Simultaneous separation of sixteen positional and optical isomers of the tryptophan family by ligand-exchange micellar electrokinetic chromatography

Summary Ligand-exchange micellar electrokinetic chromatography has been used for the simultaneous separation of 16 positional and optical isomers of the tryptophan family. The Cu(II) complex with L-hydroxyproline was used as the chiral selector. Two groups of anionic surfactant, linear alkylbenzenesulfonates (LAS) and straight-chain alkyl sulfates such as sodiumn-decyl sulfate (SDeS), sodium dodecyl sulfate (SDS) and sodiumn-tetradecyl sulfate (STS), were used for simultaneous separation. The best result was obtained by use of SDS. The influence of SDS concentration and of pH on the separation was investigated. The separation behavior in the absence of the Cu(II) complex with L-hydroxyproline was also examined. Use of organic modifiers causes the resolution to decrease. Relationships between the logarithm of the octanol-water partition coefficient (logP _OW) and resolution, and between logP _OW and migration time are discussed.     

Chiral ligand exchange micellar electrokinetic chromatography using borate anion as a central ion

Three compounds having 1,2-diol structure (1-phenyl-1,2-ethanediol, 3-phenoxy-1,2-propanediol, and 3-benzyloxy-1,2-propanediol) were enantioseparated by ligand exchange MEKC using (5S)-pinanediol (SPD) as a chiral selector and borate anion as a central ion together with SDS. When (S)-1,2-propanediol, (S)-1,2,4-butanetriol, or (S)-3-tert-butylamino-1,2-propanediol were used as the chiral ligand instead of SPD, these three compounds were not enantioseparated. When borate was replaced with 2-aminoethane-1-sulfonate or N-cyclohexyl-3-aminopropanesulfonate, no chiral separation was achieved. Therefore, the hydrophobic interaction between the chiral selector and the chiral analytes within the transient diastereomeric complex may play an important role in the enantioseparation achieved by the proposed method.     

Chiral separation of raltitrexed by cyclodextrin-modified micellar electrokinetic chromatography

A rapid and effective method was developed for the chiral separation of raltitrexed (RD) enantiomers by carboxymethyl-beta-cyclodextrin (CM-β-CD)-modified micellar electrokinetic chromatography (MEKC). Optimization of conditions including the type and concentration of the chiral selector, concentration of sodium dodecyl sulfate (SDS), pH and concentration of the background electrolyte (BGE), capillary temperature, and applied voltage was investigated. The enantiomers of raltitrexed could be separated with satisfactory resolution and linear response by using 75 mM Tris-phosphate at pH 8.0 containing 30 mM SDS and 8 mM CM-β-CD as buffer system. Furthermore, the usefulness of this method was demonstrated in a purity test of a real synthetic drug sample. Figure Chiral separation of raltitrexed by CM-β-CD MEKC was optimized and applied to test the purity of a synthetic drug sample     

S-trityl-(R)-cysteine, a powerful chiral selector for the analytical and preparative ligand-exchange chromatography of amino acids

S-trityl-(R)-cysteine [(R)-STC] is the new selector of a dynamically coated, chiral ligand-exchange stationary phase which proved to be highly effective in both analytical and preparative-scale separation of enantiomers of some natural and unnatural underivatized amino acids, with good separation and resolution factors. With the aim of identifying the best chromatographic conditions suitable for the preparative-scale separations, some parameters controlling retention, separation and resolution factors (such as the type and amount of cupric salt and the eluent pH) were investigated. The relatively easy removal of the Cu(II) ions renders this technique suitable for obtaining small amounts of enantiomerically pure samples for preliminary biological evaluations.     

Optimization of the separation of flavonoids using solvent-modified micellar electrokinetic chromatography

In this work the separation of eighteen flavonoids was attempted using reduced-flow micellar elektrokinetic chromatography (RF-MEKC) electrolytes modified by selected solvents with differing properties: methanol (MeOH), acetonitrile (ACN) and tetrahydrofuran (THF). Structural aspects such as unsaturation of the C ring, number and position of OH groups, methylation and glycosylation as well as solvent effects and their impact on the electrophoretic behavior of flavonoids were addressed. By evaluating the electropherograms obtained from mixture-designed electrolytes and searching for changes in the critical pairs, a favorable separation condition was achieved using 20 mmol/L phosphate buffer at pH 2.5 containing 50 mmol/L sodium dodecyl sulfate (SDS), 15% ACN and 5% THF (one critical pair) in less than 12 min with 1.5% coefficient of variation (CV) for retention factor and 3% CV for peak area (n = 5). The applicability of the proposed separation condition was demonstrated by the inspection of flavonoids in herbal extracts of Neem.     

