Direct Chiral Separation Research Articles

Versatile capillary electrophoresis method for the direct chiral separation of aliphatic and aromatic α-hydroxy acids, β-hydroxy acids and polyhydroxy acids using vancomycin as chiral selector

Hydroxy acids (HAs) are ubiquitous in nature and play significant roles in various industrial and biological processes. Most HAs harbor at least one chiral center, therefore the development of efficient chiral analysis techniques for HA stereoisomers is of crucial importance across a wide range of fields. A capillary electrophoresis (CE) method was developed for the chiral analysis and quantification of aliphatic and aromatic α-hydroxy acid (AHA) enantiomers, aliphatic β-hydroxy acid (BHA) enantiomers and aliphatic polyhydroxy acid (PHA) stereoisomers. Using a modified partial filling-counter current method with indirect UV detection, high resolution (Rs) was achieved with vancomycin as a chiral selector added to the background electrolyte composed of 10 mM of benzoic acid/L-histidine at pH 5 using a polyacrylamide-coated capillary. This method could be readily applied to the determination of the enantiomers of 12 aliphatic AHAs, 4 aromatic AHAs, 3 aliphatic BHAs, as well as to the determination of the stereoisomers of tartaric acid, 2,3-dihydroxybutanoic acid, 2,3,4,5-tetrahydroxypentanoic acid, and 2,3,4,5,6-pentahydroxyhexanoic acid without the need for sample derivatization. Finally, our study provides a robust and versatile strategy for the chiral and stereoselective analysis of a broad range of hydroxy acid compounds.

Direct chiral separation of abscisic acid by high-performance liquid chromatography with a phenyl column and a mobile phase containing γ-cyclodextrin.

Abscisic acid (2-cis,4-trans-abscisic acid) is a plant hormone that has an asymmetric carbon atom. We tried to separate the enantiomers of native abscisic acid by HPLC using a phenyl column and a chiral mobile phase containing γ-cyclodextrin. The optimum mobile phase conditions were found to be 0.8% (w/v) γ-cyclodextrin, 4% (v/v) acetonitrile, and 20mM phosphate buffer (pH 6.0). It was found that (R)-abscisic acid was earlier detected than (S)-abscisic acid. Since γ-cyclodextrin is hardly retained on a phenyl column, it was suggested that (R)-abscisic acid formed a more stable complex with γ-cyclodextrin than the (S)-abscisic acid. Abscisic acid in an acacia honey sample was successfully enantioseparated with the proposed method and only (S)-abscisic acid was detected. A biologically inactive 2-trans,4-trans-abscisic acid, which was prepared by irradiation of abscisic acid with a light-emitting diode lamp at 365nm, was partially enantioseparated by the proposed method. Since the irradiation of (S)-abscisic acid-induced cis-to-trans isomerization to produce one 2-trans,4-trans-abscisic acid enantiomer, it is reasonable that racemization did not proceed during the cis-to-trans isomerization. (S)-Abscisic acid and probably (S)-2-trans,4-trans-abscisic acid were detected in a honey sample, where the peak area of (S)-abscisic acid was 7 times larger than that of (S)-2-trans,4-trans-abscisic acid.

A lipase-based chiral stationary phase for direct chiral separation in capillary electrochromatography

Candida antarctica lipase B (CALB) is a natural biocatalyst with an intrinsically strong chiral environment and a high degree of enantio-selectivity, which is widely used in the separation of racemates. Here, a facile and efficient covalent immobilization approach was utilized to immobilize CALB onto the capillary inner wall as a novel chiral stationary phase to explore and broaden its application in the direct chiral separation by electrochromatography. The obtained CALB immobilized capillary column was characterized by scanning electron microscopy (SEM), fluorescence imaging and Fourier transform infrared spectroscopy (FT-IR). The enantioseparation property of the CALB immobilized capillary column was confirmed by direct chiral separation of several pairs of monoamine neurotransmitter enantiomers in OT-CEC mode. Outstanding enantioseparation performance for three types of monoamine neurotransmitter enantiomers including epinephrine, norepinephrine and phenylephrine was obtained by the CALB immobilized column. Thanks to the effectiveness of covalent bonding method and the intrinsic stability of CALB, the prepared CALB immobilized capillary columns were quite steady and reproducible. The relative standard deviations for retention times of the enantiomers were as follows: for intra-day (n = 5) runs (≤0.25%), inter-day (n = 3) runs (≤0.72%) and between-columns (n = 3) (≤2.42%). After 90 consecutive runs in CEC mode, the CALB immobilized column still exhibited desirable enantionseparation performance.

