Enantioseparation and molecular modeling study of five β‐adrenergic blockers on Chiralpak IC column

A new high‐performance liquid chromatography (HPLC) method was developed for the enantiomeric resolution of five β‐adrenergic blockers on a Chiralpak IC column (250 mm × 4.6 mm, 5.0 μm particle size) in normal phase mode. The mobile phase used was n‐hexane‐ethanol‐diethylamine in different proportions at the flow rate of 1.0 mL/min with the column temperature of 25°C using a UV detector at 230 nm. The influences of base additives and alcohol modifiers were evaluated and optimized. The maximum resolution values for bevantolol, propranolol carteolol, esmolol, and metoprolol were 4.80, 2.77, 2.09, 2.30, and 1.11, respectively. To gain a better understanding of the interaction between chiral stationary phase and analyte enantiomers, the molecular docking of chiral stationary phase with five pairs of enantiomer was carried out using AutoDock molecular docking technique. By simulation studies, the mechanism of chiral recognition was determined. According to the results, hydrogen bond interactions and π‐π interactions were the chief interactions for the chiral recognition.     

Enantioseparation of Racemic Flurbiprofen by Aqueous Two‐Phase Extraction With Binary Chiral Selectors of L‐dioctyl Tartrate and L‐tryptophan

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two‐phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two‐phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L‐dioctyl tartrate and L‐tryptophan, which were screened from amino acids, β‐cyclodextrin derivatives, and L‐tartrate esters. Factors such as the amounts of L‐dioctyl tartrate and L‐tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L‐dioctyl tartrate, 80 mg; L‐tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L‐dioctyl tartrate and L‐tryptophan, which enantioselectively recognized R‐ and S‐enantiomers in top and bottom phases, respectively. Compared to conventional liquid–liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers. Chirality 27:650–657, 2015. © 2015 Wiley Periodicals, Inc.     

Fast chiral separation of drugs using columns packed with sub‐2 μm particles and ultra‐high pressure

The use of columns packed with sub‐2 μm particles in liquid chromatography with very high pressure conditions (known as UHPLC) was investigated for the fast enantioseparation of drugs. Two different procedures were evaluated and compared using amphetamine derivatives and β‐blockers as model compounds. In one case, cyclodextrins (CD) were directly added to the mobile phase and chiral separations were carried out in less than 5 min. However, this strategy suffered from several drawbacks linked to column lifetime and low chromatographic efficiencies. In the other case, the analysis of enantiomers was carried out after a derivatization procedure using two different reagents, 2,3,4‐tri‐O‐acetyl‐α‐D ‐arabinopyranosyl isothiocyanate (AITC) and N‐α‐(2,4‐dinitro‐5‐fluorophenyl)‐L ‐alaninamide (Marfey's reagent). Separation of several amphetamine derivatives contained within the same sample was achieved in 2–5 min with high efficiency and selectivity. The proposed approach was also successfully applied to the enantiomeric purity determination of (+)‐(S)‐amphetamine and (+)‐(S)‐methamphetamine. Similar results were obtained with β‐blockers, and the separation of 10 enantiomers was carried out in less than 3 min, whereas the individual separation of several β‐blocker enantiomers was performed in 1 min or less. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Investigation of the Enantioselectivity of Tetramethylammonium L‐hydroxyproline Ionic Liquid as a Novel Chiral Ligand in Ligand‐Exchange CE and Ligand‐Exchange MEKC

Chiral ionic liquids (ILs) have drawn more and more attention in separation science; however, only a few papers focused on the application of chiral ILs as chiral ligands in LE‐CE. In this article, a novel amino acid ionic liquid (AAIL), tetramethylammonium L‐hydroxyproline ([TMA][L‐OH‐Pro]), was first applied as a chiral ligand to evaluate its enantioselectivity towards several aromatic amino acids in ligand‐exchange capillary electrophoresis (LE‐CE) and ligand‐exchange micellar electrokinetic capillary chromatography (LE‐MEKC). In the LE‐CE system, excellent separations were achieved for tryptophan (Rs = 3.03) and 3, 4‐dihydroxyphenylalanine (DOPA) (Rs = 4.35). Several parameters affecting the enantioseparation were systematically investigated, including AAIL concentration, type and concentration of central metal ion, buffer pH, as well as applied voltage. The optimum separation was obtained with 60 mM AAIL containing 30 mM Cu (II) at pH 4.5. Additionally, an LE‐MEKC system was established to further study the enantioselectivity of [TMA][L‐OH‐Pro] towards selected analytes. As observed, the separations of the enantiomers of tryptophan, phenylalanine, and histidine were all improved compared to the LE‐CE system. The results indicated that the application of AAILs as chiral ligands is a promising method in chiral separation science. Chirality 27:58–63, 2015. © 2014 Wiley Periodicals, Inc.     

