Multivariate optimization approach for chiral resolution of drugs using human serum albumin in affinity electrokinetic chromatography-partial filling technique

The enantiomeric resolution of chiral compounds using HSA by means of affinity EKC (AEKC)-partial filling technique is the result of a delicate balance between different experimental variables such as protein concentration, running pH (background electrophoretic buffer, protein and compound solutions) and protein solution plug length. In this paper multivariate optimization approaches for chiral separation of four basic drugs (alprenolol, oxprenolol, promethazine and propranolol) using HSA as chiral selector in AEKC-partial filling technique are studied. The experimental conditions to achieve maximum resolution are optimized using the Box-Behnken experimental design. Partial least squares and pareto charts are used to analyse the main effects on the resolution. The experimental resolutions observed for all compounds studied in optimum conditions agree with the estimated values based on response surface models. The results obtained show that the range of experimental conditions that provided enantioresolution narrows as hydrophobicity of analytes decreases. This fact can be explained by assuming that hydrophobicity controls the interaction of basic compounds with HSA.     

Evaluation of sulfated maltodextrin as a novel anionic chiral selector for the enantioseparation of basic chiral drugs by capillary electrophoresis

Introducing a new class of chiral selectors is an interesting work and this issue is still one of the hot topics in separation science and chirality. In this study, for the first time, sulfated maltodextrin (MD) was synthesized as a new anionic chiral selector and then it was successfully applied for the enantioseparation of five basic drugs (amlodipine, hydroxyzine, fluoxetine, tolterodine, and tramadol) as model chiral compounds using CE. This chiral selector has two recognition sites: a helical structure and a sulfated group which contribute to three corresponding driving forces; inclusion complexation, electrostatic interaction, and hydrogen binding. Under the optimized condition (buffer solution: 50 mM phosphate (pH 3.0) and 2% w/v sulfated MD; applied voltage: 18 kV; temperature: 20°C), baseline enantioseparation was observed for all mentioned chiral drugs. When instead of sulfated MD neutral MD was used under the same condition, no enantioseparation was observed which means the resolution power of sulfated MD is higher than neutral MD due to the electrostatic interaction between sulfated groups and protonated chiral drugs. Also, the countercurrent mobility of negatively charged MD (sulfated MD) allows more interactions between the chiral selector and chiral drugs and this in turn results in a successful resolution for the enantiomers. Furthermore, a higher concentration of neutral MD (approximately five times) is necessary to achieve the equivalent resolution compared with the negatively charged MD.     

Optimization of the LC enantioseparation of chiral pharmaceuticals using cellulose tris(4‐chloro‐3‐methylphenylcarbamate) as chiral selector and polar non‐aqueous mobile phases

The resolving power of a new commercial polysaccharide‐based chiral stationary phase, Sepapak‐4, with cellulose tris(4‐chloro‐3‐methylphenylcarbamate) coated on silica microparticles as chiral selector, was evaluated toward the enantioseparation of ten basic drugs with widely different structures and hydrophobic properties, using ACN as the main component of the mobile phase. A multivariate approach (experimental design) was used to screen the factors (temperature, n‐hexane content, acidic and basic additives) likely to influence enantioresolution. Then, the optimization was performed using a face‐centered central composite design. Complete enantioseparation could be obtained for almost all tested chiral compounds, demonstrating the high chiral discrimination ability of this chiral stationary phase using polar organic mobile phases made up of ACN and containing an acidic additive (TFA or formic acid), 0.1% diethylamine and n‐hexane. These results clearly illustrate the key role of the nature of the acidic additive in the mobile phase.     

Optimization of capillary electrophoretic enantioseparation for basic drugs with native beta-CD as a chiral selector

This study presents the advantages of the 20 microm inner diameter (id) capillary for the enantioseparation of ten basic drugs with native beta-CD as the chiral selector. The apparent binding constants of each enantiomeric pair were determined to calculate the optimum beta-CD concentration ([beta-CD]opt) and the optimization was subsequently carried out. Comparison of the 20 microm id with 50 microm id were made in terms of the results obtained in the optimization and detection limits. Applying the optimum conditions for each compound, reproducible results (RSD from 0-3; n>5) were obtained for the 20 microm id capillary. Although the sensitivity is lower in the 20 microm id capillary, the LOD determined using this capillary is still found to be acceptable for the ten basic drugs studied. Enhanced resolution and faster analysis times were the main advantages observed with the use of this capillary in enantioseparation.     

