Recent advances in peptide chiral selectors for electrophoresis and liquid chromatography

The application of peptides in chiral separations using techniques such as capillary electrophoresis (CE), electrokinetic capillary chromatography (EKC) and liquid chromatography is the focus of this review. Methods for finding peptide selectors using combinatorial library approaches are discussed, as well as recent advances in the use of peptides as general chiral selectors for electrophoresis and liquid chromatography. One example shows the effectiveness of polymeric dipeptide surfactants as general chiral selectors for electrophoresis. Another example shows the versatility of oligoproline chiral stationary phases, exhibiting resolution for a number of racemic analytes comparable to other well-established chiral stationary phases.     

Evaluation of the synergistic effect with amino acids for enantioseparation of basic drugs using capillary electrophoresis

The synergistic effect of two acidic amino acids, aspartic and glutamic acid, on the electrophoretic enantioseparation of four basic drugs was evaluated in the BGE containing a CD and at different pHs. Chlorpheniramine, hydroxyzine, propranolol and tramadol were used as the basic model drugs. However, no enantioseparations were achieved with a BGE containing sole amino acid, but the combined use of an acidic amino acid and a CD showed improved enantioseparations (synergistic effect) compared with the single CD system. The results demonstrated that at optimized pH, the electrostatic interactions of the anionic amino acids with the positively charged basic drugs could result in a decrease of the analyte migration velocity and it consequently improved the enantioseparation. The effective parameters such as the amino acid and chiral selector type and concentration, buffer pH, applied voltage, and capillary temperature were optimized. Favorable enantiomeric resolution and migration times of the model drugs were achieved with a 100 mM phosphate buffer solution (pH 3.0) containing 5.0 mM HP‐α‐CD/HP‐β‐CD and 20 mM aspartic acid with an 18 kV applied voltage at 25°C. ¹H NMR experiments were also carried out in a mixture of an analyte and CD in the absence and presence of aspartic acid. The NMR results were consistent with the results obtained by CE which showed the synergistic effect of amino acid.     

Chiral amines as reagents for HPLC-MS enantioseparation of chiral carboxylic acids

Mass spectrometry (MS) has become a popular analytical technique because of its high sensitivity and specificity. Therefore, the use of a chiral derivatization reagent for the MS detection seems to be efficient for the enantiomeric separation of racemates. However, the number of chiral reagents for the liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis is very limited. The applicability of commercially available chiral amines as the derivatization reagents for the enantiomeric separation of chiral carboxylic acids is reported in this paper by using non-steroidal anti-inflammatory drugs (NSAIDs), i.e. ibuprofen, flurbiprofen, and loxoprofen. The efficiency of the chiral reagents was evaluated in terms of tagging easiness, separation by reversed-phase chromatography, and detection sensitivity by electrospray ionization (ESI)-MS/MS. Among the tested eight chiral amines, i.e. (R)-(+)-4-(3-aminopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-APy), (S)-(+)-1-(2-pyrrolidinylmethyl)-pyrrolidine (PMP), L-prolinamide, (3R)-(-)-1-benzyl-3-aminopyrrolidine, (S)-(+)-1-cyclohexyl-ethylamine, (3R)-(+)-3-(trifluoroacetamido)-pyrrolidine (TFAP), (R)-(-)-1-aminoindan (AI), and (S)-(+)-tetrahydrofurfuryl-amine, DBD-APy, PMP, AI, and TFAP could be used as the chiral reagents for the enantiomeric separation of the NSAIDs. The Rs values and the detection limits of the derivatives were in the range of 1.29-3.85 and 0.57-0.96 fmol, respectively. These four reagents were applied for the determination of the NSAIDs in rat plasma.     

