Ultrashort partial-filling technique in capillary electrophoresis for infinite resolution of tramadol enantiomers and its metabolites with highly sulfated cyclodextrins

The possibility to enhance resolution to infinite value in chiral capillary electrophoresis is attained as soon as the apparent mobility of one enantiomer becomes opposite to the other. This could be achieved on the basis of the carrier ability of multiple charged chiral selectors such as highly sulfated cyclodextrin (HS-CD). With tramadol and its phase I metabolites selected as model compounds, the HS-gamma-CD was found to be the most appropriate chiral selector. The CD concentration was determined where one enantiomer still migrated as a cation while the other migrated in the opposite side. Besides the chiral selector concentration, secondary parameters such as buffer concentration appeared to be critical to reach infinite resolution. The latter was achieved with partial filling technique using ultrashort separation zones (a few mm). In order to better understand the interaction mechanism between the selected CD and the analytes, the classical affinity capillary electrophoresis method, although not fully satisfactory because of ionic strength variations within a series of mobility shift measurements, was applied to estimate complexation constants and complex mobilities. The results obtained point to the prevailing role of complex mobility differences in the enantioselectivity mechanism.     

Enantioseparation of chiral N-imidazole derivatives by electrokinetic chromatography using highly sulfated cyclodextrins: 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 by CD-EKC using highly sulfated CDs (alpha, beta, gamma highly S-CDs) as chiral selectors. The influence of the type and concentration of the chiral selectors on the enantioseparations was investigated. The highly S-CDs 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 inferior to 2.5 min and resolution factors R(s) of 3.73, 3.90, 1.40, and 4.35 for compounds 1, 2, 3, and 5, respectively, using 25 mM phosphate buffer at pH 2.5 containing either highly S-alpha-CD, highly S-beta-CD, and highly S-gamma-CD (3 or 4% w/v) at 298 K, with an applied field of 0.30 kV/cm. The determination of the enantiomer migration order for the various analytes and the study of the analyte structure-enantioseparation relationships display the high contribution of the interactions between the analytes phenyl ring and the CDs to the enantiorecognition process. The thermodynamic study of the analyte-CD affinities permits us to improve our knowledge about the enantioseparation mechanism.     

Enantioseparation of new nucleoside analogs, related to d4T and acyclovir, by chiral capillary electrophoresis using highly sulfated beta-cyclodextrins

Baseline separation of some new acyclic nucleosides which are potential antiviral agents was achieved using cyclodextrin capillary zone electrophoresis (CD-CZE). A method for the enantiomeric resolution of these compounds and determination of their enantiomeric purity was developed using anionic CDs (highly sulfated-CD or highly S-CD) as chiral selectors and capillaries, which were dynamically coated with polyethylene oxide (PEO). Operational parameters including (i) the nature and concentration of the chiral selectors, (ii) organic modifiers, (iii) temperature, and (iv) applied voltage were investigated. The use of charged CDs provides (i) a supplementary driving force for the compounds in a running buffer and (ii) enantiomeric resolution by inclusion of compounds in the CD cavity. The highly S-CD was found to be the most effective complexing agent and allowed good enantiomeric resolution. The complete resolution of five nucleoside analogs was obtained using 25 mM phosphate buffer, pH 2.5, containing either highly S-alpha-CD, S-beta-CD or S-gamma-CD at 30 degrees C with an applied field of 0.30 kV/cm. The apparent association constants of the inclusion complexes were calculated. The enantiomer migration order for the molecules investigated was determined and the detection limit of enantiomeric impurities was found to vary between 0.34 to 3.56 ng.mL(-1) for the first enantiomer.     

