Design of experiment assisted concurrent enantioseparation of bupropion and hydroxybupropion by high‐performance thin‐layer chromatography

A simple and efficient high‐performance thin‐layer chromatographic method was developed for chiral separation of rac ‐bupropion (BUP) and its active metabolite rac ‐hydroxybupropion (HBUP). Design of experiment (DoE)‐based optimization was adopted instead of a conventional trial‐and‐error approach. The Box–Behnken design surface response model was used and the operating variables were optimized based on 17 trials design. The optimized method involved impregnation of chiral reagent, L(+)‐tartaric acid, in the stationary phase with simultaneous addition in the mobile phase, which consisted of acetonitrile : methanol : dichloromethane : 0.50% L‐tartaric acid (6.75:1.0:1.0:0.25, v /v /v /v ). Under the optimized conditions, the resolution factor between the enantiomers of BUP and HBUP was 6.30 and 9.26, respectively. The limit of detection and limit of quantitation for (R)‐BUP, (S)‐BUP, (R,R)‐HBUP, and (S,S)‐HBUP were 9.23 and 30.78 ng spot⁻¹, 10.32 and 34.40 ng spot⁻¹, 12.19 and 40.65 ng spot⁻¹, and 14.26 and 47.53 ng spot⁻¹, respectively. The interaction of L‐tartaric acid with analytes and their retention behavior was thermodynamically investigated using van't Hoff's plots. The developed method was validated as per the International Conference on Harmonization guidelines. Finally, the method was successfully applied to resolve and quantify the enantiomeric content from marketed tablets as well as spiked plasma samples.     

Strategy Approach for Direct Enantioseparation of Hyoscyamine Sulfate and Zopiclone on a Chiral αl‐Acid Glycoprotein Column and Determination of Their Eutomers: Thermodynamic Study of Complexation

Rapid and simple isocratic high‐performance liquid chromatographic methods with UV detection were developed and validated for the direct resolution of racemic mixtures of hyoscyamine sulfate and zopiclone. The method involved the use of α_l‐acid glycoprotein (AGP) as chiral stationary phase. The stereochemical separation factor (?) and the stereochemical resolution factor (R_s) obtained were 1.29 and 1.60 for hyoscyamine sulfate and 1.47 and 2.45 for zopiclone, respectively. The method was used for determination of chiral switching (eutomer) isomers: S‐hyoscyamine sulfate and eszopiclone. Several mobile phase parameters were investigated for controlling enantioselective retention and resolution on the chiral AGP column. The influence of mobile phase, concentration and type of uncharged organic modifier, ionic strength, and column temperature on enantioselectivity were studied. Calibration curves were linear in the ranges of 1–10 µg mL^‐1 and 0.5–5 µg mL^‐1 for S‐hyoscyamine sulfate and eszopiclone, respectively. The method is specific and sensitive, with lower limits of detection and quantifications of 0.156, 0.515 and 0.106, 0.349 for S‐hyoscyamine sulfate and eszopiclone, respectively. The method was used to identify quantitatively the enantiomers profile of the racemic mixtures of the studied drugs in their pharmaceutical preparations. Thermodynamic studies were performed to calculate the enthalpic ΔH and entropic ΔS terms. The results showed that enantiomer separation of the studied drugs were an enthalpic process. Chirality 28:49–57, 2016. © 2015 Wiley Periodicals, Inc.     

High‐Performance Liquid Chromatographic Enantioseparation of Unusual Isoxazoline‐Fused 2‐Aminocyclopentanecarboxylic Acids on (+)‐(18‐Crown‐6)‐2,3,11,12‐Tetracarboxylic Acid‐Based Chiral Stationary Phases

The enantiomers of four unusual isoxazoline‐fused 2‐aminocyclopentanecarboxylic acids were directly separated on chiral stationary phases containing (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid as chiral selector. The nature of the alcoholic modifier (MeOH, EtOH, IPA) exerted a great effect on the retention, whereas the selectivity and resolution did not change substantially. Two types of dependence of retention on alcohol content were detected: k₁ increased continuously with increasing alcohol content or a U‐shaped retention curve was observed. A comparison of the chromatographic data obtained with HCOOH, AcOH, TFA, HClO₄, H₂SO₄, or H₃PO₄ as acidic modifier at a constant concentration demonstrated that in most cases, larger k values were obtained on the application of AcOH or HCOOH, and an increase of the acid content resulted in a decrease of retention. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes and selector. The sequence of elution of the enantiomers was determined in all cases. Chirality 24:817‐824, 2012. © 2012 Wiley Periodicals, Inc.     