Polymeric alkenoxy amino acid surfactants: V. Comparison of carboxylate and sulfate head group polymeric surfactants for enantioseparation in MEKC

In this work, six amino acid derived (L-leucinol, L-leucine, L-isoleucinol, L-isoleucine, L-valinol, and L-valine) polymeric chiral surfactants with carboxylate and sulfate head groups that were recently synthesized in our laboratory [30, 33, 35] are compared for the simultaneous enantioseparation of several groups of structurally similar analytes under neutral and basic pH conditions. The physicochemical properties of the monomers and polymers of both classes of sulfated and carboxylated surfactants are compared. In addition, cryogenic high-resolution electron microscopy showed tubular structures with distinct order of the tubes of 50-100 nm width. A Plackett-Burmann experimental design is used to study the factors that influence the chiral resolution and analysis time of ten structurally related phenylethylamines (PEAs). It is observed that increasing the number of hydroxy groups on the benzene ring of the PEAs resulted in deterioration of enantioseparation using any of the six polymeric surfactants. For all three classes of PEAs, polysodium N-undecenoxycarbonyl-L-amino acidate (poly-L-SUCAA)-type surfactants provided enhanced resolution compared to that of polysodium N-undecenoxycarbonyl-L-amino acid sulfates (poly-L-SUCAASS). Several classes of basic and neutral chiral compounds (e.g., beta-blockers benzoin derivatives, PTH-amino acids, and benzodiazepines) also provided improved chiral separations with poly-L-SUCAA. Among the poly-L-SUCAAs, polysodium N-undecenoxycarbonyl-L-isoleucine sulfate (poly--SUCL) exhibited overall the best enantioseparation capability for the investigated basic and neutral compounds, while among the poly-L-SUCAASs, polysodium N-undecenoxycarbonyl-L-isoleucine sulfate (poly-L-SUCILS), and polysodium N-undecenoxycarbonyl-L-valine sulfate (poly-L-SUCVS) proved to be equally effective for enantioseparation. This work clearly demonstrates that variation in the head group of polymeric alkenoxy amino acid surfactants has a significant effect on chiral separations.     

Enantiomeric Separation of Nefopam Hydrochloride by Affinity Electrokinetic Chromatography Using Chondroitin Sulfate A as Chiral Selector and Its Chiral Recognition Mechanism

In this study, a new approach to the enantioseparation of nefopam hydrochloride by means of affinity electrokinetic chromatography (AEKC) with chondroitin sulfate A belonging to linear ionic polysaccharides has been developed. The difference in the antinociceptive activity of the enantiomers of nefopam was demonstrated in some studies, and the method established in this paper allowed complete separation of nefopam. Especially, there are no reports concerned with the enantioselective separation of nefopam using chondroitin sulfate A as chiral selectors in CE. During the course of this work, both migration time and enantioseparation of nefopam were influenced by several parameters such as pH of the BGE, selector concentration, capillary temperature and applied voltage. Consequently, these parameters were systematically optimized in order to obtain the optimum enantioseparation of nefopam. Moreover, comparison of the influences of the studied parameters was further investigated using univariate analysis of variance as a calculation method by Statistical Product and Service Solutions (SPSS) in this paper. Finally, a mechanism of enantiorecognition in AEKC towards the enantiomers of nefopam with chondroitin sulfate A was described.     

Stereochemical resolution of enantiomeric 2-aryl propionic acid non-steroidal anti-inflammatory drugs on a human serum albumin based high-performance liquid chromatographic chiral stationary phase

Summary The native enantioselectivity in binding of human serum albumin (HSA) towards 2-aryl propionic acid non-steroidal anti-inflammatory drugs (2-APA-NSAIDs, the profens) was found to be preserved when the protein was immobilized within a commercially available diol high-performance liquid chromatographic column. High capacity factors were obtained, reflecting the previously observed extensive binding of the 2-APA-NSAIDs to free HSA. The capacity factors were modified by the addition of octanoic acid to the mobile phase. Chiral resolution of the enantiomers of all nine 2-APA-NSAIDs studied was achieved. Preliminary studies show that in addition to being a useful chiral analytical tool for this therapeutically important series of compounds, the HSA chiral stationary phase may provide useful information on the affinity and binding mechanism of small molecules to HSA.     