A novel strategy for the determination of enantiomeric compositions of chiral compounds by chemometric analysis of the UV-vis spectra of bovine serum albumin receptor-ligand mixtures

In this work, a novel strategy was constructed to determine the enantiomeric composition of chiral substances discriminated by bovine serum albumin (BSA) based on the UV-vis spectra of the receptor-ligand mixtures coupled with partial least squares (PLS-1) analysis. Taking tryptophan (Trp) enantiomer as an example, when 20 microM BSA was used, the enantiomeric composition was accurately determined with concentration of only 100 nM and the corresponding enantiomeric excess as high as 98% (or -98%), which is relatively more sensitive than in literature. Furthermore, the BSA-based approach was also used to predict the enantiomeric composition of other chiral compounds, such as phenylalanine (Phe), tyrosine (Tyr), alanine (Ala), cysteine (Cys), DOPA and propranolol (Prop). The results fully demonstrate that BSA is effective in determination of enantiomeric composition of some chiral compounds.     

Investigations of mobile phase contributions to enantioselective anion- and zwitterion-exchange modes on quinine-based zwitterionic chiral stationary phases

Novel chiral stationary phases (CSPs) based on zwitterionic Cinchona alkaloid-type low-molecular mass chiral selectors (SOs), as they have been reported recently, were investigated in HPLC towards effects on their chromatographic behavior by mobile phase composition. Mobile phase characteristics like acid-to-base ratio and type of acidic and basic additives as well as effect of type of bulk solvents in nonaqueous polar organic and aqueous reversed-phase (RP) eluent systems were varied in order to illustrate the variability and applicability of zwitterionic CSPs with regard to mobile phase aspects. Chiral SOs of the five zwitterionic CSPs investigated herein contained weak and strong cation-exchange (WCX, SCX) sites at C9- and C6′-positions of the Cinchona alkaloid scaffold which itself accommodated the weak anion-exchange (WAX) site. The study focused on zwitterion-exchange (ZX) operational mode and chiral amino acids as target analytes. Besides, also the anion-exchange (AX) mode for chiral N-blocked amino acid analytes was considered, because of the intramolecular counterion (IMCI) property available in AX mode. Overall, most general and successful conditions in ZX mode were found to be weakly acidic methanolic mobile phases. In aqueous eluents RP contributions to retention came into play but only at low organic modifier content because of the highly polar character of zwitterionic analytes. At higher acetonitrile content, HILIC-related retention phenomena were observed. When using weakly basic eluent system in AX mode remarkably fast enantiomer separations involving exclusion phenomena were possible with one enantiomer eluting before and the other after void volume.     

Chiral capillary electrophoretic determination of the enantiomeric purity of tetrahydronaphthalenic derivatives, melatoninergic ligands, using highly sulfated beta-cyclodextrins

Using cyclodextrin capillary zone electrophoresis (CD-CZE), baseline separation of synthetic tetrahydronaphthalenic derivatives, potential melatoninergic compounds, was achieved. A method for the enantioresolution of these tetralins and determination of their enantiomeric purity was developed using anionic CDs (highly sulfated-CD or highly S-CD) as chiral selectors and capillaries dynamically coated with polyethylene oxide (PEO). Operational parameters such as the nature and concentration of the chiral selectors, buffer pH, organic modifiers, temperature and applied voltage were investigated. The use of charged CDs provides a driving force for our neutral compounds in the running buffer and enantiomeric resolution by inclusion of compounds in the CD cavity. The highly S-beta-CD was found to be the most effective complexing agent, allowing good enantiomeric resolution. The complete resolution of three tetralin compounds was obtained using 25 mM phosphate buffer at pH 2.5 containing 2.5% w/v of highly S-beta-CD at 25 degrees C with an applied field of 0.25 kV/cm. The apparent association constants of the inclusion complexes were calculated. This optimized method was validated in terms of linearity, sensitivity, accuracy and recovery. The enantiomeric purity for the three molecules was determined and the detection limit of enantiomer impurities is about 0.3-0.6%.     

