Preparative enantioseparation of loxoprofen precursor by recycling countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as a chiral selector

Recycling countercurrent chromatography was successfully applied to the resolution of 2‐(4‐bromomethylphenyl)propionic acid, a key synthetic intermediate for synthesis of nonsteroidal anti‐inflammatory drug loxoprofen, using hydroxypropyl‐β‐cyclodextrin as chiral selector. The two‐phase solvent system composed of n‐hexane/n‐butyl acetate/0.1 mol/L citrate buffer solution with pH 2.4 (8:2:10, v/v/v) was selected. Influence factors for the enantioseparation were optimized, including type of substituted β‐cyclodextrin, concentration of hydroxypropyl‐β‐cyclodextrin, separation temperature, and pH of aqueous phase. Under optimized separation conditions, 50 mg of 2‐(4‐bromomethylphenyl)propionic acid was enantioseparated using preparative recycling countercurrent chromatography. Technical details for recycling elution mode were discussed. The purities of both the S and R enantiomers were over 99.0% as determined by high‐performance liquid chromatography. The enantiomeric excess of the S and R enantiomers reached 98.0%. The recovery of the enantiomers from eluted fractions was 40.8–65.6%, yielding 16.4 mg of the S enantiomer and 10.2 mg of the R enantiomer. At the same time, we attempted to enantioseparate the anti‐inflammatory drug loxoprofen by countercurrent chromatography and high‐performance liquid chromatography using a chiral mobile phase additive. However, no successful enantioseparation was achieved so far.     

Stereoisomer separation of flavanones and flavanone‐7‐O‐glycosides by means of nanoliquid chromatography employing derivatized β‐cyclodextrins as mobile‐phase additive

A nanoliquid chromatographic method for the stereoisomer separation of some flavanone aglycones and 7‐O‐glycosides has been proposed employing a C18 capillary column and a chiral mobile‐phase additive such as cyclodextrin. The chiral separation of eriodictyol, naringenin, and hesperitin was obtained by addition of carboxymethyl‐β‐cyclodextrin to the mobile phase, whereas eriocitrin, naringin, narirutin, and hesperidin diastereoisomers were resolved by using sulfobutyl ether‐β‐cyclodextrin. The influence of the composition of the mobile phase, the length of the capillary column, and the flow rate on the chiral recognition were investigated. At optimum conditions, baseline separation for the selected aglycones and glycosylated forms were achieved with a mobile phase consisting of 50 mM sodium acetate buffer pH 3 and 30% methanol containing 20 mM of carboxymethyl‐β‐cyclodextrin and 10 mM of sulfobutyl ether‐β‐cyclodextrin, respectively. Precision, linearity, and sensitivity of the method were tested. Limits of detection and quantification for the studied flavanone glycosides were in the range 1.3‐2.5 and 7.5‐12.5 µg/mL, respectively. The method was used for the determination of the diastereomeric composition of the flavanone‐7‐O‐glycosides in Citrus juices after solid‐phase extraction procedure.     

Chiral separation of brompheniramine enantiomers by recycling high‐speed countercurrent chromatography using carboxymethyl‐β‐cyclodextrin as a chiral selector

A recycling high‐speed countercurrent chromatography protocol was proposed for the enantioseparation of brompheniramine by employing β‐cyclodextrin derivatives as a chiral selector. The two‐phase solvent system of n‐hexane/isobutyl acetate/0.10 mol/L phosphate buffer solution with a volume ratio of 2:4:6 was selected by a series of extraction experiments. Factors that affected the distribution of the enantiomers over the two‐phase system (e.g., the type and concentration of β‐cyclodextrin derivatives = pH value of the aqueous solution, and the separation temperature) were also investigated. In addition, the theory of thermodynamics is applied to verify the feasibility of the enantioseparation process and the corresponding results demonstrate that this separation process is feasible. The optimized conditions include carboxymethyl‐β‐cyclodextrin concentration of 0.010 mol/L, pH of 7.5, and temperature of 5°C. Under the optimal conditions, the purities of both monomer molecules were over 99%, and the recovery yields were 88% for (+)‐brompheniramine and 85% for (–)‐brompheniramine, respectively.     