Validated chiral separation of M9, a Mannich ketone compound, by chiral ligand-exchange chromatography

Enantioseparation of the Mannich ketone M9, a potential antifungal compound, was examined using chiral ligand-exchange chromatography. The chiral mobile phase contained complexes of Cu(II) with the optically active selector L-aspartame (APM) and the organic modifier methanol. The separation was optimized with respect to the concentration of the Cu(II)-(L-APM) complexes, pH of mobile phase, methanol content, and column temperature. A baseline separation (R(s) = 3.08) was achieved for enantiomers of M9 under optimal conditions, and the analysis was accomplished in eleven minutes. The developed method was extensively validated. The sample stability, linearity, precision (method repeatability and intermediate precision) and accuracy, and the limits of detection and quantification of the developed method were studied. The proposed method was shown to be accurate and suitable for the quantitative determination of each enantiomer of M9.     

Enantiomeric Separation of Epinephrine and Salbutamol by Micellar Electrokinetic Chromatography Using β-Cyclodextrin as Chiral Additive

Introduction The development of chiral substances, especially inthe pharmaceutical field, places increasing demands onanalytical methods for the separation of these kinds ofisomers and the chiral purity control of drugs in phar-macokinetic studies. As the enantiomers of epinephrineand salbutamol have different pharmacological andtoxicological characteristics, separation and quantitationof the single enantiomers are required. Analytical methods used so far for the enantiomerseparation inclu…     

Chiral separation of trimetoquinol hydrochloride and related compounds by micellar electrokinetic chromatography using sodium taurodeoxycholate solutions and application to optical purity determination

The chiral separation of trimetoquinol hydrochloride, which is a bronchodilator (Inolin), and three related compounds by micellar electrokinetic chromatography was investigated using a bile salt as a chiral surfactant. Enantiomers of these compounds, except laudanosoline, were successfully separated within 12 min using a separation tube of effective length 500 mm × 0.05 rum i.d. and a 0.05 M sodium taurodeoxycholate solution of pH 7.0. The observed theoretical plate numbers of the peaks were ca. 150000. Chiral recognition was affected by the structure of bile salts, the pH of the buffer solutions used and the structure of the solutes. Of four kinds of bile salts, successful chiral separation was achieved only using sodium taurodeoxycholate solution under neutral conditions. The method was applied to the optical purity determination of trimetoquinol hydrochloride. The effects of surfactant concentrations and some additives to the micellar solution are briefly described.     

Chiral separation based on ligand-exchange capillary electrophoresis using a copper(II)-L-ornithine ternary complex as selector

A ligand-exchange capillary electrophoresis was explored, with L-ornithine as the ligand and copper(II) as the central ion. Its applicability was demonstrated with underivatized and dansyl amino acids, a dipeptide, and drugs with amino alcohol structure. The enantioselectivity was found to be strongly dependent on pH and copper(II)-L-Orn complex concentration. Due to the adsorption of the positively charged species onto the capillary inner walls, the chiral separation selectivity is very high while the efficiency is relatively low. Permanent 1,3-propanediamine-coated capillaries show an improved separation efficiency and theoretical plate numbers increasing from 10(4) to 10(5). Similar phenomena were observed when sodium dodecyl sulfate (SDS) micelles were added to the copper(II) complex solution. The poor separation efficiency of chiral compounds in uncoated capillaries may result from the low rate of the ligand-exchange reactions, and the high enantioselectivity may derive from the complexing process in the adsorbed phase.     

Cyclodextrin-modified micellar electrokinetic chromatography for the analysis of Esterom,a topical product consisting of hydrolyzed benzoylecgonine in propylene glycol

Esterom, a new drug currently in human clinical trials, is a mixture of compounds in a propylene glycol vehicle. It is being evaluated as a topical treatment to aid in the relief of muscle pain and to increase range of motion. Benzoylecgonine is the major component of Esterom and there are at least nine other minor constituents, including four hydroxypropyl esters that have multiple diasteriomers. The aim of the study was to develop a capillary electrophoresis method for the simultaneous separation of the main components in Esterom, including the multiple proposed diastereomers of the esters. Due to the complex sample composition, the use of micelles and cyclodextrins as buffer modifiers was evaluated. A cyclodextrin-modified micellar electrokinetic chromatography method was able to determine 7 of the 8 UV-active Esterom components, with baseline separation of 7 of the 10 diastereomers of the hydroxypropyl esters.     

