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.     

Chiral cyclodextrin-modified microemulsion electrokinetic chromatography

Cyclodextrin (CD)-modified microemulsion electrokinetic chromatography (MEEKC) or CD-MEEKC has not previously been applied to the area of chiral separations. Herein, the results of investigations of various microemulsions with CD additives are presented. Two different microemulsions are explored: an ethyl acetate sodium dodecyl sulfate microemulsion, and a chiral dodecoxycarbonylvaline (DDCV) microemulsion. Each microemulsion is paired separately with a neutral CD (hydroxypropyl-beta-CD) and an anionic CD (sulfated-beta-CD). In addition, the chiral DDCV microemulsion is investigated in both the R- and S- form. By varying simple parameters such as buffer system, applied voltage, surfactant enantiomer, and type of cyclodextrin, dramatic improvements in the chiral separations were noted. Resolution was found to be highly dependent on buffer identity and concentration, and somewhat dependent on whether the CDs used were randomly or highly sulfated. Under optimized conditions, the resolution ranged from 0.8 to 4.8, with plate counts ranging from 4000 to 26 000. Additionally, S- and R-levetiracetam, which had never before been enantioseparated via capillary electrophoresis (CE) methodologies, were separated in less than 8 min, with a resolution of 1.1.     

Enantioseparation of phenothiazines in cyclodextrin-modified micellar electrokinetic chromatography

In this study, enantioseparations of five phenothiazines in cyclodextrin (CD)-modified micellar electrokinetic chromatography (MEKC) were investigated using a citrate buffer containing tetradecyltrimethylammonium bromide (TTAB) as a cationic surfactant at low pH. Beta-cyclodextrin (beta-CD) and hydroxylpropyl-beta-CD (HP-beta-CD) were selected as chiral selectors. The results indicate that the separation window is greatly enlarged by beta-CD concentration and that the separability and selectivity of phenothiazines are remarkably influenced by the concentrations of both beta-CD and TTAB, as well as buffer pH. The interaction of thioridazine with beta-CDs is considerably reduced in the presence of TTAB micelles due to competitive complexation of thioridazine with TTAB micelles, which is pH-dependent. As a result, effective enantioseparation of thioridazine is simultaneously achievable with that of trimeprazine and promethazine or ethopropazine in MEKC with addition of either beta-CD or HP-beta-CD, respectively, to a micellar citrate buffer containing TTAB at pH 3.5. Better enantioresolution of thioridazine in MEKC than in capillary zone electrophoresis can be obtained.     

On-line preconcentration and chiral separation of propiconazole by cyclodextrin-modified micellar electrokinetic chromatography

A method for the chiral separation of propiconazole using cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) with hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) as chiral selector is reported. The use of a mixture of 30 mM HP-gamma-CD, 50mM SDS, methanol-acetonitrile 10%:5% (v/v) in 25 mM phosphate buffer solution was able to separate two enantiomeric pairs of propiconazole. Stacking- and sweeping-CD-MEKC under neutral pH (pH 7) and under acidic condition (pH 3.0) were used as two on-line preconcentration methods to increase detection sensitivity of propiconazole. Good repeatabilities in the migration time, peak area and peak height were obtained in terms of relative standard deviation (RSD). A sensitivity enhancement factor of 100-fold was achieved using sweeping-CD-MEKC at acidic pH. This is the first report on the separation of two pairs of propiconazole enantiomers and all the enantiomers of fenbuconazole and tebuconazole using sweeping-CD-MEKC. The limit of detection (S/N=3) for the three triazole fungicides ranged from 0.09 to 0.1 microg/mL, which is well below the maximum residue limits (MRL) set by Codex Alimentarius Commission (CAC). Combination of solid-phase extraction (SPE) pretreatment and sweeping-CD-MEKC procedure was applied to the determination of selected triazole fungicides in grapes samples spiked at concentration 10-40 times lower than the MRL established by the CAC. The average recoveries of the selected fungicides in spiked grapes samples were good, ranging from 73% to 109% with RSD of 9-12% (n=3).     