Enantiomeric separation of Novel Psychoactive Substances by capillary electrophoresis using (+)‐18‐crown‐6‐tetracarboxylic acid as chiral selector

In the recent years, hundreds of Novel Psychoactive Substances (NPS) have entered both the European and the global drug market. These drugs, which are mainly used for recreational matters, have caused serious social problems. Every year, the spectrum of these misused drugs is enlarged by new derivatives, which are produced by modifications of basic structures of already well‐known substances. Additionally, a lot of them possess a stereogenic center which leads to 2 enantiomeric forms. The fact that the pharmacological effects and potencies of the enantiomers of these chiral NPS may differ can be assumed from a broad spectrum of active pharmaceutical ingredients. For this reason, analytical method development regarding enantiomeric separation for these classes of substances is of great pharmaceutical and medical interest. The aim of this work was to create an easy‐to‐prepare chiral capillary electrophoresis method for the enantioseparation of NPS which contains a primary amino group by means of (+)‐18‐crown‐6‐tetracarboxylic acid as chiral selector. Novel Psychoactive Substances were purchased at various Internet stores or represent samples seized by Austrian police. The effects of selector concentration, the electrolyte composition, and the addition of organic modifiers to the background electrolyte on enantioseparation were investigated. Under optimized conditions, the use of 20‐mM (+)‐18‐crown‐6‐tetracarboxylic acid, 10‐mM Tris, and 30‐mM citric acid buffer at pH 2.10 turned out to be effective. Fifteen of 24 tested NPS were resolved in their enantiomers within 15 minutes. It was found that all NPS were traded as racemic mixtures.     

Enantiomeric assay of escitalopram S(+)‐enantiomer and its “in‐process impurities” using two different techniques

The enantiomeric purity of escitalopram oxalate ESC and its “in‐process impurities,” namely, ESC‐N‐oxide, ESC‐citadiol, and R(?)‐enantiomer were studied in drug substance and products using high‐performance liquid chromatography (HPLC)‐UV (Method I), synchronous fluorescence spectroscopy (SFS) (Method IIA), and first derivative SFS (Method IIB). Method I describes as an isocratic HPLC‐UV for the direct resolution and determination of enantiomeric purity of ESC and its “in‐process impurities.” The proposed method involved the use of α_l‐acid glycoprotein (AGP) chiral stationary phase. The regression plots revealed good linear relationships of concentration range of 0.25 to 100 and 0.25 to 10 μg mL^?1 for ESC and its impurities. The limits of detection and quantifications for ESC were 0.075 and 0.235 μg mL^?1, respectively. Method II involves the significant enhancement of the fluorescence intensities of ESC and its impurities through inclusion complexes formation with hydroxyl propyl‐β‐cyclodextrin as a chiral selector in Micliavain buffer. Method IIA describes SFS technique for assay of ESC at 225 nm in presence of its impurities: R(?)‐enantiomer, citadiol, and N‐oxide at ?λ of 100 nm. This method was extended to (Method IIB) to apply first derivative SFS for the simultaneous determination of ESC at 236 nm and its impurities: the R(?)‐enantiomer, citadiol, and N‐oxide at 308, 275, and 280 nm, respectively. Linearity ranges were found to be 0.01 to 1.0 μg mL^?1 for ESC and its impurities with lower detection and quantification limits of 0.033/0.011 and 0.038/0.013 μg mL^?1 for SFS and first derivative synchronous fluorescence spectra (FDSFS), respectively. The methods were used to investigate the enantiomeric purity of escitalopram.     