Evaluation of the enantioselectivity of capillary electrokinetic chromatography using ethanediamine‐bonded poly (glycidyl methacrylate) microspheres as the pseudostationary phases

In this work, a new capillary electrokinetic chromatography (EKC) approach using ethanediamine‐bonded poly (glycidyl methacrylate) (Ami‐PGMA) microspheres as pseudostationary phases (PSPs) for enantioseparation with a polysaccharide, chondroitin sulfate E (CSE), as the chiral selector. The CSE@Ami‐PGMA EKC system was applied to enantioseparate basic drugs, and distinct improved separations of tested enantiomers were obtained while comparing with the single CSE system (the resolution increased from 0.41 to 1.26 for nefopam, from 1.24 to 2.15 for laudanosine, and from 0.92 to 2.36 for amlodipine). The Ami‐PGMA microspheres were fully characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared (FT‐IR) spectroscopy, and the results showed Ami‐PGMA microspheres were uniform and spherical in size (1 μm). Several principal parameters were systematically investigated, and the optimal chiral separations were obtained with Tris/H₃PO₄ (20 mM, pH 2.4, and 3.4 for NEF) containing 2.5% (w/v) CSE and 20‐μg Ami‐PGMA microspheres in 20°C. Subsequently, the concentrations of Ami‐PGMA microspheres and CSE were proved to be the dominant factors for the separation in the CSE@Ami‐PGMA EKC system by Statistical Product and Service Solutions (SPSS).     

Comparison of separation performances of amylose‐ and cellulose‐based stationary phases in the high‐performance liquid chromatographic enantioseparation of stereoisomers of β‐lactams

High‐performance liquid chromatographic methods were developed for the separation of the enantiomers of 19 β‐lactams. The direct separations were performed on chiral stationary phases containing either amylose‐tris‐3,5‐dimethylphenyl carbamate, (Kromasil® AmyCoat? column) or cellulose‐tris‐3,5‐dimethylphenyl carbamate, (Kromasil® CelluCoat? column) as chiral selector. The different methods were compared in systematic chromatographic examinations. The separations were carried out with good selectivity and resolution. The AmyCoat? and CelluCoat? columns appear to be highly complementary. The best separations of bi‐ and tricyclic β‐lactam stereoisomers were obtained with the AmyCoat? column, whereas the 4‐aryl‐substituted β‐lactams were better separated on the CelluCoat? column. The elution sequence was determined in all cases; no general rule could be established. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Effect of Basic and Acidic Additives on the Separation of Some Basic Drug Enantiomers on Polysaccharide‐Based Chiral Columns With Acetonitrile as Mobile Phase

The separation of enantiomers of 16 basic drugs was studied using polysaccharide‐based chiral selectors and acetonitrile as mobile phase with emphasis on the role of basic and acidic additives on the separation and elution order of enantiomers. Out of the studied chiral selectors, amylose phenylcarbamate‐based ones more often showed a chiral recognition ability compared to cellulose phenylcarbamate derivatives. An interesting effect was observed with formic acid as additive on enantiomer resolution and enantiomer elution order for some basic drugs. Thus, for instance, the enantioseparation of several β‐blockers (atenolol, sotalol, toliprolol) improved not only by the addition of a more conventional basic additive to the mobile phase, but also by the addition of an acidic additive. Moreover, an opposite elution order of enantiomers was observed depending on the nature of the additive (basic or acidic) in the mobile phase. Chirality 27:228–234, 2015. © 2015 Wiley Periodicals, Inc.     