Enantioselective analysis of ofloxacin enantiomers by partial-filling capillary electrophoresis with bacteria as chiral selectors

The enantiomeric separation of ofloxacin enantiomers (OFLX) was achieved by using capillary electrophoresis partial-filled with Escherichia coli, Pseudomonas aeruginosa (Gram-negative), and Staphylococcus aureus (Gram-positive) as chiral selectors. Experimental parameters, including the concentration of background electrolyte, applied voltage, length of the filled bacteria plug, and pH of the buffer, were intensively investigated. Baseline separation of OFLX could be achieved within 7 min by using E. coli and P. aeruginosa as chiral selectors under the following conditions: electrophoretic buffer composed of 10 mM phosphate buffer at pH 7.4, applied voltage at 15 kV, and the bacteria (6.0 × 10(8) cells/mL) were injected into the capillary by gravity with injection height of 17.5 cm for 180 s (E. coli), 300 s (P. aeruginosa), and 300 s (S. aureus), respectively. E. coli and P. aeruginosa had better chiral selectivity for OFLX than S. aureus, which was in good agreement with OFLX having better antimicrobial activity on Gram-negative rather than Gram-positive bacteria. A novel method was developed for the enantioselective separation of enantiomers using bacteria as chiral selectors, which provides a new approach for antimicrobials enantioselective analysis, chiral pharmacodynamics, and chiral pharmacokinetics studies.     

Retention of fluorinated chiral selectors in biphasic fluorinated solvent systems and its application to the separation of enantiomers by countercurrent chromatography

Ethoxynonafluorobutane (ENFB) has been used as a component of new biphasic solvent mixtures. The suitability of several mixtures as solvent systems in countercurrent chromatography was tested. The applicability of the ENFB/2-PrOH/H₂O mixture to the separation of enantiomers, in combination with a fluorinated chiral selector (CS), was evaluated. N-Perfluoroundecanoyl-l-proline-3,5-dimethylanilide (2), analogous to the previously used N-dodecyl-l-proline-3,5-dimethylanilide (1), was synthesized for this purpose. The capacity of the new solvent system to retain the fluorinated CS in the fluorinated phase used as stationary was examined. Chiral selector 1 was applied in analogous conditions for comparative purposes. Additionally, MTBE/phosphate buffer solvent system was also used with the two CSs. The ENFB/2-PrOH/H₂O (25:35:40) mixture was found to be adequate in the enantioseparation of DNB-Leu and DNB-Leu-tBu. Enantioselectivity was maintained in the fluorinated solvent system without compromising eluting time. The complete separation of DNB-Leu-tBu was achieved and no leaks of CS to the mobile phase were detected.     

Use of chiral amino acid ester‐based ionic liquids as chiral selectors in CE

In this study, the applicability of a chiral ionic liquid (CIL) as the sole chiral selector in CE was investigated for the first time. In particular, five amino acid ester‐based CILs were synthesized and used as additives in the BGE in order to evaluate their chiral recognition ability. The performance of these CILs as the sole chiral selectors was evaluated by using 1,1′‐binaphthyl‐2,2‐diylhydrogenphosphate (BNP) as the analyte and by comparing the resolution values. Different parameters were examined, such as the alkyl group bulkiness and the configuration of the cation, the anion type of the CIL and its concentration, and the pH of the BGE, in order to optimize the separation of the enantiomers and to demonstrate the effect that each parameter has on the chiral‐recognition ability of the CIL. Baseline separation of BNP within 13 min was achieved by using a BGE of 100 mM Tris/10 mM sodium tetraboratedecahydrate (pH 8) and a chiral selector of 60 mM l ‐alanine tert butyl ester lactate. The run‐to‐run and batch‐to‐batch reproducibilities were also evaluated by computing the %RSD values of the EOF and the two enantiomer peaks. In both cases, very good reproducibilities were observed, since all %RSD values were below 1%.     

The study of enantioseparation of zolmitriptan on vancomycin-bonded chiral stationary phase

In this study, the chiral stationary phase was prepared by bonding vancomycin to 5 microm spherical silica gel according to "one-pot" synthetic strategies, and used to separate the enantiomers of zolmitriptan under polar ionic mode. The influences of mobile phase composition, such as the concentration and ratio of glacial acetic acid (HOAc) and triethylamine (TEA), on the enantioseparation were investigated, and the chiral recognition mechanism is discussed. It was found experimentally that the retention factors were increased with the increase of the HOAc/TEA concentration in a certain extent, and the ionic interactions, hydrogen bondings, and steric interactions may play key role together. The method is suitable for baseline separation of zolmitriptan enantiomers.     