Direct enantioselective gradient reversed-phase ultra-high performance liquid chromatography tandem mass spectrometry method for 3-hydroxy alkanoic acids in lipopeptides on an immobilized 1.6μm amylose-based chiral stationary phase.

3-Hydroxy fatty acids are important chiral building blocks of lipopeptides and metabolic intermediates of fatty acid oxidation, respectively. The analysis of the stereochemistry of such biomolecules has significant practical impact to elucidate and assign the enzymatic specificity of the biosynthesis machinery. In this work, a new mass spectrometry compatible direct chiral ultra high performance liquid chromatography separation method for 3-hydroxy fatty acids without derivatization is presented. The application of amylose tris(3,5-dimethylphenyl carbamate) based polysaccharide chiral stationary phase immobilized on 1.6μm silica particles (CHIRALPAK IA-U) allows the enantioseparation of 3-hydroxy fatty acids under generic electrospray ionization mass spectrometry friendly reversed phase gradient elution conditions. Adequate separation factors were achieved with both acetonitrile and methanol as organic modifiers, covering hydrocarbon chain lengths between C6 and C14 . Elution orders were derived from rhamnolipid (R-95) of which enantiomerically pure or enriched (R)-3-hydroxy fatty acids were recovered after ester hydrolysis. The S-configured acids consistently eluted before the respective R-enantiomers. The method was successfully applied for the elucidation of the absolute configuration of 3-hydroxy fatty acids originating from a novel lipopeptide with unknown structure. The work furthermore demonstrates that gradient elution is a viable option also in enantioselective (ultra)high performance liquid chromatography, even for analytes with modest separation factors, although less commonly exploited.

Stationary phase type and temperature effect on HPLC separation of lactic acid enantiomers

Abstract Lactic acid is a biologically important organic acid existing in two enantiomeric forms which are differently metabolized in the human body. In this paper, direct chiral separation of lactic acid by high performance liquid chromatography is presented. Five chiral stationary phases based on macrocyclic antibiotics were used for enantioseparation and chromatographic parameters, such as retention factors, resolution and selectivity factors, were determined under different column temperatures ranging from 5 to 45 °C. Optical isomers of lactic acid were efficiently separated using chiral stationary phases based on teicoplanin (R S = 1.9 ) and ristocetin (R S = 1.7 ) in reversed-phase separation mode at the column temperature of 25 °C.

Temperature and eluent composition effects on enantiomer separation of carvedilol by high-performance liquid chromatography on immobilized amylose-based chiral stationary phases

Carvedilol is a chiral drug with potent antihypertensive and antianginal activities. Although it is clinically used as a racemic mixture, its enantiomers show different pharmacokinetic and pharmacodynamic profiles. Here, the direct chiral separation of racemic drug by high performance liquid chromatography using two immobilized-type amylose-based chiral stationary phases is presented. Some chromatographic parameters, such as retention and selectivity, were determined under multimodal eluent conditions and different temperatures. A temperature-dependent inversion of the elution order of enantiomers was observed in the operative temperature range of chiral chromatographic support. Finally, an effective direct enantioselective method was successfully applied to the separation of the enantiomers of carvedilol on a semipreparative scale.

Enantioselective separation of RS-mandelic acid using β-cyclodextrin modified Fe3O4@SiO2/Au microspheres.

