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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Resolution of Rac‐Bambuterol via Diastereoisomeric Salt Formation with o‐Chloromandelic Acid and Differences in the Enantiomers' Pharmacodynamical Effects in Guinea Pigs and Beagles

In this study an enantioseparation method for rac‐bambuterol (5‐(2‐(tert‐butylamino)‐1‐hydroxyethyl)‐1,3‐phenylene bis(dimethylcarbamate)) via diastereoisomeric salt formation with o‐chloromandelic acid was developed. The enantiomeric excess (ee) values and chemical purities of the desired products were confirmed by high‐performance liquid chromatography (HPLC) using chiral stationary phase and reverse‐phase HPLC analyses, respectively. The ee values and the chemical purities both exceeded 99%. Animal experiments showed that (R)‐bambuterol was a potent inhibitor for histamine‐induced asthma reactions. (S)‐bambuterol was ineffective in relaxing the airways. Both enantiomers increased heart rates in beagles. Therefore, replacing rac‐bambuterol with (R)‐bambuterol could be beneficial for asthma patients. Chirality 28:306–312, 2016. © 2016 Wiley Periodicals, Inc.     

Studies on Enantioselective Liquid–Liquid Extraction of Amino‐(4‐nitro‐phenyl)‐acetic Acid Enantiomers: Modeling and Optimization

BINAP‐metal complexes were prepared as extractant for enantioselective liquid–liquid extraction (ELLE) of amino‐(4‐nitro‐phenyl)‐acetic acid (NPA) enantiomers. The influence of process variables, including types of organic solvents and metal precursor, concentration of ligand, pH, and temperature on the efficiency of the extraction, were investigated experimentally. An interfacial reaction model was established for insightful understanding of the chiral extraction process. Important parameters required for the model were determined. The experimental data were compared with model predictions to verify the model prediction, It was found that the interfacial reaction model predicted the experimental results accurately. By modeling and experiment, an optimal extraction condition with pH of 7 and host (extractant) concentration of 1 mmol/L was obtained and high enantioselectivity (α_op) of 3.86 and performance factor (pf) of 0.1949 were achieved. Chirality 26:79–87, 2014. © 2013 Wiley Periodicals, Inc.     

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.     

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.     

Preparative Enantioseparation of β‐Substituted‐2‐Phenylpropionic Acids by Countercurrent Chromatography With Substituted β‐Cyclodextrin as Chiral Selectors

Preparative enantioseparation of four β‐substituted‐2‐phenylpropionic acids was performed by countercurrent chromatography with substituted β‐cyclodextrin as chiral selectors. The two‐phase solvent system was composed of n‐hexane‐ethyl acetate‐0.10 mol L‐1 of phosphate buffer solution at pH 2.67 containing 0.10 mol L^‐1 of hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) or sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD). The influence factors, including the type of substituted β‐cyclodextrin, composition of organic phase, concentration of chiral selector, pH value of the aqueous phase, and equilibrium temperature were optimized by enantioselective liquid–liquid extraction. Under the optimum separation conditions, 100 mg of 2‐phenylbutyric acid, 100 mg of tropic acid, and 50 mg of 2,3‐diphenylpropionic acid were successfully enantioseparated by high‐speed countercurrent chromatography, and the recovery of the (±)‐enantiomers was in the range of 90–91% for (±)‐2‐phenylbutyric acid, 91–92% for (±)‐tropic acid, 85–87% for (±)‐2,3‐diphenylpropionic acid with purity of over 97%, 96%, and 98%, respectively. The formation of 1:1 stoichiometric inclusion complex of β‐substituted‐2‐phenylpropionic acids with HP‐β‐CD was determined by UV spectrophotometry and the inclusion constants were calculated by a modified Benesi‐Hildebrand equation. The results showed that different enantioselectivities among different racemates were mainly caused by different enantiorecognition between each enantiomer and HP‐β‐CD, while it might be partially caused by different inclusion capacity between racemic solutes and HP‐β‐CD. Chirality 27:795–801, 2015. © 2015 Wiley Periodicals, Inc.     

Construction and application of a model on the resolution of tropic acid enantiomers by enantioselective liquid-liquid extraction in centrifugal contactor separators

This paper reports on the construction and application of a mathematical model on multistage liquid-liquid extraction of tropic acid (TA) enantiomers in centrifugal contactor separators (CCSs). An efficient extraction system was obtained, where HE-β-CD and butyl acetate was selected as the best extractant and organic solvent. The mechanism of reactive extraction of TA enantiomers by HE-β-CD was proposed and the thermodynamic constants such as physical partition coefficient and reactive equilibrium constants were obtained, which combined with the law of mass conservation gave the basis for construction of the model. The model was applied to predict and optimize the symmetrical separation of TA enantiomers. The optimal condition was obtained as follows, O/W volume ratio of 3.0, pH of 3.00 and HE-β-CD concentration of 0.05 mol/L, where ee_eq (equal enantiomeric excess) can reach up to 42%. The simulated results reveal that the minimum number of stages for ee_eq > 97% and ee_eq > 99% was 56 and 78, respectively.     