Retention behaviour of peptides in capillary electrochromatography using an embedded ammonium in dodecacyl stationary phase

A highly water-soluble new cyclodextrin (CD) derivative 2-O-acetonyl-2-O-hydroxypropyl-beta-CD (2-AHP-beta-CD) was synthesized and tested as an effective chiral selector for the capillary zone electrophoretic resolution (Rs) of several basic and acidic analytes. The primary purpose of the research was to explore the capability of the 2-AHP-beta-CD as chiral selectors on comparison with the neutral CDs such as beta-CD, DM-beta-CD and HP-beta-CD. Substitution with 2-O-acetonyl-2-O-hydroxypropyl group at the secondary hydroxyl sites of the CD is aimed at influencing the magnitude and selectivity of analyte-CD interactions. The chiral resolution was strongly influenced by the concentration of the CDs and buffer pH. 2-AHP-beta-CD showed the best enantiomer resolution properties among the tested compounds, while the other CDs showed inferior or no performances at all.     

Use of vancomycin silica stationary phase in packed capillary electrochromatography I. Enantiomer separation of basic compounds

Chiral separation of basic compounds was achieved by using 75 or 100 microm ID fused-silica capillaries packed with a vanoomycin-modified diol silica stationary phase. The capillary was firstly packed for about 12 cm with a slurry mixture composed of diolsilica (3:1) then with the vancomycin modified diol-silica (3:1) (23 cm), and finally with diol-silica (3:1) for about 2 cm. Frits were prepared by a heating wire at the two ends of the capillary; the detector window was prepared at 8.5 cm from the end of the capillary where vancomycin was not present. The influence of the mobile phase composition (pH and concentration, organic modifier type and concentration) on the velocity of the electroosmotic flow, chiral resolution and enantioselectivity was studied. Good enantiomeric resolution was achieved for atenolol, oxprenolol, propranolol, and venlafaxine using a mobile phase composition of 100 mM ammonium acetate solution (pH 6)/water/acetonitrile (5:5:90 v/v/v) while for terbutaline a mixture of 5:15:80 v/v/v provided the best separations. The use of methanol instead of acetonitrile caused a general increase of enantiomer resolution of the studied compounds together with a reduction of efficiency and detector response. However, the combination of acetonitrile and methanol in the mobile phase (as, e.g., 10% methanol and 80% acetonitrile) allowed to improve the enantiomer resolution with satisfactory detector response.     

Kinetic method for the simultaneous chiral analysis of different amino acids in mixtures

The kinetic method has been extended to enantiomeric excess (ee) determinations on amino acids present in mixtures. Singly charged trimeric clusters [Cu(II)(ref*)(2)(A(m)) - H](+) are readily generated by electrospraying solutions containing Cu(II), a chiral reference ligand (ref*), and the amino acids (analytes A(m), m = 1-3). A trimeric cluster ion for each amino acid is individually mass-selected and then collisionally activated to cause dissociation by competitive loss of either the reference ligand or the analyte. For each analyte in the mixture, as shown from separate experiments, the logarithm of the ratio of the fragment abundances for the complex containing one enantiomer of this analyte expressed relative to that for the fragments of the corresponding complex containing the other enantiomer is linearly related to the enantiomeric composition of the amino acid. Formation and dissociation of each trimeric complex ion are shown to occur independently of the presence of other analytes. Chiral selectivity appears to be an intrinsic property and the chiral selectivity R(chiral(m)) measured from the mixture of analytes is equal to R(chiral) measured for the pure analyte. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity, characteristic of the kinetic method, allow the determination of chiral impurities of less than 2% ee in individual compounds present in mixtures by simply recording the ratios of fragment ion abundances in a tandem mass spectrum.     