Chiral magnetic microspheres show great potential in direct enantioseparation. Herein novel chiral magnetic microspheres were prepared by self-assembling β-cyclodextrin on magnetic Fe3O4@SiO2/Au composite microspheres. The prepared materials were characterized by transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), vibrating sample magnetometry (VSM) and Fourier transform infrared spectroscopy (FTIR). The results showed that the functional microspheres have a three-ply structure and high saturation magnetization. β-Cyclodextrin-immobilized magnetic Fe3O4@SiO2/Au microspheres (Fe3O4@SiO2/Au/β-CD) were applied in the direct chiral separation of RS-mandelic acid. The analysis results of HPLC revealed that the Fe3O4@SiO2/Au/β-CD magnetic microspheres exhibited stronger enantioselective adsorption for R-mandelic acid than S-mandelic acid. The chiral selector functionalized magnetic microspheres are therefore expected to be an efficient and economical chiral separation method for use in further research.

Direct Chiral Separation of Binaphthyl Derivatives Using Atroposelective Antibodies

AbstractA simple procedure for chiral separation based on the difference in the molecular weights of the antigen‐antibody complex and analyte is developed. We raise monoclonal antibodies (mAbs) highly specific for the R‐ or S‐isomer of a binaphthyl derivative (BN) by immunizing mice with the corresponding BN enantiomer. Aqueous solutions of racemic BN are mixed with mAbs. The free BN is separated by ultrafiltration. The enantiomers of BN are obtained with a high e.e. using atroposelective antibodies.

Direct chiral separations of the enantiomers of phenylpyrazole pesticides and the metabolites by HPLC.

The enantiomeric separation of the enantiomers of three phenylpyrazole pesticides (fipronil, flufiprole, ethiprole) and two fipronil metabolites (amide-fipronil and acid-fipronil) were investigated by high-performance liquid chromatography (HPLC) with a CHIRALPAK® IB chiral column. The mobile phase was n-hexane or petroleum ether with 2-propanol or ethanol as modifier at a flow rate of 1.0mL/min. The influences of mobile phase composition and column temperature between 15 and 35°C on the separations were studied. All the analytes except ethiprole obtained complete enantiomeric separation after chromatographic condition optimization. Fipronil, flufiprole, amide-fipronil, and acid-fipronil obtained complete separation with the best resolution factors of 2.40, 3.40, 1.67, and 16.82, respectively, but ethiprole showed no enantioselectivity under the optimized conditions. In general, n-hexane with 2-propanol gave better separations in most cases. The results showed decreasing temperature and content of modifier in the mobile phase resulted in better separation and longer analysis time as well. The thermodynamic parameters calculated according to linear the Van't Hoff equation indicated the chiral separations in the study were enthalpy-driven. Fipronil and its two chiral hydrolyzed metabolites obtained baseline separation simultaneously under optimized conditions.

Fabrication of chiral amino acid ionic liquid modified magnetic multifunctional nanospheres for centrifugal chiral chromatography separation of racemates

As the rapid development of nanotechnology, the magnetic nanospheres modified with special chiral selective ligands show a great potentiality in enantiomeric separation. In this study, magnetic nanospheres modified with task-specific chiral ionic liquid were designed for the separation of chiral amino acids. These modified magnetic nanospheres were effective in a direct chiral separation of five racemic amino acids (d- and l-cysteine, d- and l-arginine, d- and l-leucine, d- and l-glutamine and d- and l-tryptophan). Furthermore, a new online method for complete separation of the enantiomers via the magnetic nanospheres was established with centrifugal chiral chromatography using a spiral tube assembly mounted on a type-J coil planet centrifuge. One kind of chiral compounds, d- and l-tryptophan was resolved well using this method. These results demonstrated that the modified nanospheres display a good chiral recognition ability, and can be used as a potential material for chiral separation of various racemates.