Azithromycin as a new chiral selector in capillary electrophoresis

In capillary electrophoresis (CE), separation of enantiomers of a chiral compound can be achieved through the chiral interactions and/or complex formation between the chiral selector and the enantiomeric analytes on leaving their diastereomeric forms with different stability constants and hence different mobilities. A great number of chiral selectors have been employed in CE and among them macrocyclic antibiotics exhibited excellent enantioselective properties towards a wide number of racemic compounds. The use of azithromycin (AZM) as a chiral selector has not been reported previously. This work reports the use of AZM as a chiral selector for the enantiomeric separations of five chiral drugs and one amino acid (tryptophan) in CE. The enantioseparation is carried out using polar organic mixtures of acetonitrile (ACN), methanol (MeOH), acetic acid and triethylamine as run buffer. The influences of the chiral selector concentration, ACN/MeOH ratio, applied voltage and capillary temperature on enantioseparation are investigated. The results show that AZM is a viable chiral selector in CE for the enantioseparation of the type of chiral drugs investigated.     

Comparison of sulfobutylether- and sulfated-beta-cyclodextrins as additives for the chiral separation of basic spirobenzopyrans by capillary electrophoresis.

Three charged substituted beta-cyclodextrins (beta-CDs), sulfobutylether-beta-(SBE-beta-CD), degree of substitution (DS) 4 and 7), and sulfated-beta-(S-beta-CD) cyclodextrins, were compared as chiral additives in capillary electrophoresis for the enantiomeric separation of basic spirobenzopyran derivatives (pKa 9.9) which differ from each other by an N-alkyl group. The number of sulfobutylether groups attached to the cyclodextrin moiety significantly influences the enantioseparation of the basic drugs. SBE-beta-CD (DS 7) which is more strongly bound to cationic analyte than SBE-beta-CD (DS 4.6), requires smaller concentrations to achieve the same resolution. Besides, better enantioresolutions were obtained with S-beta-CD rather than with SBE-beta-CDs though higher concentrations are required, which led to high current values. However, both pairs of enantiomers cannot be resolved using S-beta-CD while SBE-beta-CDs make it possible to resolve simultaneous enantioseparation of such solutes slightly differing in hydrophobicity. This supports the hypothesis that hydrophobic interactions (outside of the CD cavity) between the butyl group attached to SBE-beta-CD and the N-alkyl group of spirobenzopyran play a role in the enantioseparation. On the other hand, the sulfate group of S-beta-CD was directly attached to the CD moiety which means that the S-beta-CD-drug complexation mechanism arises through the combination of electrostatic and hydrophobic (inside the CD cavity) interactions. Finally, enantiomers of spirobenzopyran drugs were satisfactorily resolved by CE using a 20 mg/mL S-beta-CD concentration (resolution 4.0), 7 mg/mL SBE-beta-CD DS 4 (resolution 1.3), or 5 mg/mL SBE-beta-CD DS 7 (resolution 3.3) added to the phosphate buffer (pH 2.6, 50 mM ionic strength).     

手性固定相AD、AS和OD的拆分性能

手性固定相-高效液相色谱法在手性药物、手性农药等的分离分析中应用广泛。本文采用3种多糖衍生物的手性固定相(即EnantioPak AD、AS和OD)对20种手性化合物开展手性分离研究,进而探讨样品分子结构、多糖骨架和衍生基团对手性分离的影响。结果表明,除化合物13外,其余化合物在EnantioPak AD上均实现基线分离,分离度多在2.0以上,在正己烷-醇流动相中加入酸碱添加剂可改善和优化酸性或碱性化合物的分离效果;芳香醇(化合物13~16)随着侧链碳数增加在色谱柱上的保留减弱,其分离度呈现增加的趋势;对比8种化合物在3种手性固定相上的分离结果可知,EnantioPak AD表现出更优的分离性能。这为深入研究和了解多糖手性固定相、拓展其手性分离应用提供了参考。     

Enantioselective separation of biologically active basic compounds in ultra-performance supercritical fluid chromatography