CE‐LIF chiral separation of aspartic acid and glutamic acid enantiomers using human serum albumin and sodium cholate as dual selectors

Sodium cholate (SC), β‐CD, hydroxypropyl (HP)‐β‐CD, HSA, and the dual mixtures of them were evaluated for the analysis of aspartic acid (Asp) and glutamic acid (Glu) enantiomers fluorescently tagged with 5‐(4,6‐dichloro‐s‐triazin‐2‐ylamino) fluorescein (DTAF) by CE with LIF detection. Among the investigated chiral selectors and the dual selector systems, the dual selector systems of HSA and SC resulted to be the most useful chiral selectors allowing relatively high chiral resolution. Several experimental parameters such as chiral reagent type and concentration, buffer concentration, and pH, type and concentration of organic modifier were studied in order to find the optimum conditions for the chiral resolution of the two derivatized amino acids in their enantiomers. The effect of different variables that affect derivatization (time, temperature, pH, and DTAF concentration) was studied. Under optimum conditions, the analytes were separated in a short 10.5 min analysis time, and the RSDs for migration time and peak area were less than 0.12 and 2.8%, respectively. The method was applied for the analysis of compound amino acids injection without interference from other amino acids in the sample matrices observed.     

Improved preparation of chiral stationary phases via immobilization of polysaccharide derivative‐based selectors using diisocyanates

The classical method for the preparation of immobilized polysaccharide‐based chiral stationary phases (CSPs) with a diisocyanate was improved. Cellulose or amylose was directly coated onto 3‐aminopropyl silica gel after it was dissolved in a mixture of N,N‐dimethylacetamide, LiCl, and pyridine, then immobilized onto silica gel with a diisocyanate, and finally allowed to react with an excess of corresponding isocyanate. Four polysaccharide derivatives, 3,5‐dimethylphenylcarbamate and 3,5‐dichlorophenylcarbamate of cellulose, and 3,5‐dimethylphenylcarbamate and 5‐chloro‐2‐methylphenylcarbamate of amylose, were immobilized onto silica gel utilizing this method. Compared with the classical diisocyanate method, the improved procedure avoided the derivatization and regeneration of 6‐hydroxyl groups of cellulose and amylose, and thus showed an advantage for simple and economical preparation. The relationships among the amount of diisocyanate used, immobilization efficiency, and enantioseparation on the cellulose‐based CSPs were investigated. Also, the solvent durability of the obtained CSPs was examined with eluents containing chloroform or THF. By utilizing these eluents, the chiral recognition abilities of the obtained CSPs for some of the tested racemates were improved.     

Advanced dress-up chiral columns: New removable chiral stationary phases for enantioseparation of chiral carboxylic acids

This paper describes the preparation of new dress-up columns featuring reproducibly removable and replaceable chiral stationary phases. After synthesizing perfluroalkylated quinine and quinidine derivatives as chiral stationary phase compounds (F-CSPs), we adsorbed them reversibly onto a fluorous LC column through pumping of their solutions. Using this dress-up chiral column and fluorophobic elution of aqueous ammonium formate/MeOH mixtures, we could enantioseparate four racemic N-acetyl amino acids, dichlorprop, and sixteen fluorescent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized amino acids. Dressing and undressing of the coated F-CSPs could be controlled by varying the fluorophilicity and fluorophobicity of the eluent. The relative standard deviations of the retention times, the retention factors, the number of theoretical plates, the enantioseparation factors, and the resolutions of each of four preparations of such dress-up columns were all less than or equal to 5.26% (from 20 repeated analyses); the reproducibilities from four different preparations were all less than or equal to 10.6%. These columns also facilitated highly sensitive and selective analyses of AQC-amino acids when detected using LC–MS/MS.     

Recent developments in chiral analysis of β‐blocker drugs by capillary electromigration techniques

The latest developments in chiral analysis of β‐blocker drugs by capillary electromigration techniques are reviewed in this article. Following the previous review by Aturki et al. [Electrophoresis 2011, 32, 2602–2628], this review includes the papers published during the period from January 2011 to December 2013. During this time, some novel chiral selectors were reported and applied to improve the enantioseparation of β‐blocker drugs and structurally related compounds. These chiral selectors include CDs and their derivatives, macrocyclic antibiotics, tartrate complexs, the monolithic molecularly imprinted polymer, and the polymeric surfactants. In addition, this article summarizes the methodological improvements for enhancing sensitivity in chiral analysis of β‐blockers and structurally related compounds by CE. The involved authors described the use of online sample preconcentration techniques to increase the detection sensitivity in the enantiomeric analysis of a broad range of samples.     