Process optimization of chiral extraction of 2,3-diphenylpropionic acid by experiment and simulation

This paper reports the process optimization of chiral extraction of 2,3-diphenylpropionic acid (2,3-2-PPA) enantiomers by experiment and simulation in centrifugal contactor separators. An efficient extraction system was obtained firstly through single-stage extraction experiments and single-stage extraction model, where sulfobutylether-β-cyclodextrin (SBE-β-CD) and 1,2-dichlorethane were selected as the optimal extractant and organic solvent, respectively. The mechanism of the extraction system was proposed and the thermodynamic constants such as physical partition coefficient and reactive equilibrium constants were obtained. Based on phase and reactive equilibrium as well as the law of mass conservation, a model describing the fractional extraction process was acquired. The process of symmetrical separation of 2,3-2-PPA enantiomers was optimized by the fractional extraction model. The optimal conditions composed of flow rate ratio (W/O) of 3.0, pH of 3.00 and SBE-β-CD concentration of 0.10 mol/L were obtained. Under this case, equal enantiomeric excess (ee_eq) can reach up to 37% at 10 stages. The simulated results reveal that the minimum series for ee_eq > 97% and ee_eq > 99% was 78 and 102, respectively.     

Copper‐Chiral Camphor β‐Amino Alcohol Complex Catalyzed Asymmetric Henry Reaction

Four novel chiral amino alcohols were synthesized from D‐(+)‐camphor and utilized as ligands in a Cu(I)‐catalyzed asymmetric Henry reaction. The reactions were carried out under mild conditions with excellent enantioselectivities and moderate yields without the exclusion of air or moisture. The highest enantioselectivity was observed up to 94% enantiomeric excess (ee) with ligand L1 in toluene at room temperature. Chirality 27:761–765, 2015. © 2015 Wiley Periodicals, Inc.     

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.     

HPLC‐PDA‐ORD Bioassay of S‐(+) and R‐(−) Clopidogrel on Rat Dried Blood Spots

A simple and rapid chiral high‐performance liquid chromatography (HPLC) method was developed and validated for bioanalysis of clopidogrel enantiomers on rat dried blood spots (DBS). Clopidogrel enantiomers were extracted from DBS using ethanol: methanol (80:20, v/v) and separated on a Chiralcel OJ‐H column containing cellulose tris (4‐methly benzoate) as a polysaccharide stationary phase using n‐hexane–ethanol‐diethylamine (70:30, 0.1 v/v) as a mobile phase at a flow rate of 1.0 mL/min. The detection was carried out at 220 nm using a photodiode array (PDA) detector while the elution order of the enantiomers was determined by a polarimeter connected to PDA in series. The effect of hematocrit on extraction of clopidogrel enantiomers from DBS was evaluated and no interference from endogenous substances was noticed. The overall accuracy of (R) and (S) enantiomers of clopidogrel from DBS were 91.6 and 89.2%, respectively. The calibration curves were linear over the concentration range of 1–500 µg/mL for both enantiomers. The results show that the method is specific, precise, and reproducible (intra‐ and interday precision relative standard deviations (RSDs) <10.0%). The stability of racemic clopidogrel was performed under all storage conditions and the results were found to be well within the acceptance limits. Chirality 26:102–107, 2014.© 2014 Wiley Periodicals, Inc.     

Enantioselective resolution of 4‐chloromandelic acid by liquid–liquid extraction using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid

A liquid–liquid extraction resolution of 4‐chloro‐mandelic acid (4‐ClMA) was studied by using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid (2‐Cl‐Z‐AA) as a chiral extractant. Important factors affecting the extraction efficiency were investigated, including the type of chiral extractant, pH value of aqueous phase, initial concentration of chiral extractant in organic phase, initial concentration of 4‐ClMA in aqueous phase, and resolution temperature. It was observed that the concentration of (R)‐4‐ClMA was much higher than that of (S)‐4‐ClMA in organic phase due to a higher stability of the complex formed between (R)‐4‐ClMA and 2‐Cl‐Z‐AA. A separation factor (α) of 3.05 was obtained at 0.02 mol/L 2‐Cl‐Z‐Valine dissolved in dichloromethane, pH of 2.0, concentration of 4‐ClMA of 0.11 mmol/Land T of 296.7K.     