Enantioseparation of chiral benzimidazole derivatives by electrokinetic chromatography using sulfated cyclodextrins

Baseline separation of 18 new substituted benzimidazole derivatives, potent AMP‐activated protein kinase (AMPK) activators, with one chiral center, was achieved by CD‐EKC using sulfated and highly sulfated CDs (SCDs and HS‐CDs) as chiral selectors. The influence of the type and concentration of the chiral selectors on the enantioseparations was investigated. The SCDs exhibit a very high enantioselectivity power since they allow excellent enantiomeric resolutions compared to those obtained with the neutral CDs. The enantiomers were resolved with analysis times around 6 min using 25 mM phosphate buffer at pH 2.5 containing either β‐S‐CD, HS‐β‐CD, HS‐γ‐CD (3 or 4% w/v) at 25°C, with a voltage of 20 kV. The apparent association constants of the inclusion complexes were calculated. The study of the solute structure‐enantioseparation relationships seems to show the high contribution of the interactions between the solutes phenyl ring and the CDs to the enantiorecognition process. The optimized method was briefly validated (LOD less than 1%) and the purity of enantiomers of compound 3 was determined. The enantiomer migration shows reversal order depending on the kind of CD.     

Enantioseparation of chiral allenic acids by micellar electrokinetic chromatography with cyclodextrins as chiral selector

Enantioseparation of chiral aryl allenic acids by micellar electrokinetic chromatography (MEKC) with cyclodextrins (CDs) as chiral selectors was described. The screen of chiral selectors (beta-CD, gamma-CD, and hydroxypropyl (HP)-gamma-CD) showed that the enantioseparation was not only dependent on the type of CD but also the presence of 2-propanol in the buffer. In order to optimize the operational parameters, the effect of the concentration of CDs, sodium dodecyl sulfate (SDS), and 2-propanol, as well as the buffer ionic strength and pH on enantioseparation were studied. It was proved that the concentration of CDs, 2-propanol, and the buffer ionic strength were the critical parameters. Under optimal conditions, baseline separations of all seven allenic acid enantiomers were achieved. Furthermore, the method validation in terms of repeatability, linearity, limit of detection (LOD), and limit of quantitation (LOQ) were performed. Using the present method, the optical purity of a nonracemic sample with the enantiomeric excess (e.e.%) value of 99.65% was determined.     

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.     

Effect of oil substitution in chiral microemulsion electrokinetic chromatography

In a previous publication (Pascoe, R., Foley, J. P., Analyst 2002, 127, 710-714), a novel chiral microemulsion based on 1.0% w/v dodecoxycarbonylvaline (DDCV), 0.50% v/v ethyl acetate and 1.2% v/v 1-butanol, was shown to provide rapid enantiomeric separations of various pharmaceutical compounds. The two deficiencies noted with this method were that the peak shapes obtained were asymmetric and the efficiencies were lower than those previously obtained using DDCV micelles (Peterson, A. G., Ahuja, E. S., Foley, J. P., J. Chromatogr. B 1996, 683, 15-28). This study examines the use of three alternative low-interfacial-tension oils (methyl acetate, methyl propionate, and methyl formate), in combination with DDCV, to characterize their effect on the elution range, efficiency, resolution, and enantioselectivity of various pharmaceutical enantiomers. The oils were evaluated in both the same volume percentage and the same molar concentration as ethyl acetate in the original DDCV microemulsion system. Including ethyl acetate, a total of seven microemulsion systems were examined. For the compounds that were separated, average enantioselectivities ranged from 1.09 to 1.28, with corresponding efficiencies of 14,000-20,000. While some interesting differences were observed, ethyl acetate still proved to be the most advantageous in terms of enantioselectivity, resolution, and elution range.     

Enantioseparation of chiral thiobarbiturates using cyclodextrin-modified capillary electrophoresis

The racemates of several chiral thiobarbiturates were separated by using different cyclodextrins in capillary electrophoresis (CE). Six neutral and negatively charged cyclodextrins 1 (CDs) were employed as chiral separators whereof five led to successful separation of enantiomeric thiobarbiturate pairs. The CDs used were the native alpha-CD, beta-CD, gamma-CD, and heptakis-(2,6-di-O-methyl)-beta-cyclodextrin (HDM) as well as heptakis-(2,3-di-O-methyl-6-sulfato)-beta-cyclodextrin (HDMS) and heptakis-(2,3-di-O-acetyl-6-sulfato)-beta-CD (HDAS). Five of the six chiral thiobarbiturates studied could be resolved at a basic pH value of 9.4 and a phosphate buffer concentration of 100 mM in a fused-silica capillary. Structurally related substances showed a similar behavior in separation: 1 and 2 bearing the center of chirality in the side chain at C5 can be best separated using gamma-CD, the N-alkyl-substituted compounds 3 and 4 as well as the N/S-dialkyl-substituted compound 5 could be resolved with HDM. Using the neutral CDs, the migration times were relatively small (< 11 min). 3 and 4 could be also resolved by means of the negatively charged HDMS. In the latter case, the migration time is twice as long as with HDM.     

