Synthesis and application of tetramethylammonium-carboxymethylated-β-cyclodextrin: A novel ionic liquid in capillary electrophoresis enantioseparation

Journal Pre-proof Synthesis and application of tetramethylammonium-carboxymethylated-␤-cyclodextrin: a novel ionic liquid in capillary electrophoresis enantioseparation Xinqi Zhu (Conceptualization) (Methodology) (Software), Cheng Chen (Writing - review and editing), Jiaquan Chen, Guangfu Xu, Yingxiang Du (Data curation) (Writing - original draft), Xiaofei Ma (Visualization) (Investigation), Xiaodong Sun (Supervision), Zijie Feng (Software) (Resources), Zhifeng Huang (Writing - review and editing)

PII:

S0731-7085(19)32518-X

DOI:

https://doi.org/10.1016/j.jpba.2019.113030

Reference:

PBA 113030

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received Date:

14 October 2019

Revised Date:

28 November 2019

Accepted Date:

4 December 2019

Please cite this article as: Zhu X, Chen C, Chen J, Xu G, Du Y, Ma X, Sun X, Feng Z, Huang Z, Synthesis and application of tetramethylammonium-carboxymethylated-␤-cyclodextrin: a novel ionic liquid in capillary electrophoresis enantioseparation, Journal of Pharmaceutical and Biomedical Analysis (2019), doi: https://doi.org/10.1016/j.jpba.2019.113030

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Synthesis and application of tetramethylammoniumcarboxymethylated-β-cyclodextrin: a novel ionic liquid in capillary electrophoresis enantioseparation

Xinqi Zhu1,2, Cheng Chen1,2 , Jiaquan Chen1,2 Guangfu Xu1,2, Yingxiang Du1, 2*,

ro of

Xiaofei Ma1,2, Xiaodong Sun1,2, Zijie Feng1,2, Zhifeng Huang1,2

Key Laboratory of Drug Quality Control and Pharmacovigilance (Ministry of

re

1

-p

Xinqi Zhu and Cheng Chen contributed equally to this work.

Education), China Pharmaceutical University, Nanjing 210009, P. R. China State Key Laboratory of Natural Medicines, China Pharmaceutical University,

lP

2

*

ur na

Nanjing 210009, P. R. China

Correspondence: Professor Yingxiang Du, China Pharmaceutical University, No.24

Tongjiaxiang, Nanjing, Jiangsu 210009, China

Jo

E-mail: [email protected] Tel./fax: +86 25 83221790

1

ro of

Graphical abstract

Highlights

 A new cyclodextrin-derived IL, TMA-CM-β-CD was synthesized

-p

for the first time.

re

 The IL was managed to separate twelve racemic drugs in CE and obtained enhancement enantioselectivity.

lP

 The recognition mechanism was firstly investigated by molecular

ur na

modeling methods.

Abstract

Jo

In this study, a carboxymethylated-β-cyclodextrin-based chiral ionic liquid (CIL), tetramethylammonium-carboxymethylated-β-cyclodextrin successfully designed and synthesized.

(TMA-CM-β-CD)

was

This cyclodextrin-based ionic liquids (ILs)

was used as the sole chiral selector in capillary electrophoresis, and it is very interesting to find that the chiral separation capability can be remarkably improved when a conventional cyclodextrin chiral selector evolved into an IL chiral selector. The ionic 2

liquid showed satisfactory separation performance towards twelve tested drugs. A series of parameters affecting the enantioseparation, such as the type and proportion of organic modifier, buffer pH, chiral selector concentration, as well as applied voltage were systematically investigated. Additionally, the molecular docking program Autodock was applied to further demonstrate the mechanism of chiral recognition and the enhanced enantioselectivity of TMA-CM-β-CD, which kept in agreement with our

ro of

experimental results.

ur na

lP

re

-p

Keywords: Chiral selector, Capillary electrophoresis, Ionic liquids, Molecular docking

1. Introduction

The separation of chiral compounds has been the center of great interest for years

Jo

[1]. And the reason is that the enantiomers of drugs usually have different physiological and pharmacological behaviors, which is a heightened awareness. To avoid the possible undesirable effects of enantiomeric impurity in chiral drug, it is necessary to develop efficient methods for difficult enantioseparation tasks [ 2-4]. Among the various analytical techniques, capillary electrophoresis (CE) is acknowledged as a useful and 3

powerful tool due to its major advantages, such as

high separation efficiency, short

analysis time, variable separation conditions, and low consumption of samples and solvents [ 5-7]. The most common method in CE involves the addition of a chiral selector [8]. So far, cyclodextrins [9- 12], polysaccharides [13], antibiotics [14], Surfactants [15], and DNA oligonucleotides [16] are widely used as the chiral selectors in enantioseparation.

ro of

Among them, cyclodextrins, which have a cavity possessing a hydrophobic internal

surface and a hydrophilic external surface, are the most popular chiral selectors in CE