Separation of epimeric aromatic acid (−)‐menthol esters by countercurrent chromatography using hydroxypropyl‐β‐cyclodextrin as an additive

The separation of ten epimeric aromatic acid (−)‐menthol esters by countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as the mobile phase additive was investigated, and methods for the analysis of all the epimeric esters by reversed‐phase high‐performance liquid chromatography were established. A biphasic solvent system composed of n‐hexane/20–70% methanol containing 50 mmol/L of hydroxypropyl‐β‐cyclodextrin (1:1, v/v) was selected, which provided high separation factors for five of the epimeric esters, and successful separations by countercurrent chromatography were achieved. The complete separation of five pairs of epimeric ester was obtained with the purity being over 98% for each peak fractions, as determined by high‐performance liquid chromatography. The recovery of each analyte from the eluted fractions reached around 80–88%.     

Stereoselective separation of (1S, 4S)‐sertraline from medicinal reaction mixtures by countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as stereoselective selector

Four stereoisomeric components were produced during the synthesis of the antidepressant drug (1S, 4S)‐sertraline hydrochloride due to the two chiral carbon centers in its chemical structure, including (1S, 4S), (1R, 4R), (1S, 4R), and (1R, 4S)‐isomer. Stereoselective separation of the target isomer (1S, 4S)‐sertraline from the medicinal reaction mixtures by countercurrent chromatography using hydroxypropyl‐β‐cyclodextrin as the stereoselective selector was investigated. A biphasic solvent system composed of n‐hexane/0.20 mol/L phosphate buffer solution with pH 7.6 containing 0.10 mol/L of hydroxypropyl‐β‐cyclodextrin (1:1, v/v) was selected for separation of cis‐sertraline and trans‐sertraline using reverse phase elution mode and (1S, 4S)‐sertraline was separated with (1R, 4R)‐sertraline using recycling elution mode. A fabricated in‐house analytical countercurrent chromatographic apparatus was used for optimization of the separation conditions. Stationary phase retention and peak resolution were investigated for separation of cis‐sertraline and trans‐sertraline by the analytical apparatus.     

Enantioseparation of pheniramine enantiomers by high‐speed countercurrent chromatography using β‐cyclodextrin derivatives as a chiral selector

The enantioselective separation of pheniramine was studied by a high‐speed countercurrent chromatography method using β‐cyclodextrin derivatives as a chiral selector. Several key variables, for instance, type of organic solvent and chiral selector, concentration of chiral selector, pH value of aqueous phase, and temperature on the enantioselectivity, were investigated systematically by liquid–liquid extraction experiments. Combining the results of extraction experiments and high‐speed countercurrent chromatography, the most suitable conditions for separation of pheniramine enantiomers were obtained with the two‐phase system that consisted of isobutyl acetate/aqueous phase, containing 0.02 mol/L carboxymethyl‐β‐cyclodextrin, pH 8.50 at 278.15 K. Under the optimal conditions, pheniramine enantiomer was successfully resolved after four cycles of high‐speed countercurrent chromatography. By using high‐performance liquid chromatography to analyze the fractions, the purities of both (+)‐pheniramine and (–)‐pheniramine were over 99% and the recovery of this method was up to 85–90%.     

Capillary electrophoretic enantioseparation of m‐nisoldipine using two different β‐cyclodextrins

The methods for the enantioseparation of m‐nisoldipine, a new 1,4‐dihydropyridine calcium ion antagonist, were developed. The elaborated methods of m‐nisoldipine enantiomers separation were successfully performed using an anionic CD–sulfobutyl ether‐β‐CD (SBE‐β‐CD) or carboxymethyl‐β‐CD as chiral selector. However, the results indicated that SBE‐β‐CD was a better chiral selector for enantioseparation of the neutral m‐nisoldipine. Furthermore, comparing the two SBE‐β‐CDs, the derivative with a higher degree of substitution (DS) of 7.0 induced better enantioresolution than the one with low DS (4.0). In addition, possible chiral recognition mechanisms of dihydropyridines were discussed.     

Enantioseparation of 3‐phenyllactic acid by chiral ligand exchange countercurrent chromatography

3‐Phenyllactic acid is an antimicrobial compound with broad‐spectrum activity against various bacteria and fungus. The observed difference in pharmacological activity between optical isomeric 3‐phenyllactic acid necessitates a method for enantioseparation. Chiral ligand exchange countercurrent chromatography was investigated for the enantioseparation of 3‐phenyllactic acid with a synthesized chiral ligand. A two‐phase solvent system was composed of n‐butanol/hexane/water (0.4:0.6:1, v/v/v) to which N‐n‐dodecyl‐l ‐hydroxyproline was added to the organic phase as chiral ligand and cupric acetate was added in the aqueous phase as a transitional metal ion. The influence factors were optimized by enantioselective liquid–liquid extraction. Baseline enantioseparation of racemic 3‐phenyllactic acid by analytical high‐speed countercurrent chromatography was achieved. The optical purities of enantiomeric 3‐phenyllactic acid reached 99.0%, as determined by chiral high‐performance liquid chromatography.     