Mixed micellar electrokinetic capillary chromatography separation of depolymerized grape procyanidins

Oligomeric procyanidins are potent antioxidant polyphenols of potential interest as disease-preventing agents. Their efficiency depends on the size and composition of their oligomeric structures. The mean degree of polymerization of these compounds is usually estimated by thiolysis with thiol-alpha-toluene followed by analysis using high-performance liquid chromatography (HPLC). We show the development of a mixed micellar electrokinetic chromatography (MEKC) method for the separation of the major components obtained after thiolysis with cysteamine (catechins and their cysteamine conjugates). MEKC studies using sodium dodecyl sulfate (SDS as pseudostationary phase led to long migration times, e.g., with 100 mM SDS, at pH 7, the solutes were separated in about 40 min), while the use of sodium cholate (SC) produced an elution window relatively short. Using a mixed micellar SC-SDS system (50 mM phosphate at pH 7 containing 40 mM SC and 10 mM SDS), it is possible to separate these compounds in less than 15 min. The proposed method is useful to separate the major components of the thiolysate in effluents from food processing (e.g., skins and seeds from grape and apple) considered as potential procyanidin sources.     

Non-ionic surfactant modified ligand exchange chromatography using copper (II) complex of N,N-dimethyl-l-phenylalanine as the chiral additive for enantioselective amino acids separation

The influence of non-ionic surfactants on the selectivity and retention in the ligand exchange chromatography for the enantioselective separation of racemic mixtures of the amino acids dl-methionine, dl-leucine, dl-valine and dl-tyrosine applying chiral mobile phases was investigated, whereas five different surfactants were tested as modifiers. The experiments were carried out using a commercially available non-chiral RP-C8 column and the copper (II) complex of N,N-dimethyl-l-phenylalanine as the chiral additive. Varying the surfactant concentrations the retention factors and the selectivity could be controlled and in general no negative influence on the separation (due to surfactant adsorption on the non-chiral stationary phase) occurred. Changing the temperature the van’t Hoff plots were obtained and the thermodynamic parameters calculated. Temperature had influence on the selectivity for each surfactant and lowered the retention times as expected.     

Enantioseparation of nuarimol by affinity electrokinetic chromatography-partial filling technique using human serum albumin as chiral selector

The present paper deals with the enantiomeric separation of nuarimol enantiomers by affinity EKC-partial filling technique using HSA as chiral selector. Firstly, a study of nuarimol interactions with HSA by CE-frontal analysis was performed. The binding parameters obtained for the first site of interaction were n(1) = 0.84; K(1) = 9.7 +/- 0.3x10(3 )M(-1) and the protein binding percentage of nuarimol at physiological concentration of HSA was 75.2 +/- 0.2%. Due to the moderate affinity of nuarimol towards HSA the possibility of using this protein as chiral selector for the separation of nuarimol using the partial filling technique was evaluated. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and plug length was carried out. Separation of nuarimol enantiomers was obtained under the following selected conditions: electrophoretic buffer composed of 50 mM Tris at pH 7.3; 160 muM HSA solution applied at 50 mbar for 156 s as chiral selector; nuarimol solutions in the range of 2-8x10(-4) M injected hydrodynamically at 30 mbar for 2 s and the electrophoretic runs performed at 30 degrees C applying 15 kV voltage. Resolution, accuracy, reproducibility speed and cost of the proposed method make it suitable for quality control of the enantiomeric composition of nuarimol in formulations and for further toxicological studies. The results showed a different affinity between nuarimol enantiomers towards HSA.     

Enantioseparation of esbiothrin by cyclodextrin‐modified microemulsion and micellar electrokinetic chromatography

A comparison between chiral cyclodextrin‐modified microemulsion electrokinetic chromatography (CD‐MEEKC) and cyclodextrin‐modified micellar electrokinetic chromatography (CD‐MEKC) for the enantiomeric separation of esbiothrin was carried out. For both methods, the separation conditions were optimized by varying CD types and concentration, running buffer pH and compositions, organic modifiers, and temperature. The optimal CD‐MEEKC conditions were 0.8% n‐heptane, 2.3% SDS, 6.6% n‐butanol, 90.3% 10 mM sodium tetraborate containing 3% (w/v, the ratio of CD mass to microemulsion volume) methyl‐β‐cyclodextrin, pH 10, 25°C. The optimized CD‐MEKC conditions were 3.3% SDS, 96.7% 10 mM sodium tetraborate containing 5% (w/v) β‐CD, pH 10, 25°C. The difference in physicochemical properties of the buffer and CDs resulted in different optimal CD type. The competitive distribution between the microemulsion (or micelle) and chiral CD contributed to the chiral separation. Both methods provided excellent separation (R_s ～? 3) with similar migration time (ca. 15 min). CD‐MEEKC provided higher separation efficiencies (>300000) than CD‐MEKC (>200000). The LODs for CD‐MEEKC and CD‐MEKC were 4.7 μg/mL and 3.2 μg/mL, respectively. The RSDs of migration time and peak area for CD‐MEEKC were slightly higher than for CD‐MEKC. Both the demonstrated CD‐MEEKC and CD‐MEKC methods provided high efficiencies, low LODs, and reproducible enantioseparations of esbiothrin.     