[17]. Although these chiral selectors can achieve enantiomeric separation in most cases,

-p

the development of new chiral selectors remains a top priority.

melting points close to or below

re

Ionic liquids (ILs) are a class of organic salts consisting of cations and anions with 100oC [ 18]. Very recently, ILs have been widely

lP

used in CE due to their excellent properties, including negligible vapor pressure, good stability, environmental friendliness, and designable properties [ 19]. What’s more, ILs

ur na

functionalized β-CDs are applied in the fields of separation and extraction process [20]. As a kind of chiral selector for CE, ILs can interact with the enantiomers of the drug and adsorb to the capillary wall, causing changes in electroosmotic flow (EOF) to

Jo

improve the separation [ 21]. Although ILs have great prospects for chiral drug separation, most of them are used as additives in CE to establish synergistic systems with chiral selectors, and only a few articles focused on ILs to be the sole chiral selector to separate chiral compounds [ 22-25]. The specific mechanism of ILs for enantioseparation in CE remains to be solved 4

and requires further study. However, molecular modeling methods, NMR spectroscopy experiments, and theoretical chemistry calculations, have proven to be powerful tools for researching the interaction between the enantiomers and the chiral selectors [ 2629]. In this paper, carboxymethylated-β-cyclodextrin (CM-β-CD) with certain chiral recognition ability was chosen as a sample, and the

ionic liquid (IL), named

tetramethylammonium-carboxymethylated-β-cyclodextrin

(TMA-CM-β-CD),

ro of

successfully synthesized. This is

was

a novel IL and is used

to separate chiral drugs.

Fortunately, when compared to CM-β-CD, TMA-CM-β-CD showed better resolution

-p

capability as a chiral selector. In addition, we have further studied its chiral recognition

Jo

ur na

lP

were consistent with the experiment.

re

mechanism with AutoDock, which proved an increase in interaction, and the results

5

2. Experimental 2.1. Chemicals and reagents CM-β-CD (purity > 98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. ( Shanghai, China); Tetramethylammonium hydroxide pentahydrate (TMAOH) and tetramethylammonium chloride (TMA-Cl) were obtained by Shanghai Macklin Biochemical Co. Ltd (Shanghai, China); Atenolol (ATE), Amlodipine (AML),

ro of

Chlorphenamine (CHL), Metoprolol (MET), S-Ofloxacin (S-OFX) and Salbutamol (SAL) were all supplied by Jiangsu Institute for Food and Drug Control (Nanjing,

Jiangsu, China); Esmolol (ESM), Sotalol (SOT) and Propranolol (PRO) were all

-p

purchased from Dalian Meilun Biotechnology Co. Ltd (Dalian, Liaoning, China);

re

Labetalol (LAB), Isoproterenol (ISO) and Ofloxacin (OFX) were all obtained by Shanghai Macklin Biochemical Co. Ltd (Shanghai, China); S-Propranolol (S-PRO) was Shanghai Aladdin Bio-Chem Technology Co., Ltd (Shanghai, China);

lP

purchased from

ur na

Bisoprolol (BIS) was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Peking, China); S-Salbutamol (S-SAL) was supplied by Toronto Research Chemicals Inc (Toronto, ON, Canada); The structures of these drugs are shown in Fig.1. Phosphoric acid (H3PO4), dihydrogen phosphate

Jo

Sodium (NaH2PO4), hydrochloric acid (HCl) and sodium hydroxide (NaOH) were analytically pure and purchased from Nanjing Chemical Reagent Co. Ltd (Nanjing, Jiangsu, China); Methanol, Ethanol, Acetonitrile, and Nylon filters (0.45 μm) were all obtained from Jiangsu Hanbon Science and Technology (Nanjing, Jiangsu, China). Double distilled water was used throughout all the experiments. 6

2.2. Apparatus and CE procedures CE experiments were performed with an Agilent 3D CE system, which includes a sampling device, a power supply, a photodiode array UV detector (wavelength range from 190 to 600 nm) and a data processor. The whole system was driven by Agilent Chem-Station software for system control, data collection and analysis. 33 cm total length (24.5 cm to detection) × 50 μm

I.D. × 365 μm O.D. and uncoatedfused-silica

ro of

capillary was purchased from Heibei Yongnian County Reafine Chromatography Ltd (Heibei China). A new capillary was flushed with 1 M NaOH, 0.1 M NaOH, and

distilled water for 20 min respectively. Between consecutive injections, the capillary

-p

was flushed with 0.1 M NaOH and distilled water for 3 min each and then with running

capillary by 50 mbar pressure for

re

buffer solution till baseline stabilization. All the samples were introduced into the 5 s. The tested drugs were separated at 20℃ and

lP

monitored at different wavelengths (210 nm for LAB, 225 nm for PRO, MET, BIS,

ur na

ESM, SOT, ATE, ISO and SAL, 237 nm for AML, 265 nm for CHL, 289 nm for OFX). A methanol aqueous solution (20%, v/v, if not stated otherwise) was used for preparation of the 40 mM NaH2PO4 buffer solution. The running background electrolyte solution (BGE) containing chiral additives, was freshly prepared by