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.     

Chiral capillary electrophoretic separation of amino acids derivatized with 9-fluorenylmethylchloroformate using mixed chiral selectors of beta-cyclodextrin and sodium taurodeoxycholate

Chiral separation of 19 pairs of amino acid (AA) enantiomers derivatized with 9-fluorenylmethylchloroformate (FMOC) was successfully conducted by capillary electrophoresis using the mixture of beta-CD and sodium taurodeoxycholate (STDC) as selectors. Resolution was considerably superior to that obtained by using either beta-CD or STDC alone. After a systematic inspection, a buffer composed of 150 mM borate and 18% v/v isopropanol at pH 8.0, dissolved with 30 mM beta-CD and 30 mM STDC, was adopted and able to generate baseline resolution (>1.50) for 17 pairs of FMOC-AA enantiomers and somewhat lower resolution for arginine (1.36) and alanine (1.18), respectively. Experimental data revealed that the addition of the second selector did not increase the mobility difference between a pair of enantiomers (Delta mu = mu(D) - mu(L) and the number of theoretical plates (N), but decreased the summed apparent mobility of a pair of enantiomers (Sigma mu = mu(D) - mu(L)), which was mainly due to the decrease of the electroosmotic flow. The variation of Sigma mu was thus the major reason responsible for the improvement of chiral resolution in this study. The result demonstrated that not only the intrinsic selectivity of the selectors was the basis of the chiral separation, but also the non-chiral effect of the selectors, the change of the electroosmotic flow, was an important factor in enhancing the enantioseparation resolution. This study could probably help to explain the reasons for resolution improvement in some dual selectors systems, which are not very clear at present.     

Study of the enantiomeric separation of the anticholinergic drugs on two immobilized polysaccharide‐based chiral stationary phases by HPLC and the possible chiral recognition mechanisms

In this work, the enantiomeric separation of ten anticholinergic drugs was first examined on two derivative polysaccharide chiral stationary phases (CSPs), i.e., Chiralpak ID and Chiralpak IA in the normal phase mode. Except for scopolamine hydrobromide, the remaining nine analytes could be completely separated with good resolutions using both columns under the optimized mobile phase conditions. And the enantiomeric discrimination ability of the studied CSPs towards nine analytes was in the order of Chiralpak ID > Chiralpak IA. The influences of organic modifier types, alcohol content, and base/acid additives on the enantiomeric separation were evaluated and optimized. According to the experimental results, the effect of the structures of analytes on enantiomeric separation was discussed. Additionally, the chiral recognition mechanisms were proposed based on the thermodynamic analysis of the experimental data.     

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.     

Influence of steric hindrance on enantioseparation of Dns-amino acids and pesticides on terguride based chiral selectors in capillary electrophoresis

Three urea derivatives of ergoline-based chiral selectors (CSs), differing in the size of the urea side chain, i.e. dimethyl- (CSI), diethyl- (CSII), and diisopropylurea (CSIII), were used to study the effect of steric hindrance on the enantioseparation of dansyl amino acids (Dns-AAs), pesticides, and mandelic acid under condition of capillary electrophoresis (CE) in linear polyacrylamide coated capillaries. A mixture of organic modifiers (MeOH/THF, 4:1 v/v) in a BGE consisting of 100 mM beta-alanine-acetate was used to increase the solubility of CSs up to 25 mM. The capillary was filled with CS (high UV absorption), and the inlet and outlet vials contained buffer solutions only. The best enantioseparation of Dns-AAs was achieved on CSI. Increased steric hindrance of the chiral binding site led to reduction of both enantioselectivity and resolution. The opposite pattern was observed for the separation of mandelic acid enantiomers, where the best enantioseparation and resolution was obtained with CSIII. Most of the pesticides studied reached maximum selectivity on the diethylurea ergoline derivative (CSII). Enantioseparation of fenoxaprop was found to be independent of steric hindrance.     