Multiple dual-mode countercurrent chromatography applied to chiral separations using a (S)-naproxen derivative as chiral selector

Countercurrent chromatography (CCC) is a liquid–liquid chromatographic technique without a solid support. Several alternative elution modes can be applied to take advantage of the special nature of the liquid stationary phase. Among these dual-mode (DM) and multiple dual-mode (MDM) consist of switching alternatively between Reversed and Normal Phase operation during the experiment (once for DM and several times for MDM). In this paper, MDM has been applied to the chiral CCC separations of two racemic mixtures, (±)-N-(3,4-cis-3-decyl-1,2,3,4-tetrahydrophenanthren-4-yl)-3,5-dinitrobenzamide and N-(3,5-dinitrobenzoyl)-(±)-leucine, using (S)-naproxen N,N-diethylamide as chiral selector (CS). Although the behaviour of the two analytes differed, improved resolution factors were successfully obtained. Results are rationalized on the basis of the distinct partition behaviour of the CS/enantiomer complexes in the biphasic system.     

离子液体对胶束电动色谱胶束微结构以及分离效果的影响

考察了离子液体对胶束电动色谱胶柬微结构以及分离效果的影响.研究结果表明,离子液体使胶束的表面电荷密度变小、粒径变大及其内核极性增大.以泼尼松、氢化可的松和泼尼松龙为分析对象,氢化可的松与泼尼松龙在十二烷基硫酸钠(SDS)胶束体系中不能实现分离.而在SDS-离子液体混合介质(20 mmol/L SDS-10 mmol/L...     

Separation of some chiral flavonoids by microbial cyclosophoraoses and their sulfated derivatives in micellar electrokinetic chromatography

Neutral cyclosophoraoses (Cys) and highly sulfated cyclosophoraoses (HS-Cys) were successfully applied as chiral selectors with SDS for the separation of some chiral flavonoids in MEKC. HS-Cys were synthesized by the chemical modification of a family of neutral Cys isolated from a soil microorganism, Rhizobium meliloti 2011. Chiral catechin was separated with a resolution (R(s)) of 0.754 by neutral Cys and SDS. In the case of isosakuranetin and neohesperidin, resolution (R(s)) values of 1.483 and 1.306 were obtained with HS-Cys and SDS, respectively.     

Chiral separation of brompheniramine enantiomers by recycling high‐speed countercurrent chromatography using carboxymethyl‐β‐cyclodextrin as a chiral selector

A recycling high‐speed countercurrent chromatography protocol was proposed for the enantioseparation of brompheniramine by employing β‐cyclodextrin derivatives as a chiral selector. The two‐phase solvent system of n‐hexane/isobutyl acetate/0.10 mol/L phosphate buffer solution with a volume ratio of 2:4:6 was selected by a series of extraction experiments. Factors that affected the distribution of the enantiomers over the two‐phase system (e.g., the type and concentration of β‐cyclodextrin derivatives = pH value of the aqueous solution, and the separation temperature) were also investigated. In addition, the theory of thermodynamics is applied to verify the feasibility of the enantioseparation process and the corresponding results demonstrate that this separation process is feasible. The optimized conditions include carboxymethyl‐β‐cyclodextrin concentration of 0.010 mol/L, pH of 7.5, and temperature of 5°C. Under the optimal conditions, the purities of both monomer molecules were over 99%, and the recovery yields were 88% for (+)‐brompheniramine and 85% for (–)‐brompheniramine, respectively.     

Chiral ligand-exchange chromatography on an RP HPLC column coated with a new chiral selector derived froml-spinacine

Summary A commercial reversed-phase (RP) C₁₈ HPLC column has been dynamically coated with the chiral selectorN ^τ-n-decyl-l-spinacine and then loaded with copper(II) ions. Several racemic mixtures of underivatized amino acids and oligopeptides were resolved on the column by chiral ligand-exchange chromatography. The most important experimental conditions affecting column efficiency, retention, and selectivity (temperature and mobile phase flow rate and composition) were extensively investigated.