Jo

dissolving appropriate amounts of CM-β-CD or other additives in the NaH2PO4 buffer solution having a specified apparent pH, and then adjusting the apparent pH exactly to a desired value by adding a small volume of phosphate acid or sodium hydroxide solution using a microsyringe. The racemic samples (0.5 mg/mL) were dissolved in distilled water. Thiourea was used as a neutral to determine the electroosmotic flow 7

(EOF). Running buffers and samples were filtered with a 0.45 μm pore membrane filter and degassed by sonication prior to use. 2.3. Synthesis of tetramethylammonium-carboxymethylated-β-cyclodextrin The CM-β-CD (2.20 g, 1.43 mM) was dissolved in water (25 mL) and then Tetramethylammonium hydroxide pentahydrate (1.65 g, 9.08 mM) was added. The reaction mixture was stirred for

10 h at room temperature. Evaporation at

45oC to

remove excess CM-β-CD. After evaporation at drying at

ro of

remove the solvent. Then, the product was dissolved with methanol, and filtered to

40oC to remove the methanol, vacuum

60oC for 12 h. The structure of the TMA-CM-β-CD is shown in Fig. 1. 1H

-p

NMR (300 MHz, D2O): δ (ppm) 5.31 (s, 7H), 5.08 (s, 7H), 4.20-4.02 (m, 14H), 3.90 (d,

re

J=17.1 Hz, 28H), 3.61 (s, 14H), 3.45 (d, J=10.5Hz, 7H), 3.17 (s, 84H). Fig.

1

lP

2.4. Molecular modeling and optimization

ur na

Molecular modeling was carried out using the ChemBioOffice 2014 software package. The ChemBio 3D ultra 14.0 as component of the ChemBioOffice software equips MM2 force field and molecular dynamics which were used for structure optimization, and the PM3 quantum mechanical method was used for geometry

Jo

optimization to adjust any spatially unreasonable bond distances and bond angles of all analytes. The structure of TMA-CM-β-CD was based on the structure of CM-β-CD and then optimized using molecular mechanics and molecular dynamics. Geometry optimization was conducted by applying the PM3 quantum mechanical method. 2.5. Molecular docking simulation 8

Molecular docking simulations were performed with automated docking software of AutoDock 4.2.3 . It employed the Lamarckian genetic algorithm (LGA) to identify binding conformation of a flexible ligand (or small molecule) to a target receptor. As preliminary preparation for optimization of TMA-CM-β-CD and drugs structures, AutoGrid

created 3D grid boxes to generate a simplified representation for target

receptor. Each atom type (called probe) of the ligand is placed at the grid points and its

ro of

interaction energy with all the atoms of the receptor is computed and assigned to the

corresponding grid points . In this study, all the drugs were docked to TMA-CM-β-CD using the following Cartesian coordinates: x = −0.281, y = 1.475, z = 0.558 and grid

-p

boxes with dimension of 40 Å × 40 Å × 40 Å, with a grid spacing of 0.375 Å. One

re

hundred LGA runs, each with 200 individuals in the population, were performed. Results differing by less than 1 Å in a positional all atom based root mean squar

lP

deviation (rmsd) were clustered together. In each group, the lowest binding energy

ur na

configuration with highest percentage frequency was selected as the group representative.

3. Results and discussion

3.1. Evaluation of the chiral recognition ability of the ionic liquid

Jo

Twelve basic drugs were tested to evaluate the enantioseparation ability of TMA-

CM-β-CD, and the results were shown in Table 1 and Fig.

2. In order to investigate

the system, six racemic drugs (PRO, MET, BIS, ISO, SAL, OFX) were selected as model analytes. Several parameters (such as ionic liquid concentration, buffer pH, applied voltage, and organic additive concentration) were systematically investigated 9

to condition optimization. Under optimal conditions (40 mM NaH2PO4 buffer solution; buffer pH 5.0; methanol concentration (20%, v/v); applied voltage, 17 kV; capillary temperature,

20oC), the best separation was obtained.

In order to verify the repeatability of the established system, the relative average deviation (RSD) of the model analytes were evaluated by performing five consecutive separations of the enamtiomers. Herein, the intra-day RSDs for migration time and

ro of

resolution (Rs) were less than 3.5% and 3.8%, respectively. The inter-day RSDs for

migration time and Rs were less than 3.9% and 4.3%, respectively. It could therefore

system.

re

Table 1

-p

be concluded that satisfactory repeatability could be provided by the TMA-CM-β-CD

Fig.