The enantioseparation of basic compounds represent a challenging task in modern SFC. Therefore this work is focused on development and optimization of fast SFC methods suitable for enantioseparation of 27 biologically active basic compounds of various structures. The influences of the co-solvent type as well as different mobile phase additives on retention, enantioselectivity and enantioresolution were investigated. Obtained results confirmed that the mobile phase additives, especially bases (or the mixture of base and acid), improve peak shape and enhance enantioresolution. The best results were achieved with isopropylamine or the mixture of isopropylamine and trifluoroacetic acid as additives. In addition, the effect of temperature and back pressure were evaluated to optimize the enantioseparation process. The immobilized amylose-based chiral stationary phase, i.e. tris(3,5-dimethylphenylcarbamate) derivative of amylose proved to be useful tool for the enantioseparation of a broad spectrum of chiral bases. The chromatographic conditions that yielded baseline enantioseparations of all tested compounds were discovered. The presented work can serve as a guide for simplifying the method development for enantioseparation of basic racemates in SFC.     

卡那霉素作为手性选择剂的毛细管电泳手性药物分离研究

建立了一种以天然易得的卡那霉素为手性添加剂，用毛细管区带电泳法快速分离市售对乙肝有良好治疗效果的药物联苯双脂衍生物的方法，拓宽了毛细管电泳中手性选择剂的范围，通过实验研究了卡那霉素、甲醇 含量PH值，磷酸盐缓冲体系和硼硝缓冲体系对手性分离的影响，以及三种有机溶剂（甲醇、乙晴、异丙醇）添加剂对手性分离的影响，结果表明，在含有3％卡那霉素，30mol/L，硼砂缓冲体系（PH＝8．0）添加30％异丙醇是最佳的分离条件。     

Enantioseparation of phenotiazines by affinity electrokinetic chromatography using human serum albumin as chiral selector: application to enantiomeric quality control in pharmaceutical formulations

Nowadays, there is a special interest within the pharmaceutical laboratories to develop single enantiomer formulations and consequently a need for analytical methods to determine the enantiomeric purity of drugs. The present paper deals with the enantiomeric separation of promethazine and trimeprazine enantiomers by affinity electrokinetic chromatography (AEKC)-partial filling technique using human serum albumin (HSA) as chiral selector. A multivariate optimization of the most critical experimental variables in enantioresolution, running pH, HSA concentration and plug length, is carried out to obtain enantioresolution of promethazine and trimeprazine. The estimated maximum and optimum resolution of trimeprazine and prometazine enantiomers (Rs = 1.74 and 2.01, respectively) corresponded to the following experimental conditions: pH 7.5; [HSA] 170 μM and plug length 190 s and pH 7.6; [HSA] 170 μM and plug length 170 s, for trimeprazine and prometazine, respectively. The developed methodologies were applied for the enantiomeric quality control of promethazine and trimeprazine enantiomers in commercially available pharmaceutical formulations. Resolution, accuracy, reproducibility, cost and sample throughput of the proposed methodologies make it suitable for quality control of the enantiomeric composition of promethazine and trimeprazine in pharmaceutical preparations.     

Enantiomeric quality control of antihistamines in pharmaceuticals by affinity electrokinetic chromatography with human serum albumin as chiral selector

The present paper deals with the enantiomeric separation of six antihistaminic enantiomers by affinity electrokinetic chromatography (AEKC)-partial filling technique using human serum albumin (HSA) as chiral selector. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and HSA plug length (SPL) was carried out since there are interactions between variables that could not be considered in an univariate optimization. The estimated and experimental resolution values obtained for antihistaminic enantiomers varied from 1.13 (for orphenadrine) to 2.15 (for brompheniramine). The optimum experimental conditions for enantioresolution of each compound were: brompheniramine, pH 8.5, [HSA] 180 μM, SPL 180 s; chlorcyclizine, pH 6.5, [HSA] 180 μM, SPL 150 s; chlorpheniramine, pH 8.25, [HSA] 160 μM, SPL 150 s; hydroxyzine, pH 7.0, [HSA] 180 μM, SPL 150 s; and orphenadrine, pH 7.8, [HSA] 160 μM, SPL 150 s. pH and the quadratic term of pH seem to be the most critical factors that determine enantioresolution of antihistamines. The validity of the developed methodologies to enantiomeric quality control of antihistamines in pharmaceutical formulations is demonstrated analyzing the content of brompheniramine, chlorpheniramine and hyroxyzine enantiomers in commercially available pharmaceutical formulations containing racemic mixtures of compounds. Resolution, accuracy, reproducibility, cost and sample throughput of the proposed methodologies make them suitable for quality control of the enantiomeric composition of antihistamines in pharmaceutical preparations.     