Enantiomeric separation of N‐protected amino acids by non‐aqueous capillary electrophoresis using quinine or Tert‐butyl carbamoylated quinine as chiral additive

A capillary electrophoretic (CE) method for the enantioseparation of N‐protected chiral amino acids was developed using quinine and tert‐butyl carbamoylated quinine as chiral selectors added to nonaqueous electrolyte solutions (NACE). A series of various N‐derivatized amino acids were tested as chiral selectands, and in order to optimize the CE enantioseparation of these compounds, different parameters were investigated: the nature of the organic solvent, the combination of different solvents, the nature and the concentration of the background electrolyte, the selector concentration, the capillary temperature, and the applied voltage. The influence of these factors on the separation of the analyte enantiomers and the electroosmotic flow was studied. Generally, with tert‐butyl carbamoylated quinine as chiral selector, better enantioseparations were achieved than with unmodified quinine. Optimum experimental conditions were found with a buffer made of 12.5 mM ammonia, 100 mM octanoic acid, and 10 mM tert‐butyl carbamoylated quinine in an ethanol–methanol mixture (60:40 v/v). Under these conditions, DNB‐Leu enantiomers could be separated with a selectivity factor (α) of 1.572 and a resolution (Rs) of 64.3; a plate number (N) of 127,000 and an asymmetry factor (As) of 0.93 were obtained for the first migrating enantiomer. Chirality 11:622–630, 1999. © 1999 Wiley‐Liss, Inc.     

Enantioselective determination of zopiclone and its metabolites in urine by capillary electrophoresis

A method has been developed for the stereoselective determination of zopiclone and its main metabolites in urine. After the addition of the internal standard zolpidem the urine samples were extracted at pH 8 with chloroform-isopropanol (9:1). Analyses were carried out using capillary electrophoresis (CE) with β-cyclodextrin as the chiral selector. The analytes were detected using UV laser-induced fluorescence detection with a He-Cd laser operated at 325 nm. Urine samples of two volunteers after oral administration of 7.5 mg zopiclone were investigated. The S-(+)-enantiomers of zopiclone and its metabolites were always excreted in higher amounts than the R-(−)-enantiomers. With the same method the zopiclone enantiomers were quantified in saliva. Compared to high-performance liquid chromatography, the CE method is very fast and simple.     

Specific optical rotation is a versatile tool for the identification of critical micelle concentration and micellar growth of tartaric acid–based diastereomeric amphiphiles

Four novel tartaric acid–based diastereomeric chiral amphiphiles, two being enantiomers of the other two, have been synthesized and investigated using chiroptical spectroscopic methods, along with tensiometry and dynamic light scattering experiments. We found that an inflection point in specific optical rotation (SOR) values at ~0.32 mM corresponds to the critical micelle concentration (CMC). The increase in magnitude of SOR values beyond CMC corresponds to the growth of aggregates. For enantiomers, oppositely signed SOR values were observed, ruling out the possibility for the presence of aggregation size mediated artefacts. SOR values did not exhibit concentration dependence for a chiral tartaric acid based non‐aggregating analogue further establishing the absence of artefacts or anomalous interaction of tartaric acid based head group with solvent. Electronic circular dichroism spectra showed no significant changes in band positions or intensities with concentration. Due to the requirement for higher concentrations (~200 mM) needed to obtain vibrational circular dichroism spectra, these measurements are not found to be useful for studying concentration dependent properties of chiral amphiphiles.     

Enantioseparation of flurbiprofen enantiomers using chiral ionic liquids by liquid‐liquid extraction

Flurbiprofen is a kind of nonsteroidal anti‐inflammatory drug, which has been widely used in clinic for treatment of rheumatoid arthritis and osteoarthritis. It has been reported that S‐flurbiprofen shows good performance on clinic anti‐inflammatory treatment, while R‐enantiomer almost has no pharmacological activities. It has important practical values to obtain optically pure S‐flurbiprofen. In this work, chiral ionic liquids, which have good structural designability and chiral recognize ability, were selected as the extraction selector by the assistance of quantum chemistry calculations. The distribution behaviors of flurbiprofen enantiomers were investigated in the extraction system, which was composed of organic solvent and aqueous phase containing chiral ionic liquid. The results show that maximum enantioselectivity up to 1.20 was attained at pH 2.0, 25°C using 1,2‐dichloroethane as organic solvent, 1‐butyl‐3‐methylimidazole L‐tryptophan ([Bmim][L‐trp]) as chiral selector. The racemic flurbiprofen initial concentration was 0.2 mmol L^?1, and [Bmim][L‐trp] concentration was 0.02 mol L^?1. Furthermore, the recycle of chiral ionic liquids has been achieved by reverse extraction process of the aqueous phase with chiral selector, which is significant for industrial application of chiral ionic liquids and scale‐up of the extraction process.     