2

lP

Two systems (CM-β-CD system and CM-β-CD + TMA-Cl system) were studied

ur na

to compare with the TMA-CM-β-CD system. The results are shown in Table S1 and Fig.S1. As observed, the TMA-CM-β-CD system exhibited significantly improved separations of all the model drugs, compared with CM-β-CD system where some drugs could only be partially separated. In TMA-CM-β-CD system, the Rs of SAL increased

Jo

from 0.59 to 1.25. And it is worth noting that complete separations of ISO, ESM, SOT, ATE were obtained. Besides, the migration times of the analytes were prolonged, probably due to the inhibited EOF caused by the adsorption of CILs cations on the internal surface of the capillary. Another possibility was the increasing viscosity of running buffer. In the synergistic system (TMA-Cl and CM-β-CD), four analytes (PRO, 10

BIS, ISO and SOT) were completely embedded overlap in solvent peaks in synergistic system, and the improvement of the remaining model drugs were much lower than the TMA-CM-β-CD system.Hence, a conclusion that CM-β-CD and tetrabutylammonium cation of TMA-CM-β-CD were not simple combinations could be made. Therefore, converting CM-β-CD into ILs could remarkably improved the chiral separation capability of the original system.

ro of

Table S1

Fig. S13.2. Effect of chiral selector concentration on enantioseparation In the chiral separation system, the selector is the main reason for the

-p

enantiomeric recognition, so the concentration of the ionic liquid is crucial for the

re

separation. In order to obtain the optimal concentration of TMA-CM-β-CD, experiments were carried out by varying the TMA-CM-β-CD concentration in the

2. As observed, when the concentration of TMA-CM-β-CD

ur na

shown in Table

lP

range of 4.5-10.5 mM while keeping other conditions constant. The results were

increases, migration times of all the model drugs were prolonged, owing to the intensive adsorption of CILs cations and the increasing viscosity of the running buffer. The Rs and

selectivity factor (α) of all model drugs increased as the TMA-CM-β-

Jo

CD concentration increased from

4.5-9 mM. The phenomenon was attributed to the

enhanced interaction between the TMA-CM-β-CD and enantiomers. However, higher concentrations (10.5 mM) led to the diminution of enantioseparation, indicating that chiral recognition tended to be saturated. It can be noted that PRO had no peak in 60 minutes at

10.5 mM, resulting from the decreased EOF. Taking into account of good 11

separation and appropriate migration time, the 9 mM TMA-CM-β-CD concentration was determined as the optimum value for separation system. Table 2 3.3. Effect of organic modifier concentration on enantioseparation It has been reported that the addition of organic solvents can change the viscosity of the background electrolyte solution and inhibit the adsorption of basic drugs on the to improve the peak shape [30]. In this study, three organic additives

ro of

capillary wall

(methanol, ethanol and acetonitrile) were added to BGE, respectively. Methanol

shows the best results among the three organic additives. Further investigation was

3, it is demonstrated that as the methanol ratio

re

separation of the enantiomers. In Fig.

-p

carried out to research the effect of methanol concentration (0-40%, v/v) on the chiral

increases, provides more opportunities for the interaction during the separation

lP

process (see Fig. 3). However, when higher methanol concentrations (20% - 40%,

ur na

v/v) were used, Rs and α decreased. Due to the peak broadening in consequence of too long analysis time. Based on the above results, 20% of methanol was finally selected for the TMA-CM-β-CD separation system. Fig.

3

Jo

3.4. Effect of running buffer pH on enantioseparation In CE chiral separation, the pH of the buffer solution is one of the most

important factors for separation. When both the analyte and the chiral selector used are charged species, the effect of pH is more pronounced. In order to study the optimal buffer pH of TMA-CM-β-CD as the chiral selector, the effect of pH on 12

enantiomeric separation was investigated over the pH range of 4.75-5.75. From Fig. S2, as the pH increased from 4.75 to 5.00 (PRO has no peak in 60 minutes at pH 4.75), the Rs and α improved simultaneously, indicating a stronger interaction between the selector and the analyte. When the pH rises from 5.00 to 5.75, Rs and α gradually decrease, which is caused by the peak broadening. Considering good

Fig.

S2

3.5. Effect of applied voltage on enantioseparation

ro of

resolution, pH 5.00 of buffer was chosen as the optimum condition

Applied voltage is another important factor affecting CE chiral separation. In

-p

general, increasing the applied voltage results in higher separation efficiency and

re

better peak shape; however, when the operating voltage is too high, excessive Joule heat will cause the peak broadening. In order to evaluate the optimal voltage

lP

conditions of the system, the effect of operating voltage on the chiral separation of the

ur na

CILs separation system in the range of 15-19 kV was investigated. The results are shown in Fig. S3. From

15 kV to 17 kV, the Rs and α of all drugs continue to

increase. However, when the operating voltage was further increased to 19 kV, the Rs and α of the model drug decreased, which probably because of extra Joule heating and

Jo

peak broadening. Therefore, we chose 17 kV as the optimal operating voltage condition.

Fig.