Direct chiral separations of third generation b-blockers through high performance liquid chromatography: a review

Direct chiral separations of third generation b-blockers through high performance liquid chromatography: a review

Multiple Response Optimization of a HPLC Method for the Determination of Enantiomeric Purity of S-Ofloxacin

The aim of the study was to develop a new HPLC method for direct chiral separation of Ofloxacin enantiomers using polar non-aqueous mobile phase by application of response surface methodology. Rotatable central composite design (CCD) with eight factorial points, six axial points and six replications in central point was used to evaluate the influence of three independent variables (concentration of methanol, diethylamine and flow rate) on the output responses (capacity factor of first peak, tailing factors of both the enantiomers, resolution between the Ofloxacin enantiomers, retention time of the last peak and chromatographic optimization function). Further, CCD data were combined with multiple response optimization in order to obtain a set of optimal experimental conditions (% methanol/hexane/acetonitrile-43.33/10/46.62 (v/v), % acetic acid/diethylamine-0.4/0.2 and flow rate as 1.4 mL min−1) leading to the most desirable compromise between resolution and analysis time. The method demonstrated good correlation between observed and predicted responses. The developed method was validated according to ICH guidelines and applied for quantitative analysis of two commercially available tablets Zenoflox (Ofloxacin) and Glevo (Levofloxacin). Good agreement was found between the assay results and the label claim of the marketed formulations by showing good %recovery and %CV. The study resulted in a better chromatographic system for the determination of Ofloxacin enantiomers.

Immobilization of HSA on polyamidoamine-dendronized magnetic microspheres for application in direct chiral separation of racemates.

Magnetic microspheres containing dendrimers and chiral selectivity show great performance for applications in pharmacology and biomedicine. In this study, a novel chiral magnetic nano-selector was prepared by immobilizing human serum albumin (HSA) on polyamidoamine dendrimer (PAMAM)-modified magnetic silica microspheres (PMSMs). The functionalized magnetic microspheres had a core-shell structure, with an average diameter of 600 nm. They exhibited strong magnetic response, with a high magnetization saturation of 46 emu g-1. UV-vis spectrophotometry and confocal laser scanning microscopy indicated that the HSA binding capacity on PMSM increased with increasing PAMAM generation. An immunoaffinity assay indicated that HSA retained a high level of activity, after immobilization on PMSM. HSA-immobilized PMSM (HSA-PMSM) was applied in the direct chiral separation of three biological racemates. The separation results show that HSA-PMSM selectively interacted with the racemates, and exhibited different activity toward each enantiomer. The results obtained by CD and CE indicated that HSA immobilized on third-generation PMSM possessed excellent chiral separation capability and biocompatibility. The chiral recognition capability of HSA-PMSM increased with increasing PAMAM generation. The chiral magnetic nano-selector is effective for the enantiomeric separation of chiral drugs, and has potential application in pharmacological and biomedical research.

Two‐dimensional liquid chromatography for direct chiral separations: a review

Separation of enantiomers remains a challenge owing to their identical physical and chemical properties in an achiral environment, and research on specialized separation techniques such as multidimensional achiral-chiral liquid chromatography continues to resolve individual enantiomers in complex samples. Recent advances in application of multidimensional liquid chromatography applied to chiral analysis are reviewed. For this reason, benefits of achiral-chiral coupling are shown, with emphasis in applications on biological and pharmaceutical fields as well as pesticide analysis. A description of standard instrumental setup in both heart-cut and comprehensive multidimensional liquid chromatography is shown. The most broadly used chiral stationary phases for multidimensional liquid chromatography are summarized. An extensive overview of different interface designs applied to complex samples is presented.

Synthesis of teicoplanin-modified hybrid magnetic mesoporous silica nanoparticles and their application in chiral separation of racemic compounds

Teicoplanin-conjugated mesoporous silica magnetic nanoparticles (TE-MSMNPs) were fabricated as novel chiral magnetic nano-selectors. Successful preparation of the functional magnetic mesoporous materials was achieved by grafting teicoplanin on N-(2-aminoethyl)-3-aminopropyltrimethoxysilane-modified mesoporous silica Fe3O4 magnetic nanoparticles (AEAPTMS-MSMNPs), and this was confirmed by various characterization techniques. The synthesized magnetic nanoparticles were regularly spherical and uniformly mesoporous with an average diameter of around 600nm and a mean pore size of about 3.9nm, respectively. These versatile magnetic nanoparticles were effective in a direct chiral separation of five racemic compounds in phosphate buffer. Much stronger interactions were observed with the (+)-enantiomers than with the (−)-enantiomers. After washing with water and ethanol by sonication, TE-MSMNPs could be reused at least three times with little efficiency loss. The functional magnetic mesoporous nanoparticles were easily separated from the racemic solutions using an external magnetic field. These magnetic nano-materials are suitable for enantiomer separations.