Evaluation of the enantioseparation capability of the novel chiral selector clindamycin phosphate towards basic drugs by micellar electrokinetic chromatography

To date, a series of chiral selectors have been utilized successfully in capillary electrophoresis (CE). Among these various chiral selectors, macrocyclic antibiotics have been demonstrated to represent powerful enantioselectivity towards many chiral compounds. Differing from macrocyclic antibiotics, the use of lincosamide antibiotics as chiral selectors has not been reported previously. In our recent work, clindamycin phosphate belonging to the group of lincosamides has been first used as a chiral selector in capillary zone electrophoresis (CZE). In this paper, a micellar electrokinetic chromatography (MEKC) method has been developed for the evaluation of enantioseparation capability of this novel chiral selector towards several racemic basic drugs. As observed during the course of this work, clindamycin phosphate allowed excellent separation of the enantiomers of nefopam, citalopram, tryptophan, chlorphenamine, propranolol and metoprolol, as well as partial enantioresolution of tryptophan methyl ester and cetirizine. In this MEKC chiral separation system, different types of anionic surfactants, organic additives and background electrolytes were tested, and satisfactory enantioseparations of basic drugs above-mentioned were achieved using sodium dodecyl sulfate (SDS) as the surfactant, isopropanol as the organic additive, and phosphate as the background electrolyte. Furthermore, both migration times and enantioseparation of the analytes were influenced by several experimental parameters such as pH of the BGE, clindamycin phosphate and SDS concentrations, phosphate and isopropanol concentrations, and applied voltage. Consequently, the effects of these factors on enantioseparations of the studied basic drugs were systematically investigated in order to evaluate the stereoselectivity of clindamycin phosphate in MEKC.     

Study on enantiomeric separation of basic drugs by NACE in methanol‐based medium using erythromycin lactobionate as a chiral selector

A wide variety of chiral selectors have been employed in CZE, and among them macrocyclic antibiotics including glycopeptides, ansamycins, aminoglycosides and polypeptides exhibited prominent enantioselective properties toward abundant racemic compounds. Compared with CZE, the use of macrocyclic antibiotics as chiral selectors in NACE has not been reported previously. In this study, an approach to the enantioseparation of basic drugs by means of NACE with erythromycin lactobionate (EL) belonging to the group of macrolide antibiotics has been investigated. Especially different from the above four classes of antibiotics, there are no reports concerned with the use of macrolides which belong to macrocyclic antibiotics as chiral selectors in CE. In this work EL is first used as a chiral selector in NACE for the enantiomeric separations of two racemic basic drugs that possess high separability consisting of propranolol and duloxetine. Furthermore, EL possesses advantages such as high solubility and low viscosity in the solvent and very weak UV absorption. The chiral separations were achieved using Tris‐boric acid as the BGE and methanol as the organic medium. In the course of this work we observed that both migration time and enantioseparation were influenced by several parameters such as the pH and composition of the BGE, EL concentration, capillary temperature and applied voltage. Consequently, these parameters were systematically optimized in order to obtain the optimum enantioseparations.     

Evaluation of enantioselective binding of basic drugs to plasma by ACE

The present paper deals with the evaluation of the stereoselective binding of antihistamines (brompheniramine, chlorpheniramine, hydroxyzine, orphenadrine and phenindamine), phenothiazines (promethazine and trimeprazine) and a local anesthetic (bupivacaine) to human plasma proteins. Since all of them are drugs highly bound to proteins, a methodology to determine the bound fraction of each drug enantiomer was proposed. This methodology includes the incubation of samples containing plasma and racemic drug, ultrafiltration of the mixture and the chiral separation of enantiomers in the bound drug fraction using affinity EKC (AEKC)-partial filling technique and HSA as chiral selector. The results shown in this paper represent the first evidence of the enantioselective binding of some antihistamines such as brompheniramine, hydroxyzine, orphenadrine and phenindamine and the phenothiazines, promethazine and trimeprazine, to human plasma proteins. The binding of phenindamine to plasma presented the highest enantioselectivity (ES) (ES = 2.5) followed by trimeprazine (ES = 1.5) and promethazine (ES = 1.4).     