S3

3.6. Investigation of enantioseparation mechanism of TMA-CM-β-CD system with molecular modeling 13

To study the chiral recognition mechanism of TMA-CM-β-CD on drug enantiomer separation, Molecular docking software AutoDock 4.2.3 was used to carry on molecular modeling. The binding free energy (ΔG) represents the thermodynamic stability of the complex of chiral selector and the drug, calculated by a semi-empirical combination with a free energy function. The greater the negative binding energy, the more stable the thermodynamics of the composite. Furthermore, the difference of ΔG

ro of

between (R) and (S)-enantiomer (|ΔΔG|) reflects the discrepancy in affinity between the chiral selector and the enantiomers.

As shown in Table S2, compared to the CM-β-CD system, the TMA-CM-β-CD

-p

system has a larger value of |ΔG| and |ΔΔG |. It indicates that the affinity between the

re

enantiomer of the drug and the chiral selector is enhanced, which means the enantioselectivity is increased. In addition, the calculated difference in binding free

lP

energy is consistent with the experimental results of α.

ur na

In addition, PRO, OFX and SAL were also used as model analytes to verify the order of enantiomeric migration, the experimental results were in good agreement with the computer simulation results. As expected, the (R)-enantiomer of PRO and SAL migrated prior to the (S)-enantiomer, and the (S)-enantiomer of OFX was first

Jo

peak, which was very consistent with the calculation of ΔG. Usually, enantioseparation in CE is attributed to multiple interactions including

hydrogen bonding interaction, host-guest inclusion, charge-charge, dipole-dipole, π interaction, etc. Apart from the binding free energy, molecular docking can also visualize some interactions between CILs and drugs during chiral recognition process. 14

Taking PRO as an example, as shown in Fig. 4, CILs and analytes form a complex with higher stability, providing π-σ attraction (in orange lines). Other than this, the hydrogen bonding interaction (in green lines) in the TMA-CM-β-CD system is more than that of the CM-β-CD system. It can therefore draw the conclusion that TMACM-β-CD can provide more interaction with the enantiomers of the drugs, which improved the chiral recognition ability and the enantioselectivity.

ro of

Table S2

Jo

ur na

lP

re

-p

Fig. 4

15

4. Conclusion In this paper, TMA-CM-β-CD as a novel IL was designed and applied for the first time in CE chiral separation. It is exciting to find that the chiral separation capability can be significantly improved when a conventional cyclodextrin chiral selector evolved into an IL chiral selector. Compared with CM-β-CD, the new selector can not only change the solubility but also strengthen the enantioselectivity. Twelve

ro of

drugs were used to verify the separation ability of the chiral selector.

Molecular modeling method was used to further demonstrate the mechanism of chiral recognition and increased ability of enantioseparation of TMA-CM-β-CD.

-p

According to the modeling outcome, the new kind of ILs could provide more

re

interactions, such as π-σ attraction and hydrogen binding, which could enhance the

lP

enantioselectivity.

CRediT author statement

ur na

Xinqi Zhu: Conceptualization, Methodology, Software. Yingxiang Du: Data curation, Writing- Original draft preparation. Xiaofei Ma: Visualization, Investigation.

Jo

Xiaodong Sun: Supervision.

Zijie Feng: Software, Resources. Zhifeng Huang: Writing- Reviewing and Editing. Cheng Chen: Writing- Reviewing and Editing.

16

Conflict of interest statement The authors have declared no conflict of interest. Acknowledgements This work was supported by the Project of National Natural Science Foundation of China (No.: 81373378) and the Natural Science Foundation of Jiangsu Province

ro of

(Program No.: BK20150697).

Reference

[1] S. Declerck, Y. Vander Heyden, D. Mangelings, Enantioseparations of pharmaceuticals with

-p

capillary electrochromatography: A review, J. Pharm. Biomed. Anal. 130 (2016) 81-99.

re

https://doi:10.1016/j.jpba.2016.04.024.

[ 2] T.J. Ward, K.D. Ward, Chiral Separations: Fundamental Review 2010, Anal. Chem. 82 (2010)

lP

4712–4722. https://doi.org/10.1021/ac1010926.

ur na

[3] G.K.E. Scriba, Chiral recognition in separation science – an update, J. Chromatogr. A 1467 (2016) 56–78. https://doi.org/doi:10.1016/j.chroma.2016.05.061. [4] B. Chankvetadze, Contemporary theory of enantioseparations in capillary electrophoresis, J. Chromatogr. A 1567 (2018) 2–25. https://doi.org/doi:10.1016/j.chroma.2018.07.041.