Study of the enantioseparation capability of chiral dual system based on chondroitin sulfate C in CE

It has been reported that chiral dual system is able to improve the enantioseparation of enantiomers in many cases. Currently, the dual systems involved in CE chiral separation are mostly dual CDs systems, and the polysaccharides‐based chiral dual system was reported in only one paper. To the best of our knowledge, the use of chondroitin sulfate C (CSC)‐based dual system for enantiomeric separation has not been reported previously. Herein, four CSC‐based chiral dual systems, namely CSC/glycogen, CSC/chondroitin sulfate A (CSA), CSC/hydroxypropyl‐β‐CD (HP‐β‐CD), as well as CSC/β‐CD (β‐CD), were evaluated for the first time for their enantioseparation capability by CE in this paper. During the course of the work, the influences of chiral selector concentration and buffer pH values on enantioseparation in dual systems were systematically investigated. Under the optimized conditions, the dual system consisting of CSC and glycogen exhibited better separations toward nefopam, duloxetine, sulconazole, atenolol, laudanosine, and cetirizine enantiomers compared to the single CSC or glycogen system. The combination of CSC and HP‐β‐CD improved the separation of amlodipine and chlorphenamine enantiomers. However, no synergistic effect was observed in the CSC/CSA and CSC/β‐CD systems.     

Thermal treatment for immobilization of chiral polysiloxanes in capillary gas chromatography

The chiral selector L -t-leucine-t-butylamide has been anchored to a polysiloxane by nucleophilic displacement of 2,2,2-trifluoroethyl ester-functionalized block polysiloxanes with L -t-leucine-t-butylamide. Attempts were made to immobilize the chiral polysiloxanes inside the capillaries by thermal treatment at 300°C for 30–90 min. The polysiloxanes were found to be more resistant than Chirasil-Val to the racemization caused by such high temperature treatment. Capillary columns coated with this type of immobilized polysiloxane have shown very high enantioselectivity and thermal stability.     

Impact of amines as co-modifiers on the enantioseparation of various amino acid derivatives on a tert-butyl carbamoylated quinine-based chiral stationary phase

A tert-butyl carbamoylated quinine-based chiral stationary phase (CSP) for direct enantiomer separation of various natural and unnatural amino acid derivatives was studied. The influence of functional groups in the amino acid side chains upon the enantioseparation is discussed with the aim of realizing contributions to their overall chiral recognition. The effects of various amines as co-modifiers upon retention and overall enantioselectivity of amino acid derivatives in polar organic solvents was systematically investigated. In general, retention times decreased with increasing amine concentrations without a distinct alteration of enantioselectivity. All analytes were rapidly resolved on the CSP with the methanol-based mobile phase containing 87 mM acetic acid and 7 mM triethylamine.     

Enantioseparation and molecular modeling study of eight psychoactive drugs on a coated polysaccharide-based chiral stationary phase

The enantioseparation of eight psychoactive drugs has been firstly performed on a coated cellulose-based chiral stationary phase (Chiralcel OJ-H). To obtain optimum separation conditions, the influences of alcohol modifiers and basic/acidic additives have been studied. As a result, except for the partial separation of oxybutynin enantiomers, the other seven drug enantiomers, including mirtazapine, sulpiride, promethazine, citalopram, oxazepam, donepezil, and cyamemazine, have been completely separated. Additionally, for gaining a better insight into the chiral recognition mechanisms, molecular docking was carried out using the Autodock software. Herein, binding energy and conformations of the chiral stationary phase complexes were provided, and it was found that the distinction in enantiomeric conformation determined the number and strength of intermolecular interactions between analytes and chiral stationary phase which resulted in the difference in binding energies of two enantiomers, and ultimately led to the different migration. These modeling results were in accordance with the observed enantioseparation results in high performance liquid chromatography experiments. At last, chiral separation mechanisms have been discussed in detail, and it has been confirmed that hydrogen bond, π–π, hydrophobic interactions, and some special interactions synergistically contributed to the enantioseparation of psychoactive drugs.