Chiral separation of rasagiline using sulfobutylether‐β‐cyclodextrin: capillary electrophoresis,NMR and molecular modeling study

Pressure‐assisted stereospecific capillary electrophoresis method was developed for the determination of enantiomeric purity of the antiparkinsonian agent (R)‐rasagiline. The optimized method, 50 mM glycine‐HCl buffer pH 2, supplied with 30 mM sulfobutylether‐β‐cyclodextrin, at 35°C, applying 12 kV in reversed polarity, and –8 mbar pressure (vacuum), short‐end injection with ‐25 mbar × 2 s, was successful for baseline separation of rasagiline enantiomers (R_s = 3.5 ± 0.1) in a short analysis time. The method was validated according to current guidelines and proved to be reliable, linear, precise and accurate for determination of 0.15% S‐enantiomer as chiral impurity in R‐rasagiline sample, as well as quantification of the eutomer. Method application was tested on a commercial tablet formulation. Determination of spatial structure of diastereomeric associates was based on ¹H and 2D ROESY NMR, indicating that the aromatic moiety of the molecule can enter the cyclodextrin cavity. NMR titration and molecular modeling revealed that S‐rasagiline formed a more stable inclusion complex with sulfobutylether‐β‐cyclodextrin, than its antipode, which is in agreement with electrophoretic results.     

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.     

Evaluation of the separation mechanism of electrokinetic chromatography with a microemulsion and cyclodextrins using NMR and molecular modeling

Electrokinetic chromatography (EKC) allows the separation of closely related substances by the detection of fine effects in analyte-separation system interactions. With the goal of understanding the fine effects involved in separation using a dual cyclodextrin-microemulsion EKC system, an integrated study of NMR and molecular modeling was carried out. The above dual cyclodextrin-microemulsion system was previously used in the separation of clemastine and its related substances and was prepared by the addition of methyl-β-cyclodextrin (MβCD) and heptakis(2,6-di-O-methyl)-β-cyclodextrin (DMβCD) to an oil-in-water microemulsion. The use of DMβCD was shown to be essential in the separation of clemastine from one of its related substance (I(B) ). A molecular modeling study allowed the different affinities of clemastine and I(B) for the two cyclodextrins to be explained. Furthermore, rotating-frame Overhauser effect spectroscopy NMR experiments clearly indicated that besides the primary pseudostationary phase, namely the ionic microemulsion, cyclodextrins acted as a secondary pseudostationary phase. In addition, it was shown that inclusion complexation of sodium dodecyl sulfate (SDS) monomers into the cyclodextrins cavity occurs; differently, the oil (n-heptane) used in the preparation of microemulsion system resulted to be not included into the macrocycle cavity. These experimental results were supported by molecular modeling, which highlighted the preferential inclusion of SDS into DMβCD. On the basis of these results, it was confirmed that, besides its primary role as the ionic carrier in EKC, SDS is involved in inclusion equilibria toward CDs, which can be effective in increasing the system selectivity.     

Chiral separation in capillary electrophoresis using dual neutral cyclodextrins: theoretical models of electrophoretic mobility difference and separation selectivity

Simple equations and theoretical models, related to enantioselectivity (kappa) and C, have been developed for prediction of electrophoretic mobility difference (Deltamu) and separation selectivity (alpha) for enantiomers in CE using dual CDs, where alpha and kappa are defined as the ratio of mu and the ratio of binding constant (K) for enantiomers to each CD, respectively, C the CD concentration, and the average K for enantiomers and each CD. Experiments were carried out using dual CDs as beta-CD and dimethyl-beta-cyclodextrin (DM-beta-CD) and test analytes as five pairs of amphetamine drug enantiomers. A change in observed Deltamu and alpha of enantiomers in dual CDs was found to be in excellent agreement with the theoretical models. For example, in comparison with single CD1, dual CDs can enhance Deltamu and alpha up to the maximum value when enantiomers migrate with the same order in CD1 and CD2, and have the value of rho > 1.0, where rho is the enantioselectivity ratio for CD2 to CD1, while worse Deltamu and alpha are obtained for enantiomers with rho < 1.0.     