Analyses of tobacco alkaloids by cation-selective exhaustive injection sweeping microemulsion electrokinetic chromatography

In this study, an on-line concentration method which coupled cation-selective exhaustive injection (CSEI) sweeping technology with microemulsion electrokinetic chromatography (MEEKC) was used to detect and analyze several tobacco alkaloids (nornicotine, anabasine, anatabine, nicotine, myosmine and cotinine) that are commonly found in various tobacco products. First, the effects of microemulsion compositions (oil, cosurfactant and solution pH) were examined in order to optimize the alkaloid separations in conventional MEEKC. The pH value and the injection length of basic plug were found to be the predominant influences on the alkaloid stacking. This optimal CSEI sweeping MEEKC method provided approximately 180- to 540-fold increase in detection sensitivity in terms of peak height without any loss in separation efficiency when compared to normal MEEKC separation. Furthermore, this proposed CSEI sweeping MEEKC method was applied successfully for the detection of the minor alkaloids nornicotine, anabasine and anatabine in tobacco products.     

Determination of the chiral and achiral related substances of methotrexate by cyclodextrin-modified micellar electrokinetic chromatography

A cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method for the determination of the most important potential impurities of methotrexate (MTX): 2,4-diamino-6-(hydroxymethyl)pteridine, aminopterine hydrate, 4-[N-(2-amino-4-hydroxy-6-pteridinylmethyl)-N-methylamino] benzoic acid, 4-[N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino] benzoic acid, and the distomer D-MTX is presented. The MEKC separation of these compounds was optimized by applying a step-by-step approach. The addition of beta-CD to a conventional MEKC system, based on sodium dodecyl sulfate (SDS) as surfactant, showed to be essential for the enantioresolution of racemic MTX as well as for the separation of the achiral impurities. To achieve high-resolution factor between the peaks adjacent to the main component (L-MTX), as required in the analysis of related impurities, the separation conditions were stressed; in particular, the addition of methanol to the CD-MEKC system resulted in a very effective choice. Under the optimized final conditions (100 mM SDS and 45 mM beta-CD in a mixture of 50 mM borate buffer, pH 9.30-methanol (75:25 v/v)), the method was validated showing a general adequate accuracy (93-106% recovery) in the determination of L-MTX related substances at the impurity level of 0.12% w/w with a relative standard deviation (RSD)% lower than 8% (n = 4). The method was successfully applied to the analysis of pharmaceuticals (tablets and injections) which showed to contain the distomer D-MTX as major impurity and aminopterine hydrate as a further related substance in the commercial tablets.     

Temperature effects on chiral microemulsion electrokinetic chromatography employing the chiral surfactant dodecoxycarbonylvaline

Dodecoxycarbonylvaline (DDCV) microemulsions (1% and 4%, w/v) were employed to evaluate the retention mechanism of a series of enantiomers over a temperature range of 15-35 degrees C. From the acquired retention data, van't Hoff plots were constructed and enthalpy and entropy of transfer were calculated from the slope and intercept, respectively. Resolution, enantioselectivity, distribution coefficients and Gibb's free energy were also calculated, as well as between enantiomer differences in enthalpy, entropy and Gibb's free energy. Finally, comparisons were made between the microemulsion thermodynamic data and a corresponding set of micellar data. While the 4% DDCV microemulsion did not provide a linear van't Hoff relationship, the 1% DDCV microemulsion was linear over a temperature range of 15-30 degrees C. For the 1% DDCV microemulsion, the enthalpic contribution to retention was consistently favorable (deltaH < 0), whereas the entropic contribution varied from compound to compound. Finally, while the achiral attraction of the analytes was greater for the micellar phase, the microemulsion seemed to provide a suitable difference in entropy (and Gibb's free energy) between enantiomers to achieve chiral discrimination.     

Enantioseparation of linear and cyclic chiral bis(phenethyl)amines by means of cyclodextrin-modified capillary electrophoresis

For two years drugs introduced to the market have had- to be enantiomerically pure. Rapid and cheap methods of high reproducibility must, therefore, be available for evaluation of enantiomeric purity. Within the framework of a larger project dealing with chiral recognition of phenethylamines by means of native and derivatized cyclodextrins it was intended to find capillary electrophoresis methods suitable for separation of the enantiomers of chiral bis(phenethyl)amines and their corresponding cyclic analogues, within 10 min, using small amounts of a chiral selector, to save time and money. Heptakis(2,3-O-diacetyl-6-sulfato)beta-CD was found to be the most promising candidate most often fulfilling these requirements.     