Jo

[5] Q. Zhang, Y. Du, S. Du, Evaluation of ionic liquids-coated carbon nanotubes modified chiral separation system with chondroitin sulfate E as chiral selector in capillary electrophoresis, J. Chromatogr. A 1339 (2014) 185–191. https://doi.org/10.1016/j.chroma.2014.02.003. [6] R. Pérez-Míguez, M.L. Marina, M. Castro-Puyana, Enantiomeric separation of non-protein amino acids by electrokinetic chromatography, J. Chromatogr. A 1467 (2016) 409–416. 17

https://doi.org/10.1016/j.chroma.2016.06.058. [7] L. Lv, L. Wang, J. Li, Y. Jiao, S. Gao, J. Wang, H. Yan, Enantiomeric separation of seven βagonists by NACE—Study of chiral selectivity with diacetone-D-mannitol–boric acid complex, J. Pharm. Biomed. Anal. 145 (2017) 399–405. https://doi.org/10.1016/j.jpba.2017.06.044. [8] I. J. Stavrou, E. A. Agathokleous, C. P. Kapnissi-Christodoulou, Chiral selectors in CE:

819. https://doi.org/ 10.1002/elps.201600322.

ro of

Recent development and applications (mid-2014 to mid-2016), Electrophoresis 38 (2017) 786-

[ 9] G. Benkovics, I. Fejős, A. Darcsi, E. Varga, M. Malanga, É. Fenyvesi, T. Sohajda, L. Szente, S. Béni, J. Szemán, Single-isomer carboxymethyl- γ-cyclodextrin as chiral resolving agent for

-p

capillary electrophoresis , J. Chromatogr. A 1467 (2016) 445–453.

re

https://doi.org/10.1016/j.chroma.2016.06.083.

[ 10] F. Melani, B. Pasquini, C. Caprini, R. Gotti, S. Orlandini, S. Furlanetto, Combination of

lP

capillary electrophoresis, molecular modeling and NMR to study the enantioselective

ur na

complexation of sulpiride with double cyclodextrin systems , J. Pharm. Biomed. Anal. 114 (2015) 265–271. https://doi.org/10.1016/j.jpba.2015.05.031. [11] J.M. Saz, M.L. Marina, Recent advances on the use of cyclodextrins in the chiral analysis of drugs by capillary electrophoresis, J. Chromatogr. A 1467 (2016) 79–94.

Jo

https://doi.org/doi:10.1016/j.chroma.2016.08.029. [12] P. Řezanka, K. Navrátilová, M. Řezanka, V. Král, D. Sýkora, Application of cyclodextrins in chiral capillary electrophoresis, Electrophoresis 35 (2014) 2701–2721. https://doi.org/doi:10.1002/elps.201400145. [13] F. Zhu, Y. Du, J. Chen, B. Chen, Y. Zhu, X. Zhai, S. Xu, W. Zhou, Enantioselective 18

Separation of Basic Drugs by CE with Polygalacturonic Acid as a Novel Chiral Selector, Chromatographia 69 (2009) 1315–1320. https://doi.org/10.1365/s10337-009-1043-x. [14] M. Greño, M. Castro-Puyana, M. Á. García, M. L. Marina, Analysis of antibiotics by CE and CEC and their use as chiral selectors: An update, Electrophoresis 39 (2018) 235–259. https://doi.org/doi:10.1002/elps.201700306. [15] S. A. A. Rizvi, S.A. Shamsi, Synthesis, Characterization and Application of Chiral Ionic 78

ro of

Liquids and Their Polymers in Micellar Electrokinetic Chromatography , Anal Chem. (2006) 7061–7069. https://doi.org/10.1021/ac060878u.

Oligonucleotides. Anal. Chem.

87 (2015) 5491−5495.

re

https://doi.org/10.1021/acs.analchem.5b01252.

-p

[16] L. Tohala, F. Oukacine, C. Ravelet, E. Peyrin, Chiral Resolution Capabilities of DNA

[17] Y. Dai, S. Wang, J. Zhou, Y. Liu, D. Sun, J. Tang, W. Tang, Cationic cyclodextrin as versatile

lP

chiral selector for enantiomeric separation in capillary electrophoresis, J. Chromatogr. A 1246

ur na

(2012) 98–102. https://doi.org/10.1016/j.chroma.2012.02.065. [ 18] S. Tang, S. Liu, Y. Guo, X. Liu, S. Jiang, Recent advances of ionic liquids and polymeric ionic liquids in capillary electrophoresis and capillary electrochromatography, J. Chromatogr. A

Jo

1357 (2014) 147-157. https://doi.org/10.1016/j.chroma.2014.04.037.

[19] Q. Zhang, Ionic liquids in capillary electrophoresis for enantioseparation, Trends Anal. Chem. 100 (2018) 145–154. https://doi.org/10.1016/j.trac.2018.01.001.

[20] C. Zhou, J. Deng, G. Shi, T. Zhou, β-cyclodextrin-ionic liquid polymer based dynamically coating for simultaneous determination of tetracyclines by capillary electrophoresis,

19

Electrophoresis 38 (2017) 1060–1067. https://doi.org/10.1002/elps.201600229. [ 21] M. López-Pastor, B.M. Simonet, B. Lendl, M. Valcárcel, Ionic liquids and CE combination, Electrophoresis 29 (2008) 94–107. https://doi.org/10.1002/elps.200700645. [ 22] Q. Zhang, Y. Du, S. Du, J. Zhang, Z. Feng, Y. Zhang, X. Li, Tetramethylammoniumlactobionate: A novel ionic liquid chiral selector based on saccharides in capillary electrophoresis. Electrophoresis 36 (2015) 1216–1223. https://doi.org/10.1002/elps.201400358.

ro of

[ 23] Z. Ma, L. Zhang, L. Lin, P. Ji, X. Guo, Enantioseparation of rabeprazole and omeprazole by nonaqueous capillary electrophoresis with an ephedrine-based ionic liquid as the chiral selector , Biomed. Chromatogr.