Enantioselective separation of twelve pairs of enantiomers on polysaccharide‐based chiral stationary phases and thermodynamic analysis of separation mechanism

The enantioseparation of twelve pairs of structurally related 1‐aryl‐1‐indanone derivatives was studied in the normal‐phase mode using three different polysaccharide‐type chiral stationary phases, namely Chiralpak IB, Chiralpak IC, and Chiralpak ID. n‐Hexane/2‐propanol and n‐hexane/ethanol were employed as mobile phases. Among all the investigated chiral columns, Chiralpak IC exhibited the most universal and the best enantioseparation ability toward all the racemates, particularly with the mobile phase composed of n‐hexane/2‐propanol (90/10, v/v). Then the effects of column temperature on retention and enantioselectivity were examined in the range of 25–40°C. Satisfactory enantioseparation was obtained at ambient temperature. The natural logarithm of retention and separation factors (ln k and ln α) versus the reciprocal of absolute temperature (1/T) (Van't Hoff plots) were found to be linear for all racemates, indicating that the retention and separation mechanisms were independent of temperature in the range investigated. Then, the thermodynamic parameters (ΔΔH°, ΔΔS°, and ΔΔG°) were calculated from Van't Hoff plots. These values indicated that the solute transfer from the mobile to stationary phase was enthalpically favorable, and the process of enantioseparation was mainly enthalpy controlled. At last, the impact of small changes in molecular structures of the tested 1‐indanone derivatives on enantioseparation was also discussed.     

Simultaneous enantioseparation of methcathinone and two isomeric methylmethcathinones using capillary electrophoresis assisted by 2‐hydroxyethyl‐β‐cyclodextrin

Methcathinone (ephedrone), 4‐methylmethcathinone (mephedrone), and 3‐methylmethcathinone (metaphedrone) are toxicologically‐important cathinone derivatives used commonly as designer drugs. In this work we show the first method allowing to separate simultaneously all these molecules in a chiral medium, ensuring good resolution between all enantiomers. Eight cyclodextrins have been tested as potential chiral selectors, the best results were obtained with 2‐hydroxyethyl‐β‐cyclodextrin, unreported so far for efficient separation of cathinones. After optimization, the method was calibrated and validated with and without the use of internal standard. The addition of standard improved an overall repeatability and precision, the use of electrophoretic mobility ratio was especially favorable (RSD < 1%). It was demonstrated that the method may be easily extended by introducing the additional cathinone‐related drugs to the sample, maintaining satisfactory separation efficiency.     

Mono‐6A‐(4‐methoxybutylamino)‐6A‐β‐cyclodextrin as a chiral selector for enantiomeric separation

A new member of the family of methoxylalkylamino monosubstituted β‐cyclodextrins, mono‐6^A‐(4‐methoxybutylamino)‐6^A‐β‐cyclodextrin, has been developed as a chiral selector for enantioseparation in capillary electrophoresis. This amino cyclodextrin exhibited good enantioselectivities for 16 model acidic racemates including three dansyl amino acids at an optimum pH of 6.0. Excellent chiral resolutions over six were obtained for α‐hydroxy acids and 2‐phenoxypropionic acids with 3.0 mM chiral selector. The good chiral recognition for α‐hydroxyl acids was attributed to inclusion complexation, electrostatic interactions, and hydrogen bonding. The hydrogen‐bonding‐enhanced chiral recognition was revealed by NMR spectroscopy. The chiral separation of acidic racemates was further improved with the addition of methanol (≤10 vol%) as an organic additive.     

Enantioseparation tuned by solvent polarity on a β‐cyclodextrin clicked chiral stationary phase

The efficient enantioseparation of 26 racemates has been achieved with the perphenylcarbamoylated cyclodextrin clicked chiral stationary phase by screening the optimum composition of mobile phase in high‐performance liquid chromatography. The chromatographic results indicate that both the retention and chiral resolution of racemates are closely related to the polarity of the mobile phases and the structures of analytes. The addition of alcohols can significantly tune the enantioseparation in normal‐phase high‐performance liquid chromatography. The addition of methanol and the ratio of ethanol/methanol or isopropanol/methanol played a key role on the resolution of flavonoids in ternary eluent systems. The chiral separation of flavonoids with pure organic solvent as mobile phase indicates the preferential order for chiral resolution is methanol>ethanol>isopropanol>n‐propanol>acetonitrile.     