Precolumn o‐Phthalaldehyde‐N‐acetyl‐L‐cysteine Derivatization Followed by RP‐HPLC Separation and Fluorescence Detection of Sitagliptin Enantiomers in Rat Plasma

An indirect reversed‐phase high‐performance liquid chromatographic separation and fluorescence detection of sitagliptin enantiomers in rat plasma was developed and validated. Deproteinized rat plasma containing racemic sitagliptin was derivatized with o‐phthalaldehyde and N‐acetyl‐L‐cysteine under alkaline conditions, converted to diastereomers, and separated on a Lichrospher 100 RP‐18e column using 20 mM phosphate buffer and methanol (45:55 v/v) as a mobile phase under isocratic mode of elution at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 330 and 450 nm as excitation and emission wavelengths, respectively. The method was linear in the range of 50–5000 ng/ mL for both enantiomers. The intra‐ and interday accuracy and precision were within the predefined limits of ≤15% at all concentrations. The method was successfully applied to a pharmacokinetic study of sitagliptin after 5 mg/kg oral administration to Wistar rats. Robustness of the method was evaluated using design of experiments. Chirality 25:883–889, 2013. © 2013 Wiley Periodicals, Inc.     

Analysis of Oxcarbazepine and the 10‐Hydroxycarbazepine Enantiomers in Plasma by LC‐MS/MS: Application in a Pharmacokinetic Study

Oxcarbazepine is a second‐generation antiepileptic drug indicated as monotherapy or adjunctive therapy in the treatment of partial seizures or generalized tonic–clonic seizures in adults and children. It undergoes rapid presystemic reduction with formation of the active metabolite 10‐hydroxycarbazepine (MHD), which has a chiral center at position 10, with the enantiomers (S)‐(+)‐ and R‐(?)‐MHD showing similar antiepileptic effects. This study presents the development and validation of a method of sequential analysis of oxcarbazepine and MHD enantiomers in plasma using liquid chromatography with tandem mass spectrometry (LC‐MS/MS). Aliquots of 100 μL of plasma were extracted with a mixture of methyl tert‐butyl ether: dichloromethane (2:1). The separation of oxcarbazepine and the MHD enantiomers was obtained on a chiral phase Chiralcel OD‐H column, using a mixture of hexane:ethanol:isopropanol (80:15:5, v/v/v) as mobile phase at a flow rate of 1.3 mL/min with a split ratio of 1:5, and quantification was performed by LC‐MS/MS. The limit of quantification was 12.5 ng oxcarbazepine and 31.25 ng of each MHD enantiomer/mL of plasma. The method was applied in the study of kinetic disposition of oxcarbazepine and the MHD enantiomers in the steady state after oral administration of 300 mg/12 h oxcarbazepine in a healthy volunteer. The maximum plasma concentration of oxcarbazepine was 1.2 µg/mL at 0.75 h. The kinetic disposition of MHD is enantioselective, with a higher proportion of the S‐(+)‐MHD enantiomer compared to R‐(?)‐MHD and an AUC^0‐12 _{S‐(+)/R‐(?)} ratio of 5.44. Chirality 25:897–903, 2013. © 2013 Wiley Periodicals, Inc.     

Enantiopurity Determination of the Enantiomers of the Triple Reuptake Inhibitor Indatraline

The present study describes the development of two approaches for the determination of the enantiopurity of both enantiomers of indatraline. Initially, a method was developed using different chiral solvating agents (CSAs) for diastereomeric discrimination regarding signal separation in ¹H nuclear magnetic resonance (NMR) spectroscopy, revealing MTPA as a promising choice for the differentiation of the indatraline enantiomers. This CSA was also tested for its ideal molar ratio, temperature, and solvent. Optimized conditions could be achieved that made determination of enantiopurity for (1R,3S)‐indatraline up to 98.9% enantiomeric excess (ee) possible. To quantify even higher enantiopurities, a high‐performance liquid chromatography (HPLC) method based on a modified β‐cyclodextrine phase was established. The influence of buffer type, concentration, pH value, percentage and kind of organic modifier, temperature, injection volume as well as sample solvent on chromatographic parameters was investigated. Afterwards, the reliability of the established HPLC method was demonstrated by validation according to the ICH guideline Q2(R1) regarding specificity, accuracy, precision, linearity, and quantitation limit. The developed method proved to be strictly linear within a concentration range of 1.25–1000 μM for the (1R,3S)‐enantiomer and 1.25‐750 μM for its mirror image that enables a reliable determination of enantiopurities up to 99.75% ee for the (1R,3S)‐enantiomer and up to 99.67% ee for the (1S,3R)‐enantiomer. Chirality 25:923–933, 2013. © 2013 Wiley Periodicals, Inc.     

