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Electrophoresis
Tetramethylammonium-lactobionate: a novel ionic liquid chiral selector based on saccharides in capillary electrophoresis Qi Zhang a, Yingxiang Du a, b, c, *, Shuaijing Du d, Jinjing Zhang a, Zijie Feng a, Yanjie Zhang a, Xiaoqi Li a
a
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, P. R. China
b
Key Laboratory of Drug Quality Control and Pharmacovigilance (Ministry of Education), China Pharmaceutical University, Nanjing 210009, P. R. China
c
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P. R. China
d
*
College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China
Correspondence: Professor Yingxiang Du, Department of Analytical Chemistry, China
Pharmaceutical University, No. 24 Tongjiaxiang, Nanjing, Jiangsu 210009, P. R. China E-mail: [email protected] Tel./fax: +86-25-83221790
Received: 22-Jul-2014; Revised: 09-Feb-2015 ; Accepted: 24-Feb-2015 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/elps.201400358. This article is protected by copyright. All rights reserved.
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Abstract： Chiral ionic liquids (ILs) have aroused widespread interest in separation science; however, only a few papers have reported the application of chiral ILs in capillary electrophoresis (CE) for enantioseparation, and the use of chiral ILs as the sole chiral selector in an electrophoretic or a chromatographic system was reported in only three papers. In this study,
we
designed
a
lactobionic
acid
(LA)-based
ionic
liquid,
tetramethylammonium-lactobionate (TMA-LA), and it is very interesting to find that the chiral separation capability can be remarkably improved when a conventional saccharide chiral selector evolved into an ionic liquid chiral selector. A comparative study of the enantiorecognition capability of three separation systems (Single LA system, LA + tetramethylammonium-chloride (TMA-Cl) system, and TMA-LA ionic liquid system) was also conducted, and the results turned out that the use of TMA-LA ionic liquid as the sole chiral selector exhibited a remarkable superiority. A series of parameters affecting the enantioseparation, such as the type and proportion of organic modifier, buffer composition and pH, chiral selector concentration, as well as applied voltage were systematically investigated. The best enantioseparation was obtained at pH 7.6 using a 40 mM borax buffer with 40% v/v methanol, 200 mM TMA-LA, and 20 kV applied voltage. It is the first time that a saccharide-based ionic liquid is evaluated as a sole chiral selector in CE, and we hope this study would provide a new direction for the development of novel ILs chiral selectors based on conventional chiral selectors. Keywords: Capillary electrophoresis; Chiral selector; Enantioseparation; Ionic liquids; Tetramethylammonium-lactobionate This article is protected by copyright. All rights reserved.
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Electrophoresis
1. Introduction The development of methods for enantiomer separation is attracting increasing interest [1]. This interest stems from the fact that enantiomers of a racemic drug usually display markedly
different
physiological
activities,
as
well
as
different
even
opposite
pharmacokinetic and pharmacodynamic effects [2, 3]. Various analytical techniques have been developed for chiral separation. Among them, Capillary electrophoresis (CE) has been regarded as an attractive technique over the last few decades due to its several advantages such as high separation efficiency, short analysis time, convenient change in separation condition and extremely small volume requirements for sample and separation media [4-7]. The most common approach for CE enantioseparation involves the addition of different chiral selectors to the running buffer. Thus, various kinds of chiral selectors have been developed, including cyclodextrins and their derivatives [8-10], antibiotics [11, 12], proteins [13], saccharides [14], etc. Even though a majority of enantiomeric separations could be achieved with these different kinds of conventional chiral additives, the development of novel chiral selector remains an urgent priority for heavy enantioseparation tasks. Ionic liquids (ILs), which are a group of organic salts with melting point close to or below room temperature, have been studied extensively in recent years. They have many unique physicochemical characters, such as negligible vapor pressure, good thermal stability, relatively high ionic conductivity and, especially, the designable properties [15-18]. ILs have successfully been applied to various areas, such as replacing conventional organic solvent in organic or inorganic synthesis, electrochemical reactions, stationary phases in GC and mobile This article is protected by copyright. All rights reserved.
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phases additive in HPLC [19-24]. In CE, ILs have already been used as electrolyte [25], running buffer modifiers [26], or supported coatings on the capillary wall [27]. Chiral ILs, which have either a chiral cation or a chiral anion, or both, are particularly attractive for their potential applications to chiral discrimination. The designable properties of chiral ILs open a way of developing new chiral recognition material for enantiomeric separation. However, only a few papers have reported the application of chiral ILs in CE for enantioseparation [28-38], and the use of chiral ILs as the sole chiral selector in an electrophoretic or a chromatographic system was reported in only three papers [36-38]. Recently, we have reported the use of clarithromycin lactobionate (CL) as a novel chiral selector for enantioseparation [39]. In the present study, we first found that when lactobionic acid (LA) was used alone, it also showed some enantiorecongnition capability. In order to investigate what would happen when a conventional saccharide chiral selector evolved into an ionic liquid chiral selector, we further designed a LA-based ionic liquid, tetramethylammonium-lactobionate
(TMA-LA).
Interestingly,
the
TMA-LA showed
markedly improved enantioseparation capability as an ionic liquid chiral selector compared to LA. It is the first time that a saccharide-based ionic liquid is evaluated as a sole chiral selector in CE, and we hope this study would provide a new direction for the development of novel ionic liquid chiral selectors based on conventional chiral selectors.