Direct chiral separation of azelnidipine by HPLC with Pirkle-type column

Direct chiral separation of azelnidipine by HPLC with Pirkle-type column

Enantioseparation of Novel Amino Analogs of Indole Phytoalexins on Macrocyclic Glycopeptide-Based Chiral Stationary Phase

Direct chiral separation of the enantiomers of spirobrassinin, 1-methoxyspirobrassinin and ten novel cis- and trans-diastereoisomers of 2-amino analogs of indole phytoalexin 1-methoxyspirobrassinol methyl ether on macrocyclic glycopeptide-based chiral stationary phase (CSP) with teicoplanin (Chirobiotic T) was studied. Normal phase eluents containing n-hexane with modifiers ethanol and 2-propanol were used. The effects of mobile phase composition, structure of the analytes and temperature were investigated. Chiral resolution on teicoplanin CSP was achieved only in the case of trans-diastereoisomers. The van’t Hoff plots were found to show linear behavior in all cases. It was found that studied normal phase enantioseparations were enthalpy driven. The elution order of the enantiomers was determined in some cases.

Development and validation of a direct enantiomeric separation of pregabalin to support isolated perfused rat kidney studies

Pregabalin (Lyrica™) is the first compound approved to treat the neural pain associated with fibromyalgia. Pregabalin is the S-enantiomer of a γ-amino acid analogue and chiral separation from its R-enantiomer must be achieved to support metabolic studies. The direct chiral separation of pregabalin from its R-enantiomer has been developed and HPLC/MS/MS assays have been validated to support isolated perfused rat kidney studies. The separation was developed through serial coupling of various macrocyclic glycopeptide stationary phases until partial separation of the enantiomers was achieved. Identification of the resolving stationary phase followed by optimization of the mobile phase enabled the baseline resolution of the enantiomers using mass spectrometry compatible solvents and modifiers. Assays were developed and validated for quantitation of the enantiomers from rat urine, isolated rat kidney perfusate, and isolated rat kidney perfusate ultrafiltrate to support pregabalin metabolic studies.

The role of cyclodextrins in chiral capillary electrophoresis

The members of the enantiomeric pairs frequently show rather different biological effects, so their chiral selective synthesis, pharmacological studies and analysis are necessary. CE has unique advantages in chiral analysis. The most frequently used chiral selectors are CDs in this field. This paper gives a short view on the advantages on CE in direct chiral separations, emphasizing the role of CDs. The reason for the broad selectivity spectra of CDs is discussed in detail. The physical background of chiral selective separations is briefly shown in CE. Their interaction mechanisms are shortly defined. The general trend of their use is statistically evaluated. Most frequently used CDs and CD derivatives are characterized. Advantages of ionizable CDs and single-isomer derivatives are shown. The general trend of their use is established.

Direct Chiral Separation of Four Indole Derivatives by High Performance Liquid Chromatography with Chiral Stationary Phase

A chiral stationary phase (CSP) was prepared by coating cellulose-tris(3,5-dimethylphenyl carbamate) (CDMPC) onto spherical silica gel. Four newly synthesized chiral indole derivatives were directly resolved on the CDMPC-CSP by a normal-phase system. The influences of structure and proportion of the alcohol in mobile phases on the separation by high-performance liquid chromatography with chiral stationary phase (HPLC) were investigated to optimize the chromatographic conditions. N-butano1, ethanol and ethanol were suitable for the separation of sample I, III, and IV as the modifiers, respectively, whereas the mixture of ethanol and n-propanol was suitable for sample I. Under the optimal conditions, excellent enantioseparation was achieved and the enantiomer excess values of all the four samples were determined. The satisfactory results indicated that the method of HPLC is ideal for the separation of indole derivatives.