HPLC‐Fluorescence Method for the Enantioselective Analysis of Propranolol in Rat Serum Using Immobilized Polysaccharide‐Based Chiral Stationary Phase

A stereoselective high‐performance liquid chromatographic (HPLC) method was developed and validated to determine S‐(?)‐ and R‐(+)‐propranolol in rat serum. Enantiomeric resolution was achieved on cellulose tris(3,5‐dimethylphenylcarbamate) immobilized onto spherical porous silica chiral stationary phase (CSP) known as Chiralpak IB. A simple analytical method was validated using a mobile phase consisted of n‐hexane‐ethanol‐triethylamine (95:5:0.4%, v/v/v) at a flow rate of 0.6 mL min^‐1 and fluorescence detection set at excitation/emission wavelengths 290/375 nm. The calibration curves were linear over the range of 10–400 ng mL^‐1 (R = 0.999) for each enantiomer with a detection limit of 3 ng mL^‐1. The proposed method was validated in compliance with ICH guidelines in terms of linearity, accuracy, precision, limits of detection and quantitation, and other aspects of analytical validation. Actual quantification could be made for propranolol isomers in serum obtained from rats that had been intraperitoneally (i.p.) administered a single dose of the drug. The proposed method established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology. Molecular modeling studies including energy minimization and docking studies were first performed to illustrate the mechanism by which the active enantiomer binds to the β‐adrenergic receptor and second to find a suitable interpretation of how both enantiomers are interacting with cellulose tris(3,5‐dimethylphenylcarbamate) CSP during the process of resolution. The latter interaction was demonstrated by calculating the binding affinities and interaction distances between propranolol enantiomers and chiral selector. Chirality 26:194–199, 2014. © 2014 Wiley Periodicals, Inc.     

Novel Stereoselective High‐Performance Liquid Chromatographic Method for Simultaneous Determination of Guaifenesin and Ketorolac Enantiomers in Human Plasma

A novel method was developed for the simultaneous determination of guaifenesin (GUA) and ketorolac tromethamine (KET) enantiomers in plasma samples. Since GUA probably increases the absorption of coadministered drugs (e.g., KET), it would be extremely important to monitor KET plasma levels for the purpose of dose adjustment with a subsequent decrease in the side effects. Enantiomeric resolution was achieved on a polysaccharide‐based chiral stationary phase, amylose‐2, as a chiral selector under the normal phase (NP) mode and using ornidazole (ORN) as internal standard. This innovative method has the advantage of the ease and reliability of sample preparation for plasma samples. Sample clean‐up was based on simply using methanol for protein precipitation followed by direct extraction of drug residues using ethanol. Both GUA and KET enantiomers were separated using an isocratic mobile phase composed of hexane/isopropanol/trifluoroacetic acid, 85:15:0.05 v/v/v. Peak area ratios were linear over the range 0.05–20 µg/mL for the four enantiomers S (+) GUA, R (–) GUA, R (+) KET, and S (–) KET. The method was fully validated according to the International Conference on Harmonization (ICH) guidelines in terms of system suitability, specificity, accuracy, precision, robustness, and solution stability. Finally, this procedure was innovative to apply the rationale of developing a chiral high‐performance liquid chromatography (HPLC) procedure for the simultaneous quantitative analysis of drug isomers in clinical samples. Chirality 26:629–639, 2014. © 2014 Wiley Periodicals, Inc.     

Synthesis and study on the binding of thiazol‐2(3H)‐ylidine derivative with human serum albumin using spectroscopic and molecular docking methods

In this article, a facile and convenient synthesis of thiazol‐2(3H)‐ylidine derivatives of fatty acid ( 3a – c ) is described. The binding of N′‐(4,5‐dimethyl‐3‐penylthiazol‐2(3H)‐ylidine)octadec‐9‐enehydrazide ( 3a ) with human serum albumin (HSA) is explored using various spectral methods and molecular docking. Fluorescence quenching results show that 3a induces conformational changes in HSA and the polarity around the tryptophan residues is increased. Stern–Volmer quenching plots at different temperatures (298, 305 and 312 K) show that the fluorescence quenching mechanism is static quenching. Synchronous fluorescence, 3D fluorescence spectra, circular dichroism and Fourier transform infrared spectroscopy are used to determine the structural change in HSA on interaction with 3a . Förster resonance energy transfer analysis shows that the binding distance (r₀ = 2.78 nm) between HSA (Trp214) and 3a is within the of range 2–8 nm for quenching to occur. The molecular docking study also confirms that 3a is located in subdomain IIA (site I) of HSA and is stabilized by hydrogen bonding and hydrophobic forces.     