Determination of the enantiomeric purity of epinephrine by HPLC with circular dichroism detection

Several hundred drug substances approved by the U.S. Food and Drug Administration are chiral molecules. For the enantiomeric purity assessment, current practice is to develop separation techniques using chiral columns or mobile phase modifiers to separate enantiomers before detection. An alternative approach is to use currently accepted HPLC assay methods and use chiral-specific detectors to confirm whether the correct enantiomer is present. In this paper, adding a circular dichroism (CD) detector to an achiral HPLC method from the US Pharmacopeia (USP) is shown to be amenable for the determination of the enantiomeric purity of epinephrine, a substance used to treat anaphylaxis. This HPLC-UV-CD approach was able to detect the inactive D-(+) enantiomer at 1% of the total epinephrine composition. The linearity, accuracy, and precision of HPLC-UV-CD were evaluated and compared to analyses using a chiral HPLC method. Additionally, an epinephrine drug product was analyzed for assay (concentration) and enantiomeric purity. The results from achiral and chiral methods were identical within the experimental error. Overall, achiral chromatography performed using a USP method with CD detection may serve as a general means of determining chiral drug enantiomer purity and avoids the need for the development of additional chiral-specific methods for each individual drug.     

Fast enantioseparation of arylglycine amides by capillary electrophoresis with highly sulfated-beta-cyclodextrin as a chiral selector

Nine racemic arylglycine amides were synthesized and successfully enantioseparated by capillary electrophoresis (CE) using highly sulfated beta-cyclodextrin (HS-beta-CD) as a chiral selector. Baseline enantioseparation of the analytes was obtained around neutral pH but not in the acidic conditions that are commonly used. HS-beta-CD content, buffer pH, type and concentration, and organic modifier concentration were studied and optimized for fast and efficient separation. A chiral CE separation system composed of 1.5% (w/v) HS-beta-CD, 0 to 10% (v/v) methanol and 20 mM 3-(N-morpholino)propanesulfonic acid at pH 6.5 was shown suitable for baseline enantioseparation of the mentioned amides within 6 min, including simultaneous enantioseparation of three positional isomer series (methyl-, methoxyl or chloro-substituted). By using this system, D-enantiomers migrated ahead of the L-enantiomers and the enantiomeric resolution order of arylglycine amides was more or less parallel to the pK(a), order of the analytes.     

手性溶剂-NMR法测定2,4-滴丙酸对映体纯度的定量分析

研究了手性溶剂法测定2,4-滴丙酸的^1H、^13C谱,在满足NMR准确定量所要求的分离度和信噪比的条件下,能准确测定手性化合物的对映体纯度。比较了以对映体百分含量（R％）和对映体过量（ee）表示手性农药的对映体纯度的差别,发现以对映体百分含量代替对映体过量来表示手性农药的对映体纯度更为准确。     

Separation of chiral furan derivatives by liquid chromatography using cyclodextrin-based chiral stationary phases

The enantiomeric separation of a set of 30 new chiral furan derivatives has been achieved on native and derivatized beta-cyclodextrin stationary phases using high performance liquid chromatography (HPLC). The hydroxypropyl-beta-cyclodextrin (Cyclobond RSP), the 2,3-dimethyl-beta-cyclodextrin (Cyclobond DM), and the acetyl-beta-cyclodextrin (Cyclobond AC) stationary phases are the most effective chiral stationary phases (CSPs) for the separation of these racemates in the reverse phase mode. No enantioseparations have been observed on the native beta-cyclodextrin chiral stationary phase (Cyclobond I 2000) and only a few separations have been attained on the S-naphthylethyl carbamate beta-cyclodextrin (Cyclobond SN) and 3,5-dimethylphenyl carbamate beta-cyclodextrin (Cyclobond DMP) chiral stationary phases in the reverse phase mode. The polar organic and the normal phase mode on these CSPs are not effective for separation of these compounds. The characteristics of the analytes, including steric bulk, hydrogen bonding ability, and geometry, play an important role in the chiral recognition process. The pH affects the enantioseparation of compounds with ionizable groups and the addition of 0.5% methyl tert-butyl ether to the mobile phase significantly enhances the separation efficiency for some highly retained compounds.     