Inverse cationic ITP for separation of methadone enantiomers with sulfated β‐cyclodextrin as chiral selector

Chiral ITP of the weak base methadone using inverse cationic configurations with H⁺ as leading component and multiple isomer sulfated β‐CD (S‐β‐CD) as leading electrolyte (LE) additive, has been studied utilizing dynamic computer simulation, a calculation model based on steady‐state values of the ITP zones, and capillary ITP. By varying the amount of acidic S‐β‐CD in the LE composed of 3‐morpholino‐2‐hydroxypropanesulfonic acid and the chiral selector, and employing glycylglycine as terminating electrolyte (TE), inverse cationic ITP provides systems in which either both enantiomers, only the enantiomer with weaker complexation, or none of the two enantiomers form cationic ITP zones. For the configuration studied, the data reveal that only S‐methadone migrates isotachophoretically when the S‐β‐CD concentration in the LE is between about 0.484 and 1.113 mM. Under these conditions, R‐methadone migrates zone electrophoretically in the TE. An S‐β‐CD concentration between about 0.070 and 0.484 mM results in both S‐ and R‐methadone forming ITP zones. With >1.113 mM and < about 0.050 mM of S‐β‐CD in the LE both enantiomers are migrating within the TE and LE, respectively. Chiral inverse cationic ITP with acidic S‐β‐CD in the LE is demonstrated to permit selective ITP trapping and concentration of the less interacting enantiomer of a weak base.     

Chiral Auxiliary Based Reductive Alkylation of α,α‐Diallkyl β‐Phosphonyl Esters

Acyclic α,α‐disubstituted β‐phosphonyl esters containing chiral alcoholic auxiliaries were efficiently prepared and evaluated for the lithium naphthalenide‐mediated asymmetric reductive alkylation. Among which, the best diastereoselectivity was received from the substrates bearing a (?)‐phenylmenthyl group in leading to alkylated esters with up to 83:17 dr. The diastereoselectivity is proven to be controlled by the π‐facial differentiation created by the chiral ester as well as the geometry of tetrasubstituted enolates generated by the reductive cleavage of C‐P bond.     

Influence of substituent position and cavity size of the regioisomers of monocarboxymethyl‐α‐, β‐, and γ‐cyclodextrins on the apparent stability constants of their complexes with both enantiomers of Tröger's base

Enantiomers of Tröger's base were separated by capillary electrophoresis using 2^I‐O‐, 3^I‐O‐, and 6^I‐O‐carboxymethyl‐α‐, β‐, and γ‐cyclodextrin and native α‐, β‐, and γ‐cyclodextrin as chiral additives at 0–12 mmol/L for β‐cyclodextrin and its derivatives and 0–50 mmol/L for α‐ and γ‐cyclodextrins and their derivatives in a background electrolyte composed of sodium phosphate buffer at 20 mmol/L concentration and pH 2.5. Apparent stability constants of all cyclodextrin–Tröger's base complexes were calculated based on capillary electrophoresis data. The obtained results showed that the position of the carboxymethyl group as well as the cavity size of the individual cyclodextrin significantly influences the apparent stability constants of cyclodextrin–Tröger's base complexes.     

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

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

Enantiomeric separation of oxybutynin by recycling high‐speed counter‐current chromatography with hydroxypropyl‐β‐cyclodextrin as chiral selector

Recycling high‐speed counter‐current chromatography was successfully applied to the preparative separation of oxybutynin enantiomers. The two‐phase solvent system consisted of n‐hexane, methyl tert‐butyl ether, and 0.1 mol/L phosphate buffer solution (pH = 5.0) with the volume ratio of 6:4:10. Hydroxypropyl‐β‐cyclodextrin was employed as the chiral selector. The influence of factors on the chiral separation process, including the concentration of chiral selector, the equilibrium temperature, the pH value of the aqueous phase were investigated. Under optimum separation conditions, 15 mg of oxybutynin racemate was separated with the purities of both the enantiomers over 96.5% determined by high‐performance liquid chromatography. Recovery for the target compounds reached 80–82% yielding 6.00 mg of (R)‐oxybutynin and 6.15 mg of (S)‐oxybutynin. Technical details for recycling elution mode were discussed.     