24 (2010) 1332–1337. https://doi.org/10.1002/bmc.1445.

-p

[ 24] I.J. Stavrou, C.P. Kapnissi-Christodoulou, Use of chiral amino acid ester-based ionic

https://doi.org/10.1002/elps.201200469.

re

liquids as chiral selectors in CE , Electrophoresis 34 (2013) 524–530.

lP

[25] M. Greño, M.L. Marina, M. Castro-Puyana, Enantioseparation by Capillary Electrophoresis

ur na

Using Ionic Liquids as Chiral Selectors, Crit. Rev. Anal. Chem. 48 (2018) 429–446. https://doi.org/10.1080/10408347.2018.1439365. [ 26] A. Salgado, E. Tatunashvili, A. Gogolashvili, B. Chankvetadze, F. Gago, Structural rationale for the chiral separation and migration order reversal of clenpenterol enantiomers in

Jo

capillary electrophoresis using two different β-cyclodextrins , Phys. Chem. Chem. Phys. 19 (2017) 27935-27939. https://doi.org/10.1039/c7cp04761d. [ 27] L. Li, C. Wu, Y. Ma, S. Zhou, Z. Li, T. Sun, Effectively enhancing the enantioseparation ability of β-cyclodextrin derivatives by de novo design and molecular modeling, Analyst 142 (2017) 3699-3706. https://doi.org/10.1039/C7AN00986K. 20

[ 28] F.B. De Sousa, Â.M. Leite Denadai, I.S. Lula, C.S. Nascimento Jr., N.S.G. Fernandes Neto, A.C. Lima, W.B. De Almeida, R.D. Sinisterra,

Supramolecular Self-Assembly of Cyclodextrin

and Higher Water Soluble Guest: Thermodynamics and Topological Studies, J. Am. Chem. Soc. 130 (2008) 8426–8436. https://doi.org/10.1021/ja801080v. [29] S. Krait, A. Salgado, B. Chankvetadze, F. Gago, G.K.E. Scriba, Investigation of the complexation between cyclodextrins and medetomidine enantiomers by capillary electrophoresis,

ro of

NMR spectroscopy and molecular modeling, J. Chromatogr. A 1567 (2018) 198–210. https://doi.org/10.1016/j.chroma.2018.06.010.

[30] X. Ma, Y. Du, X. Sun, J. Liu, Z. Huang, Synthesis and application of amino alcohol-derived

-p

chiral ionic liquid, as additives for enantioseparation in capillary electrophoresis, J. Chromatogr. A

Jo

ur na

lP

re

1601 (2019) 340–349. https://doi.org/10.1016/j.chroma.2019.04.040.

21

Figure captions

Jo

ur na

lP

re

-p

ro of

Fig.1 Chemical structures of twelve studied drug enantiomers and TMA-CM-β-CD.

Fig. 2 The typical electrophoregrams of chiral separation of twelve drugs in TMACM-β-CD separation system. Conditions: fused-silica capillary, 33 cm (24.5 cm effective length) × 50 μm I.D. × 365 μm O.D.; capillary temperature, 20°C; applied 22

voltage, 17 kV; electric current, 60 μA; background electrolyte, 40 mM NaH2PO4 buffer (20% methanol, v/v) containing 9 mM TMA-CM-β-CD; buffer pH, 5.0; other

lP

re

-p

ro of

conditions as in Section 2.

24

ur na

Fig. 3 The effect of organic modifier on enantiomer resolution. 24 Conditions: fusedsilica capillary, 33 cm (24.5 cm effective length) × 50 μm I.D. × 365 μm O.D.; capillary temperature, 20°C; applied voltage, 17 kV; electric current, 60 μA; background electrolyte, 40 mM NaH2PO4 buffer (20% methanol, v/v) containing 9

Jo

mM TMA-CM-β-CD; buffer pH, 5.0; other conditions as in Section 2.

23

ro of -p re lP

2424

Fig. 4 Molecular docking configuration (vertical-view and side-view) for PRO enantioseparation in CM-β-CD and TMA-CM-β-CD systems; the above is R-

ur na

enantiomer and the below is S-enantiomer. The hydrogen binding is indicated by green

Jo

lines. The π-σ attraction is indicated by orange lines. O red, N blue, H white, C grey.