Effect of different immobilization strategies on chiral recognition properties of Cinchona‐based anion exchangers

In the enantiomeric separation of highly polar compounds, a traditionally challenging task for high‐performance liquid chromatography, ion‐exchange chiral stationary phases have found the main field of application. In this contribution, we present a series of novel anion‐exchange‐type chiral stationary phases for enantiomer separation of protected amino phosphonates and N‐protected amino acids. Two of the prepared selectors possessed a double and triple bond within a single molecule. Thus, they were immobilized onto silica support employing either a thiol‐ene (radical) or an azide‐yne (copper(I)‐catalyzed) click reaction. We evaluated the selectivity and the effect of immobilization proceeding either by the double bond of the Cinchona alkaloid or a triple bond of the carbamoyl moiety on the chromatographic performance of the chiral stationary phases using analytes with protecting groups of different size, flexibility, and π‐acidity. The previously observed preference toward protecting groups possessing π‐acidic units, which is a typical feature of Cinchona‐based chiral stationary phases, was preserved. In addition, increasing the bulkiness of the selectors’ carbamoyl units leads to significantly reduced retention times, while very high selectivity toward the tested analytes is retained.     

Characterization of highly sulfated cyclodextrins

A class of highly sulfated cyclodextrins (HS-CDs) was developed for enantiomeric separation of chiral compounds by capillary electrophoresis (CE). The HS-CDs were produced by a facile single-step direct sulfation of cyclodextrin using sulfur trioxide-trimethylamine complex in dimethylformamide. Characterization of the HS-CDs by electrospray ionization mass spectrometry and by CE using a well-established indirect detection method indicated the species have very narrow heterogeneity in terms of degree of sulfation. Elemental analysis of the HS-alpha-, beta- and gamma-CDs showed that the average sulfate contents were 11, 12, and 13 per CD molecule, respectively. The 13C NMR of HS-CDs is consistent with the structural assignment of nearly complete sulfation at C-6 primary hydroxyl groups and partial sulfation at the C-2 secondary hydroxyls (>70%), while the C-3 hydroxyls remain unsubstituted. Enantiomeric separation by CE using the HS-CDs as chiral selectors showed that HS-alpha-, beta- and gamma-CDs complement each other by exhibiting different chiral selectivities, resulting in resolution of many chiral neutral, acidic and basic compounds of greatly varying structural features. The part of HS-CD that interacts with the guest molecule during complexation and, therefore, the receiving end of the cyclodextrin hydrophobic bucket was surrounded with largely regiospecifically substituted C-2 sulfates and intact C-3 hydroxyls, both at the equatorial positions. Such global regiospecific structural arrangement in HS-CDs provides differential diasteroisomeric complexation is proposed to be the principal contributing factor in the resolving racemates.     

Peak splitting in the CE separation of enantiomers caused by organic solvents in the sample

Two robust chiral standard separation systems were developed for the analysis of the chiral purity of chemically different model compounds applied in homogeneous asymmetric hydrogenation catalysis. Sulfated CDs were used as chiral selectors as they allow the analysis of neutral, acidic as well as basic compounds in the same electrophoretic system. Poorly water-soluble amines were dissolved in different organic solvent/buffer mixtures. Reproducibly, depending on the amount of organic solvent in the sample solution, peak splitting occurred and/or more peaks than expected were observed, implying impure model compounds. The dependence of the "chiral purity" on experimental parameters, e.g., kind and amount of sample solvent, length of injection plug, inner surface modification of the capillary, kind of sulfated CD, hydrophobicity, and basicity of the analytes, etc. was investigated. It is gathered that different equilibrium constants of the strong binding basic analytes and highly sulfated CD complex in the organic phase of the injection plug and the aqueous electrolyte phase are resulting in two different mobility zones for each enantiomer. It follows that each enantiomer is showing two peaks instead of one. Experimental strategies are shown to avoid these peak splitting/artificial impurity effects and obtain the "real" chiral purity picture of the samples.     