Separation of phenylsuccinic acid enantiomers using biphasic chiral recognition high‐speed countercurrent chromatography

High‐speed countercurrent chromatography (HSCCC) combined with biphasic chiral recognition was successfully applied to the resolution of phenylsuccinic acid enantiomers. d ‐Isobutyl tartrate and hydroxypropyl‐β‐cyclodextrin were employed as lipophilic and hydrophilic selectors dissolved in the organic stationary phase and aqueous mobile phase, respectively. The two‐phase solvent system was made up of n‐hexane/methyl tert‐butyl ether/water (0.5:1.5:2, v/v/v). Impacts of the type and concentration of chiral selectors, the pH value of the aqueous phase solution as well as the temperature on the separation efficiency were investigated. By means of preparative HSCCC, pure enantiomer was obtained by separating 810 mg of racemate with a purity >99.5% and a recovery rate between 82 and 85%. The experimental results indicate that biphasic recognition HSCCC provide a promising means for efficient separation of racemates.     

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

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

Enantioseparation of chiral aromatic acids by multiple dual mode counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin as chiral selector

This work concentrates on extending the utilization of multiple dual mode (MDM) counter‐current chromatography in chiral separations. Two aromatic acids, 2‐(6‐methoxy‐2‐naphthyl)propionic acid (NAP) and 2‐phenylpropionic acid (2‐PPA), were enantioseparated by MDM counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as chiral selector. The two‐phase solvent systems consisting of n‐hexane/ethyl acetate 0.1 mol/L phosphate buffer pH 2.67 containing 0.1 mol/L HP‐β‐CD (7.5:2.5:10 for NAP and 7:3:10 for 2‐PPA, v/v/v) were used. Conventional MDM and modified MDM were compared according to peak resolution under current separation mechanism. The influence of elution time after the first‐phase inversion and number of cycles for MDM were investigated. Peak resolution of NAP and 2‐PPA increased from 0.62 to 1.05 and 0.72 to 0.84, respectively, using optimized MDM conditions. Being an alternative elution method for counter‐current chromatography, MDM elution greatly improved peak resolution in chiral separations.     

Synergetic mechanism and enantioseparation of aromatic β‐amino acids by biphasic chiral high‐speed counter‐current chromatography

A biphasic chiral recognition system based on chiral ligand exchange with Cu(II)‐N‐n‐dodecyl‐L‐proline and hydroxypropyl‐β‐cyclodextrin as an additive was developed to enantioseparate aromatic β‐amino acids by high‐speed counter‐current chromatography. The biphasic chiral recognition system was established with an n‐butanol/water (1:1, v/v) solvent system by adding N‐n‐dodecyl‐L‐proline and Cu(II) ions to the organic phase and hydroxypropyl‐β‐cyclodextrin to the aqueous phase. Several separation parameters, such as temperature, pH value, and chiral selector concentration, were systematically investigated by enantioselective liquid–liquid extraction. Under the optimal separation conditions, 54.5 mg of (R,S)‐β‐phenylalanine and 74.3 mg of (R,S)‐β‐3,4‐dimethoxyphenylalanine were baseline enantioseparated. More importantly, the synergistic enantiorecognition mechanism, based on the Cu(II)‐N‐n‐dodecyl‐L‐proline and hydroxypropyl‐β‐cyclodextrin, was discussed for the first time.     

Liquid chromatography with mass spectrometry enantioseparation of pomalidomide on cyclodextrin‐bonded chiral stationary phases and the elucidation of the chiral recognition mechanisms by NMR spectroscopy and molecular modeling