HPLC‐method for determination of permethrin enantiomers using chiral β‐cyclodextrin‐based stationary phase

The liquid chromatographic separation of permethrin enantiomers on chiral β‐cyclodextrin‐based stationary phase has been investigated. All four enantiomers are obtained by using simple methanol and water mobile phase, under gradient mode. The method was optimized and validated. The relationship between temperature and chromatographic parameters: k′ (capacity factor), α (separation factor) and Rs (resolution factor) was studied. Van't Hoff's curves for each enantiomer were plotted for temperature range 288–318 K. It was noticed that the response factor ratio of permethrin isomers differ and calculated value is found to be 1.66 (cis/trans, for n = 5). This method has been used for determining permethrin enantiomer ratio for a few samples of working standards and one formulation. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Enantioseparations with cellulose tris(3-chloro-4-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography

Cellulose tris(3-chloro-4-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSPs) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the chiral selector loading onto silica, mobile phase composition and pH, as well as separation variables on separation of enantiomers was studied. It was found that CSPs based on cellulose tris(3-chloro-4-methylphenylcarbamate) can be used for preparation of very stable capillary columns useful for enantioseparations in nano-LC and CEC in combination with polar organic mobile phases.     

Simultaneous determination of guaifenesin enantiomers and ambroxol HCl using 50‐mm chiral column for a negligible environmental impact

Chiral stationary phases are conveniently used for enantiomeric separation of drugs by liquid chromatography. Consumption of large volumes of hazardous solvents is considered as a common challenge for the sustainability of this technique. To this end, a columnar chromatography has been adopted using 50‐mm‐length stationary phases. The study comprised five Phenomenex Lux cellulose‐ and amylose‐based columns for the separation of guaifenesin (GUA) enantiomers. In addition, an experimental design was used to optimize the gradient profile for the separation of racemic GUA and ambroxol HCl (AMB) binary mixture. The chromatographic method was achieved using Lux Cellulose‐1 (50 × 4.6 mm) as a chiral stationary phase and ethanol/water as a mobile phase with linear gradient elution of 20% to 70% ethanol in 6 minutes at a flow rate of 1.0 mL min^?1 and UV detection at 270 nm. Linearity ranges were found to be 50 to 1000 μg mL^?1 and 15 to 450 μg mL^?1 for each GUA enantiomer and AMB, respectively. Environmental, health and safety tool was used to assess and compare greenness of the proposed and reported methods. Short column indeed reduces the environmental impact by decreasing waste by about 60% and utilizing only 1‐mL ethanol in the mobile phase. The proposed method is a safer alternative for the simultaneous determination of drugs in their combined pharmaceutical formulation. The method has been validated and compared favorably with a reported one.     

GC Separation of Enantiomers of Alkyl Esters of 2‐Bromo Substituted Carboxylic Acids Enantiomers on 6‐TBDMS‐2,3‐di‐alkyl‐ β‐ and γ‐Cyclodextrin Stationary Phases

The gas chromatographic separation of enantiomers of 2‐Br carboxylic acid derivatives was studied on four different 6‐TBDMS‐2,3‐di‐O‐alkyl‐ β‐ and ‐γ‐CD stationary phases. The differences in thermodynamic data {ΔH and –ΔS} for the 15 structurally related racemates were evaluated. The influence of structure differences in the alkyl substituents covalently attached to the stereogenic carbon atom, as well as in the ester group of the homologous analytes, and the selectivity of modified β‐ and γ‐ cyclodextrin derivatives was studied in detail. The cyclodextrin cavity size, as well as elongation of alkyl substituents in positions 2 and 3 of 6‐TBDMS‐β‐CD, also affected their selectivity. The quality of enantiomeric separations is influenced mainly by alkyl chains of the ester group of the molecule and this appears to be independent of the CD stationary phase used. In some cases the separations occur as the result of external adsorption rather than inclusion complexations with the chiral selector. It was found that the temperature dependencies of the selectivity factor were nonlinear. Chirality 26:279–285, 2014. © 2014 Wiley Periodicals, Inc.     