Simultaneous, sequential quantitative achiral-chiral analysis by two-dimensional liquid chromatography

In general, chromatographic analysis of chiral compounds involves a minimum of two methods; a primary achiral method for assay and impurity analysis and a secondary chiral method for assessing chiral purity. Achiral method resolves main enantiomeric pairs of component from potential impurities and degradation products and chiral method resolves enantiomeric pairs of the main component and diastereomer pairs. Reversed-phase chromatographic methods are preferred for assay and impurity analysis (high efficiency and selectivity) whereas chiral separation is performed by reverse phase, normal phase, or polar organic mode. In this work, we have demonstrated the use of heart-cutting (LC-LC) and comprehensive two-dimensional liquid chromatography (LC × LC) in simultaneous, sequential achiral and chiral analysis and quantitation of minor, undesired enantiomer in the presence of major, desired enantiomer using phenylalanine as an example. The results were comparable between LC-LC and LC × LC with former offering better sensitivity and accuracy. The quantitation range was over three orders of magnitude with undesired D-phenylalanine detected at approximately 0.3% in the presence of predominant, desired L-phenylalanine (99.7%). The limit of quantitation was comparable to conventional high-performance liquid chromatography. A reversed-phase C18 achiral column in the primary and reversed-phase Chirobiotic Tag chiral column in the secondary dimension were used with a compatible mobile phase.     

Enantiomeric separation of furan derivatives and fused polycycles by cyclodextrin-modified micellar capillary electrophoresis

The enantiomeric separations of highly hydrophobic furan derivatives and polycycles were performed and optimized using CD-modified micellar CE. The most effective chiral selector for the enantiomeric separation of these analytes was hydroxypropyl-gamma-CD. The effects of CD and SDS concentration and organic modifier were examined in order to optimize the separation conditions. The ratio of CD to surfactant concentration affected the enantiomeric separation significantly, with increases in the derivatized CD concentration generally enhancing resolution. Addition of an organic solvent modifier to the run buffer served to increase the analytes' solubility and enhance the separation efficiency. A highly acidic pH was necessary to effectively suppress the EOF when operating in the reverse polarity mode.     

手性化合物的毛细管胶束电动色谱分离研究

以4种不同的N-长链烷酰-L-氨基酸胶束为手性选择剂,对3种不同性质的手性化合物(α-氯代丙酰替苯胺,2-氨基-3-对硝基苯基-1,2-丙二醇和华法林)的毛细管胶束电动色谱分离进行研究.结果表明,手性表面活性剂中不同的氨基酸残基和烷基链的长度对分离影响较大;随手性表面活性剂浓度增加,溶质保留时间增大,分离度增加,不同溶质的最佳分离浓度在100～150mmol/L之间;pH对电中性手性化合物分离影响不大,但对酸性或碱性手性化合物的分离影响较大.在选定的条件下,3种样品均在20min内完全分离,分离柱效达1×10⁵理论板数/m.     

Rapid stereoselective separations of amphetamine derivatives with highly sulfated gamma-cyclodextrin

The highly sulfated gamma-CD (HS-gamma-CD) is a chiral selector widely used in CE for the enantioseparation of pharmaceutical compounds. This paper investigated different approaches to reduce the stereoselective analysis time of amphetamine (AT) derivatives according to the chiral selector concentration in the BGE. With high HS-gamma-CD concentration, tested analytes were separated in 3.5 min as anionic complexes with short-end injection technique in reversed polarity mode. However, this procedure presented some limitations in terms of efficiency and resolution, excessive Joule heating and poor compatibility with MS detection. With low HS-gamma-CD concentration, compounds were separated as cations. Conventional approaches to reduce CE analysis time demonstrated critical resolution between some analytes. Therefore, the use of the partial-filling technique compatible with MS detection was carried out. Under optimized conditions, the analysis time for the chiral separation of seven AT like compounds was reduced to 6 min. Moreover, sensitivity of CE-MS was sufficient for the determination of ATs in plasma following a simple liquid-liquid extraction.