A sensitive and validated liquid chromatography with mass spectrometry method was developed for the enantioseparation of the racemic mixture of pomalidomide, a novel, second‐generation immunomodulatory drug, using β‐cyclodextrin‐bonded stationary phases. Four cyclodextrin columns (β‐, hydroxypropyl‐β‐, carboxymethyl‐β‐, and sulfobutyl‐β‐cyclodextrin) were screened and the effects of eluent composition, flow rate, temperature, and organic modifier on enantioseparation were studied. Optimized parameters, offering baseline separation (resolution = 2.70 ± 0.02) were the following: β‐cyclodextrin stationary phase, thermostatted at 15°C, and mobile phase consisting of methanol/0.1% acetic acid 10:90 v/v, delivered with 0.8 mL/min flow rate. For the optimized parameter at multiple reaction monitoring mode 274.1–201.0 transition with 20 eV collision energy and 100 V fragmentor voltage the limit of detection and limit of quantitation were 0.75 and 2.00 ng/mL, respectively. Since enantiopure standards were not available, elution order was determined upon comparison of the circular dichroism signals of the separated pomalidomide enantiomers with that of enantiopure thalidomide. The mechanisms underlying the chiral discrimination between the enantiomers were also investigated. Pomalidomide‐β‐cyclodextrin inclusion complex was characterized using nuclear magnetic resonance spectroscopy and molecular modeling. The thermodynamic aspects of chiral separation were also studied.     

Chiral capillary electrophoresis with cationic pyrrolidinium‐β‐cyclodextrin derivatives as chiral selectors

New single‐isomer, cationic β‐cyclodextrins, including mono‐6‐deoxy‐6‐pyrrolidine‐β‐cyclodextrin chloride (pyCDCl), mono‐6‐deoxy‐6‐(N‐methyl‐pyrrolidine)‐β‐cyclodextrin chloride (N‐CH₃‐pyCDCl), mono‐6‐deoxy‐6‐(N‐(2‐hydroxyethyl)‐pyrrolidine)‐β‐cyclodextrin chloride (N‐EtOH‐pyCDCl), mono‐6‐deoxy‐6‐(2‐hydroxymethyl‐pyrrolidine)‐β‐cyclodextrin chloride (2‐MeOH‐pyCDCl) were synthesized and used as chiral selectors in capillary electrophoresis for the enantioseparation of carboxylic and hydroxycarboxylic acids and dansyl amino acids. The unsubstituted pyCDCl exhibited the greatest resolving ability. Most analytes were resolved over a wide range of pH from 6.0 to 9.0 with this chiral selector. In general, increasing pH led to a decrease in resolution. The effective mobilities of all the analytes were found to decrease with increasing CD concentration. The optimal concentration for most carboxylic acids and dansyl amino acid was in the range 5–7.5 mM and >15 mM for hydroxycarboxylic acids. ¹H NMR experiments provided direct evidence of inclusion in the CD cavity.     

高效液相色谱手性流动相添加法拆分阿卓乳酸对映体

采用C₁₈反相色谱柱,以磺丁基醚-β-环糊精（SBE-β-CD）作为手性流动相添加剂,建立了阿卓乳酸对映体的高效液相色谱拆分方法。考察了环糊精衍生物类型、手性添加剂浓度、流动相pH、流速和柱温对手性分离的影响,同时探讨了高效液相色谱采用磺丁基醚-β-环糊精分离阿卓乳酸对映体的分离机制及包结常数,确定了色谱条件为YMC-Pack ODS-A C₁₈色谱柱（250 mm×4.6 mm,5 μm）,流动相为含25 mmol/LSBE-β-CD的0.1 mol/L磷酸盐缓冲液（pH 2.68）-乙腈（90：10,v/v）,流速为0.6 mL/min,柱温为30 ℃,紫外检测波长为220 nm。对映体的保留时间分别为26.65和28.28 min,分离度为1.68。本方法分离度好,简便易行,且比使用手性固定相分离更加经济。     

Efficient preparative separation of 6‐(4‐aminophenyl)‐5‐methyl‐4, 5‐dihydro‐3(2H)‐pyridazinone enantiomers on polysaccharide‐based stationary phases in polar organic solvent chromatography and supercritical fluid chromatography

6‐(4‐Aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone is a key synthetic intermediate for cardiotonic agent levosimendan. Very few studies address the use of chiral stationary phases in chromatography for the enantioseparation of this intermediate. This study presents two efficient preparative methods for the isolation of (R)(?)‐6‐(4‐aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone in polar organic solvent chromatography and supercritical fluid chromatography using polysaccharide‐based chiral stationary phases and volatile organic mobile phases without additives in isocratic mode. Under optimum conditions, Chiralcel OJ column showed the best performance (α = 1.71, Rs = 5.47) in polar organic solvent chromatography, while Chiralpak AS column exhibited remarkable separations (α = 1.81 and Rs = 6.51) in supercritical fluid chromatography with an opposite enantiomer elution order. Considering the sample solubility, runtime and solvent cost, the preparations were carried out on Chiralcel OJ column and Chiralpak AS column (250 × 20 mm i.d.; 10 µm) in polar organic mode and supercritical fluid chromatography mode with methanol and CO₂/methanol as mobile phases, respectively. By utilizing the advantages of chromatographic techniques and polysaccharide‐based chiral stationary phases, this work provides two methods for the fast and economic preparation of (R)(?)‐6‐(4‐aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone, which are suitable for the pharmaceutical industry.     