24

25

ro of

-p

re

lP

ur na

Jo

Table 1. Chiral separation of twelve drugs in TMA-CM-β-CD separation systems Rs

α

t1 (min)

t2 (min)

t3(min)

t4(min)

BIS

3.14

1.21

25.46

30.86

——

——

OFX

4.79

1.25

7.72

9.63

——

——

ISO

3.84

1.25

22.14

27.78

——

——

SAL

1.25

1.03

6.95

7.15

——

——

MET

2.79

1.19

25.31

30.20

——

——

LAB

1.39/3.24/1.92

1.03/1.07/1.04

44.09

45.50

48.82

50.76

PRO

7.34

1.39

33.21

46.04

——

——

ESM

1.59

1.10

12.85

14.13

——

——

SOT

1.56

1.05

10.81

11.29

——

——

ATE

1.65

1.04

8.48

8.83

——

——

AML

8.07

1.21

13.03

CHL

4.12

1.27

17.74

ro of

Drugs

15.75

——

——

22.54

——

——

Conditions: fused-silica capillary, 33 cm (24.5 cm effective length) × 50 μm I.D. × 365 μm O.D.; background electrolyte, 40 mM

NaH2PO4

-p

capillary temperature, 20°C; applied voltage, 17 kV;

re

buffer (20% methanol, v/v) containing 9 mM TMA-CM-β-CD; buffer pH, 5.0. “BIS”, Bisoprolol; “OFX”, Ofloxacin; “ISO”, Isoproterenol; “SAL”, Salbutamol; “MET”,

lP

Metoprolol; “LAB”, Labetalol; “PRO”, Propranolol; “ESM”, Esmolol; “SOT”, Sotalol; “ATE”, Atenolol; “AML”, Amlodipine; “CHL”, Chlorphenamine; “——”, no peak.

ur na

The resolution (Rs) and selectivity factor (α) of the model drugs were calculated by using the following equations :

Rs =2 (t2 - t1) / (w1 + w2)

Jo

α = t2 / t1

Where t1, t2 represent the migration times of the two enantiomers and w1, w2 are the baseline peak width of corresponding enantiomers.

26

f oo

Table

2. Effect of TMA-CM-β-CD concentration on the enantioseparation of PRO, MET, BIS, ISO, OFX and SAL.

4.5mM

6mM

7.5mM

9mM

10.5mM

μeofa

13.432

12.626

6.549

6.017

Drugs

t1

e-

pr

TMA-CM-β-CD concentration

Rs

α

t1

t2

Rs

α

t1

Pr

t2

9.304

t2

Rs

α

t1

t2

Rs

α

t1

t2

Rs

α

—

—

—

—

10.76

11.81

1.42

1.10

17.01

21.78

2.63

1.28

25.14

33.72

4.81

1.34

33.21

46.04

7.34

1.39

MET

10.45

11.25

0.90

1.08

14.81

16.96

1.74

1.15

18.42

21.14

2.01

1.15

25.31

30.20

2.79

1.19

29.86

34.97

2.42

1.17

BIS

7.42

7.42

0.00

1.00

9.80

10.33

1.37

1.05

11.82

12.47

1.52

1.06

25.46

30.86

3.14

1.21

32.37

38.44

2.87

1.19

ISO

8.52

8.65

0.52

1.01

9.36

10.01

1.70

1.07

15.05

18.00

2.40

1.20

22.14

27.78

3.84

1.25

26.43

29.63

1.99

1.12

OFX

4.98

5.08

0.52

1.02

5.31

5.41

0.72

1.02

6.11

6.27

0.99

1.03

6.95

7.15

1.25

1.03

7.44

7.61

0.95

1.02

SAL

5.17

5.83

2.50

1.13

5.43

6.19

2.74

1.14

6.99

8.24

4.20

1.18

7.72

9.63

4.79

1.25

8.51

10.60

4.11

1.25

Jo ur

na l

PRO

Conditions: fused-silica capillary, 33 cm (24.5 cm effective length) × 50 μm I.D. × 365 μm O.D.; capillary temperature, 20℃; applied voltage, 17 kV; BGE, 40 mM NaH2PO4 buffer (20% methanol, v/v) containing 9 mM TMA-CM-β-CD, buffer pH, 5.0; 27

f oo

“ep μeof”, electroosmotic mobility (2-1-1-5 m2V-1s-1×10-9); “PRO”, Propranolol; “MET”, Metoprolol; “BIS”, Bisoprolol; “ISO”, Isoproterenol; “OFX”, Ofloxacin;

The electroosmotic flow (EOF) mobility was expressed by the equation:

e-

μeof = (L×l) / (V×t0)

pr

“SAL”, Salbutamol.

Pr

Where L, l, V and t0 represent total capillary length, effective capillary length, applied voltage, and migration time of thiourea (a neutral marker) respectively. The resolution (Rs) and selectivity factor (α) of the model drugs were calculated by using the following equations :

α = t2 / t1

na l

Rs =2 (t2 - t1) / (w1 + w2)

Jo ur

Where t1, t2 represent the migration times of the two enantiomers and w1, w2 are the baseline peak width of corresponding enantiomers.

28