, the resolution between eight consecutive peak pairs ranged from 1.9 to 11.8. Due to the absence of a UV-active chromophore in the metabolites, they were derivatized with 2-nitrobenzaldehyde (2-NBA). In order to mimic a proposed extraction procedure for the analysis of both NFA and/or derivatized NFM in a sample, aqueous samples (prederivatized with 2-NBA) were extracted by using C(18) SPE cartridges. After washing with H(2)O, the cartridges were eluted with a small portion of organic solvent with weak elution characteristics to remove excess 2-NBA (hexane was chosen). Target analytes were then recovered with ACN. Excellent reproducibility of migration time (t(mig)) was achieved for all analytes using the developed MECC approach, with absolute t(mig) <1% RSD and t(mig) ratio <0.2% RSD, and peak area ratio was 4% RSD. The LOD for each compound, calculated by extrapolating to an S/N of 3, were found to be 0.19-2.0 microg/mL.     

Application of capillary zone electrophoresis to the screening of some angiotensin II receptor antagonists

A capillary zone electrophoretic (CZE) method was optimized for the separation of five angiotensin II receptor antagonists (Losartan, Irbesartan, Valsartan, Telmisartan and Eprosartan) and two of their metabolites (EXP 3174 and Candesartan M1) by means of experimental design methodologies. The aim of this study was to define rapidly experimental conditions under which the analytes can be resolved for quantitation. The effects of the buffer (pH, concentration and composition), the organic modifier and voltage were studied. Critical factors were identified in a screening design (fractional factorial design) and sequentially an optimization design (central composite design) was used to choose optimal conditions for separation. The most favorable electrophoretic conditions were found by setting the resolution at a threshold value (Rs < or = 1.5) and minimizing, if possible, analysis time. Successful results were obtained with a 50 mM potassium dihydrogen phosphate:boric acid (25:75 v/v) buffer at pH 5.5 in the presence of 5% methanol and application of a 25 kV voltage. Analysis time was 8 min in a conventional fused-silica capillary (50 cm effective length) in a normal cationic mode (anode at the inlet and cathode at the outlet) after hydrostatical sample injection for 30 s.     

Separation of dansylated amino acid enantiomers by chiral ligand-exchange CE with a zinc(II) L-arginine complex as the selecting system

A facile chiral ligand-exchange capillary electrophoretic method has been explored for the enantioseparation and UV detection of dansyl-amino acids with Zn(II) L-arginine complex as a chiral selecting system. Successful enantioseparation of 17 pairs of amino acid enantiomers has been achieved with a buffer of 100 mM boric acid, 5 mM ammonium acetate, 3 mM ZnSO4 and 6 mM L-Arg at pH 8.0, of which 10 pairs were fully resolved with resolution in between 1.59 and 4.21. This new method was shown to be applicable to the separation of some mixed pairs of amino acids and to the quantitative analysis of some real samples such as rice vinegars, with a linear range between 0.8 and 150 microg/mL, correlation coefficient above 0.99 and recovery in between 90.1 and 112.4%. It was found that amino acids with low resistance side chain(s), low tendency to form intramolecular hydrogen bond or high tendency to form intermolecular hydrogen bonds are more easily enantioseparated than those with extra carboxyl and/or phenyl groups. By the use of the suggested buffer, the running pH should be selected at 7.4-9.0 to compromise the resolution and elution speed.     

Effect of the Buffer Concentration on the Separation of Lactate Dehydrogenase Isoenzymes from Different Sources by Electrophoresis

Abstract

The separation of lactate dehydrogenase isoenzymes by zone electrophoresis using cellulose acetate strips as support was dependent on the concentration of the buffer used (5 mM and 50 mM, pH 7.4) and on the source of the material (chicken liver or guinea-pig liver).

In three different 5 mM buffer systems, pH 7.4 (phosphate, veronal and Tris-HC1) the four lactate dehydrogenase isoenzymes present in chicken liver cytosol: M₃H, M₂H₂, MH₃ and H₄ were resolved into four separated bands. M₃H and M₂H₂ isoenzymes migrated towards the cathode whereas the other two isoenzymes showed anodic mobilities. In 50 mM buffers, pH 7.4 all enzyme activity appeared as a single band with anodic mobility similar to that of H₄. Guinea-pig liver isoenzymes were well resolved in both buffer conditions and appeared as five bands with anodic mobilities.

The different behaviour of the lactate dehydrogenase isoenzymes in 5 mM and 50 mM buffers can not be assigned to ionic strength effects but it may explained by assuming the binding of buffer anions to the different isoenzymes. The binding would increase with the molar concentration of the buffer and reduce charge differences among the isoenzymes to different extents depending on the source of the enzyme, chicken or guinea-pig liver.     

电渗流与缓冲液浓度之间数学关系的线性表达

It was believed that electroosmotic mobility μeo is inversely proportional to the square root of the ionic strength L But the linear relationship for regression analysis was expressed differently in different papers. The paper studied the linear expression of the mathematical relationship between μeo and c （background buffer concentration） by mathematical transform and real experimental data.μeo values of fused silica capillary were determined in four buffer systems. Their experimental conditions were controlled carefully for decreasing temperature difference AT and pH difference ApH in 50 μm ID capillary, in which no double layer overlap existed. The linear relationship between the reciprocal of electroosmotic mobility and the square root of concentration （or ionic strength） was derived by mathematical method. The regression analysis of experimental data was shown to well correspond to the relationship. The constants in regression equation could be well defined and the calculated results were acceptable.     

Separation of etodolac enantiomers by capillary electrophoresis. Validation and application of the chiral method to the analysis of commercial formulations

Separation of etodolac enantiomers, which exhibit different biological activity and pharmacokinetic profiles, has been achieved using the randomly substituted (2-hydroxy)propyl-beta-cyclodextrin (HP-beta-CD) as chiral selector in capillary electrophoresis. The selection of this CD was made after screening of different CD derivatives of neutral and anionic nature. The effect on the enantioresolution of the buffer concentration and of the degree of substitution (DS) and concentration of the CD as well as of instrumental parameters, such as the capillary temperature and the separation voltage, were studied. The highest resolution of etodolac enantiomers was around 2.5 using 100 mM phosphate buffer (pH 7.0) with 20 mM HP-beta-CD (DS approximately 4.2) and UV detection at 225 (10) nm with a reference wavelength at 360 (50) nm. Validation of the chiral method in terms of selectivity, linearity, precision (instrumental repeatability, method repeatability, intermediate precision), and the limits of detection and quantitation allowed to evaluate its quality to the analysis of etodolac enantiomers in different pharmaceutical preparations containing racemic etodolac.