Evaluation of the enantioselectivity of glycogen-based dual chiral selector systems towards basic drugs in capillary electrophoresis

Several chiral reagents including cyclodextrins (CDs) and derivatives, crown ethers, proteins, chiral surfactants and polymers have been involved in dual selector systems for enantioseparation of a series of chiral compounds by capillary electrophoresis (CE). In comparison to the chiral reagents above-mentioned, there is no report concerning the use of polysaccharides in dual chiral CE system. In this paper we first investigate the enantioselectivity of polysaccharide-based dual selector systems towards some chiral drugs. During our recent work, glycogen belonging to the class of branched polysaccharides has been used as a novel chiral selector in CE. In this study, three glycogen-based dual chiral CE systems have been established for enantiomeric separations of several racemic basic drugs consisting of duloxetine, cetirizine, citalopram, sulconazole, laudanosine, amlodipine, propranolol, atenolol and nefopam. These three dual systems combined glycogen (neutral polysaccharide) with chondroitin sulfate A (CSA, ionic polysaccharide), β-CD and HP-β-CD, respectively. It was found that the dual system of glycogen/CSA exhibited good enantioselective properties toward the tested drugs. More importantly, compared to the single selector systems, synergistic effect was observed when glycogen was used with CSA for most of the analytes. This indicated the enhancement of enantioseparation observed for these analytes in glycogen/CSA system might be due to some favorable interaction effects between glycogen and CSA. Moreover, in order to evaluate the stereoselectivity of glycogen/CSA, the influences of buffer pH and selector concentration on enantioseparation of the studied drugs were also investigated.     

淀粉及纤维素三(3-三氟甲基苯基氨基甲酸酯)手性固定相的制备及其性能评价

为了扩展多糖类手性固定相的种类,制备了基于淀粉及纤维素三(3-三氟甲基苯基氨基甲酸酯)的涂敷型手性固定相,以正己烷-异丙醇混合液为流动相,对8种手性化合物进行了高效液相色谱拆分。研究表明: 虽然与应用最广泛的分别以淀粉及纤维素三(3,5-二甲基苯基氨基甲酸酯)为手性选择因子的商品化手性柱Chiralpak AD和Chiralcel OD相比,所制备的手性固定相的手性分离能力较低,但纤维素三(3-三氟甲基苯基氨基甲酸酯)手性固定相显示出特异的手性识别能力,一些手性化合物在此固定相上得到了比在Chiracel OD上更好的分离;所制备的手性固定相的手性识别能力随流动相中异丙醇含量的降低而变好,当流动相中正己烷与异丙醇的体积比为95:5时所制备的手性固定相显示出相对较高的手性识别能力;总体来说,淀粉三(3-三氟甲基苯基氨基甲酸酯)手性固定相的手性识别能力稍强于纤维素三(3-三氟甲基苯基氨基甲酸酯)手性固定相,同时两种手性固定相的手性识别能力具有一定的互补性。     

Chiral covalent organic framework incorporated organic polymer monolithic capillary column for enantioseparations

A chiral covalent organic framework was synthesized, characterized, and incorporated into organic polymer monolithic capillary columns to provide chiral stationary phases for enantioseparations. The prepared monolithic capillary columns were characterized by scanning electron microscopy and elemental analysis. To obtain better enantioseparations, the columns’ preparation conditions, and enantioseparation conditions were optimized. Baseline resolutions of several chiral compounds were obtained with good reproducibility and stability. Furthermore, the mechanism of chiral recognition was investigated using molecular docking with AutoDock. Docking results showed that the enantioselectivity factor rather than resolution is correlated with the binding free energy difference between enantiomers with the chiral covalent organic framework. And abundant acetoxy and nitrile groups as well as benzene rings in the chiral covalent organic framework are responsible for the enantioseparation ability of the chiral monolithic capillary columns.