Combined use of l‐alanine tert butyl ester lactate and trimethyl‐β‐cyclodextrin for the enantiomeric separations of 2‐arylpropionic acids nonsteroidal anti‐inflammatory drugs

In this study, a new CE method, employing a binary system of trimethyl‐β‐CD (TM‐β‐CD) and a chiral amino acid ester‐based ionic liquid (AAIL), was developed for the chiral separation of seven 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs (NSAIDs). In particular, the enantioseparation of ibuprofen, ketoprofen, carprofen, indoprofen, flurbiprofen, naproxen, and fenoprofen was improved significantly by supporting the BGE with the chiral AAIL l ‐alanine tert butyl ester lactate (l ‐AlaC₄Lac). Parameters, such as concentrations of TM‐β‐CD and l ‐AlaC₄Lac, and buffer pH, were systematically examined in order to optimize the chiral separation of each NSAID. It was observed that the addition of the AAIL into the BGE improved both resolution and efficiency significantly. After optimization of separation conditions, baseline separation (R_s>1.5) of five of the analytes was achieved in less than 11 min, while the resolution of ibuprofen and flurbiprofen was approximately 1.2. The optimized enantioseparation conditions for all analytes involve a BGE of 5 mM sodium acetate/acetic acid (pH 5.0), an applied voltage of 30 kV, and a temperature of 20°C. In addition, the results obtained by computing the %‐RSD values of the EOF and the two enantiomer peaks, demonstrated excellent run‐to‐run, batch‐to‐batch, and day‐to‐day reproducibilities.     

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

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

The degree of substitution affects the enantioselectivity of sulfobutylether‐β‐cyclodextrin chiral stationary phases

Three chiral stationary phases were prepared by dynamic coating of sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) with different degrees of substitution, onto strong anion‐exchange stationary phases. The enantioselective potential and stability of newly prepared chiral stationary phases were examined using a set of structurally different chiral analytes. Measurements were performed in RP‐HPLC. Mobile phases consisted of methanol/formic acid, pH 2.10, and methanol/10 mM ammonium acetate buffer, pH 4.00, in various volume ratios. SBE‐β‐CDs with degrees of substitution (DS) 4, 6.3, and 10 proved suitable for the enantioseparation of 14, 11, and 8 analytes, respectively. The SBE‐β‐CD DS 4 based chiral stationary phase enabled the enantioseparation of the nearly all basic and neutral compounds. Chiral stationary phases with higher sulfobutylether‐β‐cyclodextrin substitution (especially DS 10) yielded higher enantioresolution values for acidic compounds.     

New single isomer negatively charged β‐cyclodextrin derivatives as chiral selectors in capillary electrophoresis

To improve resolution power of chiral selector and enantiomeric peak efficiency in CE, single isomer negatively charged β‐CD derivatives, mono(6‐deoxy‐6‐sulfoethylthio)‐β‐CD (SET‐β‐CD) bearing one negative charge and mono[6‐deoxy‐6‐(6‐sulfooxy‐5,5‐bis‐sulfooxymethyl)hexylthio]‐β‐CD (SMHT‐β‐CD) carrying three negative charges, were synthesized. The structure of these two β‐CD derivatives was confirmed by ¹H NMR and MS. SET‐β‐CD and SMHT‐β‐CD successfully resolved the enantiomers of several basic model compounds. SMHT‐β‐CD provided for a significantly greater enantioseparation than SET‐β‐CD at lower concentrations. This appears to be due to the higher binding affinity of SMHT‐β‐CD to the model compounds and the wider separation window resulting from an increased countercurrent mobility of the selector. Overall, the new chiral selectors provided enantioseparations with high peak efficiency while avoiding peak distortion due to polydispersive and electrodispersive effects. The information obtained from an apparent binding constant study suggested that the enantioseparation of the model compounds followed the predictions of charged resolving agent migration model and that the observed degree of enantioseparation difference were due to the magnitude of differences in both enantiomer‐chiral selector binding affinities (ΔK) and the mobilities of the complexed enantiomers (Δμ_c).