Preparation of β‐cyclodextrin‐gold nanoparticles modified open tubular column for capillary electrochromatographic separation of chiral drugs

In this paper, β‐cyclodextrin (β‐CD) modified gold nanoparticles (AuNPs) coated open tubular column (OT column) was prepared for capillary electrochromatography. The open tubular column was constructed through self‐assembly of gold nanoparticles on 3‐mercaptopropyl‐trimethoxysilane (MPTMS) prederivatized capillary and subsequent modification of thiols β‐cyclodextrin (SH‐β‐CD). Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and ultraviolet visible spectroscopy were carried out to characterize the prepared open tubular column and synthesized gold nanoparticles. By comparing different coating times of gold nanoparticles and thiols β‐cyclodextrin, we got the optimal conditions for preparing the open tubular column. Also, the separation parameters were optimized including buffer pH, buffer concentration and applied voltage. Separation effectiveness of open tubular column was verified by the separation of four pairs of drug enantiomers including bifonazole, fexofenadine, omeprazole and lansoprazole, and satisfactory separation results were achieved for these analytes studied. In addition, the column showed good stability and repeatability. The relative standard deviation values less than 5% were obtained through intra‐day, inter‐day, and column‐to‐column investigations.     

Application of bromoacetate‐substituted β‐CD‐bonded silica particles as chiral stationary phase for HPLC

Bromoacetate‐substituted [3‐(2‐O‐β‐cyclodextrin)‐2‐hydroxypropoxy]propylsilyl‐appended silica particles (BACD‐HPS), an important and useful synthetic intermediate for preparation of novel types of macrocycles‐capped β‐CD‐bonded silica particles including crown ether/cyclam/calix[4]arene‐capped β‐CD‐bonded silica particles, have been prepared and used as chiral stationary phase for HPLC. This synthetic stationary phase is characterized by means of elemental analysis. For the first time, the chromatographic behavior of BACD‐HPS was systematically evaluated with several disubstituted benzenes and some chiral drug compounds under both normal and RP conditions in HPLC. The results show that BACD‐HPS has excellent selectivity for the separation of aromatic positional isomers and chiral isomers of some drug compounds when used as stationary phase in HPLC.     

Effect of analyte lipophilicity on the resolution of α‐ and β‐amino acids on liquid chromatographic ligand exchange chiral stationary phases

Separation of the two enantiomers of racemic α‐ and β‐amino acids on two ligand exchange chiral stationary phases (CSPs) prepared previously by covalently bonding sodium N‐((S)‐1‐hydroxymethy‐3‐methylbutyl)‐N‐undecylaminoacetate or sodium N‐((R)‐2‐hydroxy‐1‐phenylethyl)‐N‐undecylaminoacetate on silica gel was studied with variation of the organic modifier (methanol) concentration in the aqueous mobile phase. In particular, the variation of retention factors with changing organic modifier concentration in the aqueous mobile phase was found to be strongly dependent on both the analyte lipophilicity and the stationary phase lipophilicity. In general, the retention factors of relatively lipophilic analytes on relatively lipophilic CSPs tend to increase with increasing organic modifier concentration in the aqueous mobile phases while those of less lipophilic or hydrophilic analytes tend to increase. However, only highly lipophilic analytes show decreasing retention factors with increasing organic modifier concentration in the aqueous mobile phase on less lipophilic CSPs. The contrasting retention behaviors on the two CSPs were rationalized by the balance of the two competing interactions, viz. hydrophilic interaction of analytes with polar aqueous mobile phase and the lipophilic interaction of analytes with the stationary phase.