Chiral separation of tryptophan enantiomers by liquid chromatography with BSA-silica stationary phase

The separation of tryptophan enantiomers was carried out with medium-pressure liquid chromatography using BSA (bovine serum albumin)-bonded silica as a chiral stationary phase. The influence of various experimental factors such as pH and ionic strength of mobile phase, separation temperature, and the presence of organic additives on the resolution was studied. In order to expand this system to preparative scale, the loadability of sample and the stability of stationary phase for repeated use were also examined. The separation of tryptophan enantiomers was successful with this system. The data indicated that a higher separation factor (α) was obtained at a higher pH and lower temperature and ionic strength in mobile phase. Addition of organic additives (acetonitrile and 2-propanol) in mobile phase contributed to reduce the retention time of L-tryptophan. About 30% of the separation factor was reduced after 80 days of repeated use.     

Liquid chromatographic separation and thermodynamic investigation of lorcaserin hydrochloride enantiomers on immobilized amylose–based chiral stationary phase

A novel liquid chromatographic method was developed for enantiomeric separation of lorcaserin hydrochloride on Chiralpak IA column containing chiral stationary phase immobilized with amylose tris (3.5‐dimethylphenylcarbamate) as chiral selector. Baseline separation with resolution greater than 4 was achieved using mobile phase containing mixture of n‐hexane/ethanol/methanol/diethylamine (95:2.5:2.5:0.1, v/v/v/v) at a flow rate of 1.2 mL/min. The limit of detection and limit of quantification of the S‐enantiomer were found to be 0.45 and 1.5 μg/mL, respectively; the developed method was validated as per ICH guideline. The influence of column oven temperatures studied in the range of 20°C to 50°C on separation was studied; from this, retention, separation, and resolution were investigated. The thermodynamic parameters ΔH°, ΔS°, and ΔG° were evaluated from van't Hoff plots,(Ink′ _versus 1/T) and used to explain the strength of interaction between enantiomers and immobilized amylose–based chiral stationary phase     

Preparation and Studies of Chiral Stationary Phases Containing Enantiopure Acridino‐18‐Crown‐6 Ether Selectors

The enantiomeric separation ability of the newly prepared chiral stationary phases containing acridino‐18‐crown‐6 ether selectors was studied by high‐performance liquid chromatography (HPLC). The chiral stationary phases separated the enantiomers of selected protonated primary aralkylamines efficiently. The best results were found for the separation of the mixtures of enantiomers of NO₂‐PEA. Chirality 26:651–654, 2014. © 2014 Wiley Periodicals, Inc.     

Enantioselective liquid chromatographic-electrospray mass spectrometric assay of beta-adrenergic blockers: application to a pharmacokinetic study of sotalol in human plasma

An enantioselective high performance liquid chromatographic-electrospray ionization mass spectrometric (HPLC-ESI-MS) method for the direct determination of several beta-adrenergic blockers was developed and validated. The method is based on the direct separation of the enantiomers of drugs on a laboratory-made chiral stationary phase (CSP) containing covalently bonded teicoplanin (TE) as chiral selector. Detection of the effluent was performed by electrospray ionization mass spectrometry, run in the selected-ion recording (SIR) mode. The method was applied to the pharmacokinetic monitoring of sotalol (STL) in the plasma of five young healthy volunteers, dosed with racemic drug. The limits of quantitation (LOQ) reached 4 ng/ml for both sotalol enantiomers. Such a method, fully validated, offers a novel, fast and very efficient tool for the direct determination of sotalol enantiomers in human plasma, and can be generally applied to the beta-adrenergic blockers stereoselective pharmacokinetics.     

Enantioseparation of ofloxacin and its four related substances with ligand exchange–micellar electrokinetic chromatography using copper(II)‐L‐isoleucine complex as chiral selector

A ligand‐exchange micellar electrokinetic capillary electrophoresis system with copper(II)‐L‐isoleucine complexes as the chiral selector incorporated in micelles of sodium dodecyl sulfate (SDS) was developed for the enantioseparation of ofloxacin and its four related substances (impurities A, C, E, and F). The effects of important parameters affecting separation such as buffer pH, SDS concentration, chiral selector concentration, and organic additive were investigated in detail. Under optimum experimental conditions, enantioseparation of ofloxacin, impurities A, C, E, and F enantiomers was accomplished with resolutions of 4.28, 2.83, 3.40, 3.58, and 2.46, respectively. Further, simultaneous separation of impurities A, C, E, and F enantiomers was achieved using 10 mmol/L NH₄OAc as the running buffer containing 4 mmol/L copper sulfate,20 mmol/L L‐isoleucine, 20 mmol/L SDS, and 5% methanol at pH 8.5. To the best of our knowledge, the simultaneous enantioseparation of four impurities of ofloxacin has not been reported previously.     