Enantioseparation of palonosetron hydrochloride by micellar electrokinetic chromatography with sodium cholate as chiral selector

The enantioseparation of four stereoisomers of palonosetron hydrochloride by micellar electrokinetic chromatography using sodium cholate as chiral surfactant was described. Sodium cholate was shown to be effective in separating palonosetron hydrochloride stereoisomers. For method optimization, several parameters such as sodium cholate concentration, buffer pH and concentration, the types and concentration of organic modifiers and applied voltage, on the enantioseparation were evaluated and the optimum conditions were obtained as follows: 30 mM borate buffer (pH 9.40) containing 70 mM sodium cholate and 20% (v/v) methanol with an applied voltage of 20 kV. Under these conditions, baseline separation of palonosetron hydrochloride stereoisomers was achieved within 18 min.     

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     

Separation of enantiomers in microemulsion electrokinetic chromatography using chiral alcohols as cosurfactants

A novel chiral microemulsion, which involved the use of chiral alcohols as cosurfactants, was demonstrated for the enantiomeric separation of a number of pharmaceutical drugs in microemulsion electrokinetic chromatography (MEEKC). The chiral alcohols investigated were optically active 2-alkanols, with the alkyl chain length having carbon number ranging from 4 to 7. The data indicated that, except for R-(-)-2-butanol, the use of R-(-)-2-pentanol, R-(-)-2-hexanol or R-(-)-2-heptanol as the chiral cosurfactant resulted in the baseline or partial resolution of most of the test solutes, i.e., (+/-)-norephedrine, (+/-)-ephedrine, DL-nadolol, and DL-propranolol. In addition to the chain length of the chiral 2-alkanols, the effects of other experimental conditions, such as the concentration and chirality of the 2-alkanols, as well as the pH of the run buffer and the oil phase of the microemulsion, on the enantiomeric separation of the test solutes were also investigated. An interesting finding was that the water-immiscible organic solvent (oil core) within the microemulsion droplets appeared to play an important role in the chiral separation mechanism. Also, the importance of hydrogen bonding between the test solutes ((+/-)-ephedrine and related compounds) and the chiral microemulsion was demonstrated, as it was not possible to resolve a pair of enantiomers which lacked a beta-amino proton (i.e., (+/-)-N-methyl ephedrine) under optimized run buffer conditions (e.g., 5.0% R-(-)-2-hexanol, 0.8% n-octane, and 3.5% SDS in 90.7% borate buffer at pH 9.2).     

结合阳离子选择性耗尽-扫集的微乳毛细管电泳法测定吴茱萸生物碱

本文建立了一种毛细管在线富集技术,通过将阳离子选择性耗尽(CSEI)-扫集(sweeping)技术与微乳毛细管电泳相结合,实现了吴茱萸中四种生物碱(1.吴茱萸碱;2.吴茱萸次碱;3.吴茱萸新碱;4.1-甲基-2-十一烷基-4(1H)-喹诺酮)的同时定量检测和分析。通过表面活性剂、有机改性剂、p H等条件的考察得到最佳微乳体系组成为:100 mmol·L-1SDS,1%(体积比)乙酸乙酯,15%(体积比)正丁醇,15%(体积比)乙腈,2.5%(体积比)甲酸,p H 2.00。通过基底缓冲p H和长度、水塞长度考察,CSEI-sweeping最佳富集条件为,水塞0.5psi水动引入2 s,p H 9.00基底缓冲0.5 psi水动引入90 s,+10 k V电动进样90 s。富集倍数达到了8~30倍。将此方法应用于实际样品中四种生物碱的定量,获得满意结果。研究结果表明,所建立CSEI-sweeping与MEEKC联用法能快速、准确地应用于吴茱萸四种生物碱的定量分析。     

Effect of microemulsion component purity on the chromatographic figures of merit in chiral microemulsion electrokinetic chromatography

Numerous combinations of one-, two-, and three-chiral-component microemulsions have been previously prepared in our group, using N-dodecoxycarbonylvaline (DDCV), 2-hexanol, and ethyl acetate, dibutyl tartrate, or diethyl tartrate. A few results of the various formulations investigated suggested the possible presence of minor impurities in one or more components of the microemulsion. In this study, the purity of the current lots of R- and S-surfactant were measured, as was the subsequent effect of minor impurities on the relevant chromatographic figures of merit (CFOMs) that describe a chiral separation, i.e., efficiency, enantioselectivity, retention, migration window (elution range), and resolution. Two related methods are proposed for correcting enantioselectivities measured in the presence of chiral impurities in the chiral microemulsion.