Chromatographic profiles of tryptophan and kynurenine enantiomers derivatized with (S)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole using LC–MS/MS on a triazole‐bonded column

d ‐ and l ‐Tryptophan (Trp) and d ‐ and l ‐kynurenine (KYN) were derivatized with a chiral reagent, (S )‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS), and were separated enantiomerically by high‐performance liquid chromatography (HPLC) equipped with a triazole‐bonded column (Cosmosil HILIC) using tandem mass spectrometric (MS/MS) detection. Effects of column temperature, salt (HCO₂NH₄) concentration, and pH of the mobile phase in the enantiomeric separation, followed by MS detection of (S )‐DBD‐PyNCS‐d ,l ‐Trp and ‐d ,l ‐KYN, were investigated. The mobile phase consisting of CH₃CN/10 mM ammonium formate in H₂O (pH 5.0) (90/10) with a column temperature of 50–60 °C gave satisfactory resolution (R s) and mass‐spectrometric detection. The enantiomeric separation of d ,l ‐Trp and d ,l ‐KYN produced R s values of 2.22 and 2.13, and separation factors (α) of 1.08 and 1.08, for the Trp and KYN enantiomers, respectively. The proposed LC–MS/MS method provided excellent detection sensitivity of both enantiomers of Trp and KYN (5.1–19 nM).     

Asymmetric Addition of Triethylaluminium to Aromatic Aldehydes Catalyzed by Titanium‐(5,5’‐Biquinoline‐6,6’‐Diol) Complexes

A novel convenient procedure for the resolution of 5,5’‐biquinoline‐6,6’‐diol (BIQOL) was achieved by separating the corresponding diastereomeric mixture of (S)‐(+)‐camphorsulfonates on a semiprepared XDB‐C8 column followed by hydrolysis. The efficient asymmetric addition of triethylaluminium to aromatic aldehydes catalyzed by Ti‐(+)/(–)BIQOL complexes under mild conditions is described. The reactions led to the formation of 1‐arylpropan‐1‐ol in up to 87.5% ee. Chirality 26:268‐271, 2014. © 2014 Wiley Periodicals, Inc.     

Enantioselective Liquid–Liquid Extraction of Zopiclone With Mandelic Acid Ester Derivatives

Enantioselective liquid–liquid extraction of zopiclone was conducted by employing a series of (R)‐mandelic acid esters as chiral extractants. The effects of concentration of extractant, concentration of zopiclone, type of organic solvent, pH value, and temperature on the extraction efficiency were investigated. (R)‐o‐chloromandelic acid propyl ester was demonstrated to be an efficient chiral extractant for zopiclone resolution with a maximum enantioselectivity of 1.6. Chirality 25:952–956, 2013. © 2013 Wiley Periodicals, Inc.     

Enzymatic Resolution of α‐Methyleneparaconic Acids and Evaluation of their Biological Activity

Both enantiomers of three biologically relevant paraconic acids—MB‐3, methylenolactocin, and C75—were obtained with enantioselectivities up to 99% by kinetic enzymatic resolutions. Good enantiomeric excesses were obtained for MB‐3 and methylenolactocin, using α‐chymotrypsin and aminoacylase as enantiocomplementary enzymes, while C75 was resolved with aminoacylase. They all were evaluated for their antiproliferative, antibacterial, and antifungal activities, showing weak effects and practically no difference between enantiomers in each case. At high concentrations (16–64 µg/mL), (–)‐ C75 acted as an antimicrobial agent against Gram‐positive bacteria. Chirality 27:239–246, 2015. © 2015 Wiley Periodicals, Inc.     

Use of a Novel Sub‐2 µm Silica Hydride Vancomycin Stationary Phase in Nano‐Liquid Chromatography. II. Separation of Derivatized Amino Acid Enantiomers

A novel vancomycin silica hydride stationary phase was synthesized and the particles of 1.8 µm were packed into fused silica capillaries of 75 µm internal diameter (I.D.). The chiral stationary phase (CSP) was tested for the separation of some derivatized amino acid enantiomers by using nano‐liquid chromatography (nano‐LC). Some experimental parameters such as the type and the content of organic modifier, the pH, and the concentration of the buffer added to the mobile phase were modified and the effect on enantioselectivity, retention time, and enantioresolution factor was studied. The separation of selected dansyl amino acids (Dns‐AAs), e.g., Asp, Glu, Leu, and Phe in their enantiomers was initially achieved utilizing a mobile phase containing 85% (v/v) methanol (MeOH) and formate buffer measuring the enantioresolution factor and enantioselectivity in the range 1.74–4.17 and 1.39–1.59, respectively. Better results were obtained employing a more polar organic solvent as acetonitrile (ACN) in the mobile phase. Optimum results (Rs 1.41–6.09 and α 1.28–2.36) were obtained using a mobile phase containing formate buffer pH 2.5/water/MeOH/ACN 6:19:12.5:62.5 (v/v/v/v) in isocratic elution mode at flow rate of 130 nL/min. Chirality 27:767–772, 2015. © 2015 Wiley Periodicals, Inc.