Comparative Enantioseparation of Ketoprofen with Trimethylated α‐, β‐, and γ‐Cyclodextrins in Capillary Electrophoresis and Study of Related Selector–Selectand Interactions Using Nuclear Magnetic Resonance Spectroscopy

The enantiomers of ketoprofen were separated by capillary electrophoresis using the (2,3,6‐tri‐O‐methyl)‐derivatives of α‐, β‐, and γ‐cyclodextrin (CyD) as chiral selectors. The affinity pattern of the ketoprofen enantiomers toward these CyDs changed depending on their cavity size. Thus, with hexakis (2,3,6‐tri‐O‐methyl)‐α‐CyD and heptakis (2,3,6‐tri‐O‐methyl)‐β‐CyD, the R enantiomer of the drug migrated first, whereas the enantiomer migration order was reversed in the presence of octakis(2,3,6‐tri‐O‐methyl)‐γ‐CyD. The change in the migration order was rationalized on the basis of changes in the structure of the complexes between the ketoprofen enantiomers and the chiral selectors as derived from nuclear magnetic resonance spectroscopy experiments. Chirality, 25:79–88, 2013.© 2012 Wiley Periodicals, Inc.     

Determination of absolute configuration in 4‐aryl‐3,4‐dihydro‐2(1H)‐pyrimidones by high performance liquid chromatography and CD spectroscopy

The absolute configuration of three 4‐aryl‐3,4‐dihydro‐2(1H)‐pyrimidones (Biginelli compounds, DHPMs) was established by comparison of the typical circular dichroism (CD) spectra of individual enantiomers with reference samples of known absolute configuration. The enantiomers were obtained by semipreparative separation of racemic mixtures on a Chiralcel OD‐H chiral stationary phase. The method was used to establish the enantiopreference of various lipases in biocatalytic kinetic resolution experiments employing activated DHPM esters. Chirality 11:659–662, 1999. © 1999 Wiley‐Liss, Inc.     

Enantiomeric resolution and modeling of DL‐alanine‐DL‐tryptophan dipeptide on amylose stationary phase

The enantiomeric resolution of DL‐alanine‐DL‐tryptophan dipeptide is described on amylose stationary phase. The eluent used was CH₃OH─CH₃COONH₄ (10mM)─CH₃CN (50: 40, 10) at 0.8‐mL/min flow, 230‐nm detection, 25‐minute run time, and 25°C ± 1°C temperature. The chiral phase was amylose [AmyCoat RP (15 cm × 0.46 cm × 5 micron)]. The magnitudes of the retention factors (k) were 2.71, 3.52, 5.11, and 7.75. The magnitudes of separation factor (α) were 1.19, 1.57, and 1.51 while the resolution factors (Rs) were 3.25, 14.84, and 15.76. The limits of detection and quantitation were of 2.5 to 5.4 and 12.8 to 27.5 μg/mL. The enantiomeric resolution is controlled by hydrogen, hydrophobic, π‐π, steric, etc interactions. The elution order of the enantiomer was supported by the modeling data. The described method is fast, reproducible, precise, and selective, which can be used successfully for evaluating the enantiomers of the reported dipeptide.     

A surface molecularly imprinted polymer as chiral stationary phase for chiral separation of 1,1′‐binaphthalene‐2‐naphthol racemates

Acrylamide (AM) was copolymerized with ethylene glycol dimethacrylate (EGDMA) in the presence of (R )‐1,1′‐binaphthalene‐2‐naphthol (BINOL) as the template molecules on the surface of silica gel by a free radical polymerization to produce a chiral stationary phase based on the surface molecularly imprinted polymer (SMIP‐CSP). The SMIP‐CSP showed a much better separation factor (α = 4.28) than the CSP based on the molecularly imprinted polymer (MIP‐CSP) without coating on the silica gel (α = 1.96) during the chiral separation of BINOL enantiomers by high‐performance liquid chromatography. The influence of the pretreatment temperature and the content of the template molecule ((R )‐BINOL) of the SMIP‐CSP, and the mobile phase composition on the separation of the racemic BINOL were systematically investigated.