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.     

Simultaneous chiral analysis of amphetamine‐type stimulants and ephedrine by capillary electrophoresis coupled to time‐of‐flight mass spectrometry

This study focused on the chiral characteristics of methamphetamine seizures in Shanghai for inferring the synthetic pathways of drugs. Capillary electrophoresis coupled to time‐of‐flight mass spectrometry was used for simultaneous chiral separation of amphetamine‐type stimulants and ephedrine, including S(+)‐amphetamine/R(?)‐amphetamine, S(+)‐methamphetamine/R(?)‐methamphetamine, (±)‐MDA (3,4‐methylenedioxyamphetamine), (±)‐MDMA (3,4‐methylenedioxymethamphetamine), (±)‐MDEA (3,4‐methylenedioxy‐N‐ethylamphetamine), d,l‐N‐ethylamphetamine, methylephedrine/methylpseudoephedrine, and 1S,2R(+)‐ephedrine/(?)‐ephedrine. The running buffer was 50‐mM ammonium formate (pH 2.2 was adjusted by 1‐M formic acid) containing 0.26% highly sulfated γ‐cyclodextrin as the chiral selector. All enantiomers were well resolved within 40 minutes by capillary electrophoresis at 20 kV in an uncoated fused‐silica capillary (50‐μm I.D. × 375‐μm O.D. × 90‐cm length) and detected by micro time‐of‐flight mass spectrometry. Twenty seized methamphetamine samples were determined by the established method. They were classified into two groups through their chiral characteristics.     

Liquid and subcritical fluid chromatographic enantioseparation of Nα‐Fmoc proteinogenic amino acids on Quinidine‐based zwitterionic and anion‐exchanger type chiral stationary phases. A comparative study

Stereoselective high‐performance liquid chromatographic and subcritical fluid chromatographic separations of 19 N^α‐Fmoc proteinogenic amino acid enantiomers were carried out by using Quinidine ‐based zwitterionic and anion‐exchanger‐type chiral stationary phases Chiralpak ZWIX(−) and QD‐AX. For optimization of retention and enantioselectivity, the ratio of bulk solvent components (MeOH/MeCN, H₂O/MeOH, or CO₂/MeOH) and the nature and concentration of the acid and base additives (counter‐ and co‐ions) were systematically varied. The effect of column temperature on the enantioseparation was investigated and thermodynamic parameters were calculated from the van't Hoff plots ln α vs. 1/T. The thermodynamic parameters revealed that the enantioseparations were enthalpy‐driven. The elution sequence was determined in all cases and with the exception of Fmoc‐Cys(Trt)‐OH, it was identical on both chiral stationary phases whereby the L‐enantiomers eluted before the D‐enantiomers.     

Experimental and Model Studies on Continuous Separation of 2‐Phenylpropionic Acid Enantiomers by Enantioselective Liquid–Liquid Extraction in Centrifugal Contactor Separators

Multistage enantioselective liquid–liquid extraction (ELLE) of 2‐phenylpropionic acid (2‐PPA) enantiomers using hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as extractant was studied experimentally in a counter‐current cascade of centrifugal contactor separators (CCSs). Performance of the process was evaluated by purity (enantiomeric excess, ee) and yield (Y). A multistage equilibrium model was established on the basis of single‐stage model for chiral extraction of 2‐PPA enantiomers and the law of mass conservation. A series of experiments on the extract phase/washing phase ratio (W/O ratio), extractant concentration, the pH value of aqueous phase, and the number of stages was conducted to verify the multistage equilibrium model. It was found that model predictions were in good agreement with the experimental results. The model was applied to predict and optimize the symmetrical separation of 2‐PPA enantiomers. The optimal conditions for symmetric separation involves a W/O ratio of 0.6, pH of 2.5, and HP‐β‐CD concentration of 0.1 mol L^?1 at a temperature of 278 K, where ee_eq (equal enantiomeric excess) can reach up to 37% and Y_eq (equal yield) to 69%. By simulation and optimization, the minimum number of stages was evaluated at 98 and 106 for ee_eq > 95% and ee_eq > 97%. Chirality 28:235–244, 2016. © 2016 Wiley Periodicals, Inc. Research highlights are as follows:

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Enantioselective liquid‐liquid extraction of 3‐chloro‐phenylglycine enantiomers using (S,S)‐DIOP as extractant

(S,S)‐DIOP, a common catalyst used in asymmetric reaction, was adopted as chiral extractant to separate 3‐chloro‐phenylglycine enantiomers in liquid‐liquid extraction. The factors affecting extraction efficiency were studied, including metal precursors, organic solvents, extraction temperature, chiral extractant concentration, and pH of aqueous phase. (S,S)‐DIOP‐Pd exhibited good ability to recognize 3‐chloro‐phenylglycine enantiomers, and the operational enantioselectivity (α) is 1.836. The highest performance factor (pf) was obtained under the condition of extraction temperature of 9.1°C, (S,S)‐DIOP‐Pd concentration of 1.7 mmol/L, and pH of aqueous phase of 7.0. In addition, the possible recognition mechanism of (S,S)‐DIOP‐Pd towards 3‐chloro‐phenylglycine enantiomers was discussed.     

Enantiomeric separation of β‐blockers and tryptophan using heparin as stationary and pseudostationary phases in capillary electrophoresis

The separation methods of the enantiomers of two β‐blockers and tryptophan were studied using capillary electrochromatography with heparin covalently as well as non‐covalently, bonded onto the capillary inner wall as stationary phase and electrokinetic chromatography with heparin as pseudostationary phase. In the case of heparin, used as a stationary phase, the method was unable to resolve enantiomers in both cases β‐blockers and tryptophan. On the other hand, when heparin was used as a pseudostationary phase, the resolution of the enantiomers was obtained only with 3‐aminopropyltriethoxysilane which were immobilised onto the inner phase of the capillary. The results of this study let us infer that the electrostatic, hydrophobic, and steric interactions were involved in the separation mechanisms. The separation was achieved in less than 10 minutes under the optimized conditions: 30 mM phosphate buffer (pH 2.5) with the adding of 15 mg/mL of heparin at 15°C and 10 kV. The usefulness of heparin as a chiral selector both in electrokinetic chromatography using 3‐aminopropyltriethoxysilane attached to the capillary was demonstrated for the first time. The developed method was powerful, sensitive, and fast, and it could be considered an important alternative to conventional methods used for chiral separation.     

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     

Chiral Separation of the Clinically Important Compounds Fucose and Pipecolic Acid Using CE: Determination of the Most Effective Chiral Selector

In this study, simple electrophoretic methods were developed for the chiral separation of the clinically important compounds fucose and pipecolic acid. In recent years, these analytes, and particularly their individual enantiomers, have attracted considerable attention due to their role in biological functions and disorders. The detectability and sensitivity of pipecolic acid and fucose were improved by reacting them with fluorenylmethyloxycarbonyl chloride (FMOC‐Cl) and 5‐amino‐2‐naphthalene‐sulfonic acid (ANSA), respectively. The enantioseparation conditions were optimized by initially investigating the type of the chiral selector. Different chiral selectors, such as polymeric surfactants and cyclodextrins, were used and the most effective ones were determined with regard to resolution and analysis time. A 10‐mM β‐cyclodextrin was able to separate the enantiomers of ANSA‐DL‐fucose and the polymeric surfactant poly(sodium N‐undecanoyl‐LL‐leucine‐valinate) was able to separate the enantiomers of FMOC‐DL‐pipecolic acid, with resolution values of 3.45 and 2.78, respectively. Additional parameters, such as the concentration and the pH of the background electrolyte (BGE), the concentration of the chiral selector, and the addition of modifiers were examined in order to optimize the separations. The addition of the chiral ionic liquid D‐alanine tert‐butyl ester lactate into the BGE was also investigated, for the first time, in order to improve resolution of the enantiomers. Chirality 25:556–560, 2013. © 2013 Wiley Periodicals, Inc.