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2. Experimental 2.1. Chemicals and reagents TMA-LA ionic liquid (purity>99%) was custom made by Shanghai Cheng Jie Chemical Co., Ltd. (Shanghai, China). LA (purity>99%, the LA used in this work was from the same batch used for the synthesis of TMA-LA ionic liquid) was purchased from Shanghai Cheng Jie Chemical Co., Ltd. (Shanghai, China), their structures are shown in Fig. 1. Atenolol hydrochloride (ATE, pKa 9.6), metoprolol tartrate (MET, pKa 9.6), nefopam hydrochloride (NEF, pKa 9.0) and duloxetine hydrochloride (DUL, pKa 9.7) were supplied by Jiangsu Institute for Food and Drug Control (Nanjing, China). Bisoprolol fumarate (BIS, pKa 9.5) was purchased from National Institute for the Control of Pharmaceutical and Biological Products. Propranolol hydrochloride (PRO, pKa 9.5) was purchased from Sigma (St. Louis, MO, USA). All these drug samples are racemic mixtures. Nylon filters (0.45 mm), methanol, ethanol and acetonitrile (ACN), all of HPLC grade, were purchased from Jiangsu Hanbon Sci. & Tech. Co., Ltd. (Nanjing, China). Sodium hydroxide, hydrochloric acid and sodium tetraborate decahydrate, all of analytical grade, were purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Double distilled water was used throughout all the experiments. Figure 1
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2.2. Apparatus Electrophoretic experiments were performed with a Beckman P/ACE MDQ capillary electrophoresis system with UV detection (Beckman Instruments, Fullerton, CA, USA). The whole system was driven by 32 karat software (Version 7.0) for instrument control, data collection and analysis. It was equipped with a 48.5 cm (38.5 cm effective length) ×50 µm id uncoated fused-silica capillary (Hebei Yongnian County Reafine Chromatography Ltd., Hebei, China). Samples were introduced into the capillary by a 5 s, 0.7 psi pressure injection. All separations were carried out at 15℃ using a voltage in the range of 15–25 kV. The wavelength for detection was 220 nm (ATE, BIS, MET and PRO), 230 nm (DUL), or 215 nm (NEF). The CE system was operated in the conventional mode with the anode at the injector end of the capillary. A new capillary was first rinsed with 1.0 M NaOH (30 min), followed by 0.1 M NaOH (20 min) and water (20 min), respectively. At the beginning of each day, the capillary was flushed with 0.1 M NaOH (10 min) followed by water (10 min). Between consecutive injections, the capillary was rinsed with 0.1 M NaOH, water and running buffer for 3 min each.
2.3. Procedures A methanol aqueous solution (40% v/v, if not stated otherwise) was used for preparation of the 40 mM borax buffer solution. The running background electrolyte (BGE) containing chiral additives, was freshly prepared by dissolving appropriate amounts of TMA-LA or other additives in the borax buffer solution having a specified apparent pH, and then adjusting the apparent pH exactly to a desired value by adding a small volume of hydrochloric acid or sodium hydroxide solution using a microsyringe. Thiourea was used as a neutral marker to detect the electroosmotic flow. The racemic samples (0.6 mg/ml) were dissolved in distilled water. Running buffers and samples were filtered with a 0.45 µm pore membrane filter and degassed by sonication prior to use.
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2.4. Calculations The resolution (Rs) and selectivity factor (α) of the enantiomers were calculated from Rs 2(t 2 t1 ) /( w1 w2 ) and t 2 / t1 , where t1 and t2 are the migration times of the two
enantiomers, and w1 and w2 are the widths of their peaks at the baseline.
3. Results and discussion 3.1. Separation performance of three chiral systems To demonstrate the superiority of the TMA-LA ionic liquid chiral selector, we investigated the enantioseparation performance of three chiral systems, including LA system, TMA-LA ionic liquid system, and LA + TMA-Cl system. Several racemic drugs (ATE, BIS, DUL, MET, NEF and PRO) were selected as model analytes. As seen in Table 1, the enantiomers of ATE, BIS, MET and PRO were baseline or partially resolved in the LA system with the resolution values in the range of 1.31 to 1.89. However, poor separation was obtained for DUL (Rs
Page 1
Electrophoresis
Tetramethylammonium-lactobionate: a novel ionic liquid chiral selector based on saccharides in capillary electrophoresis Qi Zhang a, Yingxiang Du a, b, c, *, Shuaijing Du d, Jinjing Zhang a, Zijie Feng a, Yanjie Zhang a, Xiaoqi Li a
a
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, P. R. China
b
Key Laboratory of Drug Quality Control and Pharmacovigilance (Ministry of Education), China Pharmaceutical University, Nanjing 210009, P. R. China
c
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P. R. China
d
*
College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China
Correspondence: Professor Yingxiang Du, Department of Analytical Chemistry, China
Pharmaceutical University, No. 24 Tongjiaxiang, Nanjing, Jiangsu 210009, P. R. China E-mail: [email protected] Tel./fax: +86-25-83221790
Received: 22-Jul-2014; Revised: 09-Feb-2015 ; Accepted: 24-Feb-2015 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/elps.201400358. This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 2
Electrophoresis
Abstract： Chiral ionic liquids (ILs) have aroused widespread interest in separation science; however, only a few papers have reported the application of chiral ILs in capillary electrophoresis (CE) for enantioseparation, and the use of chiral ILs as the sole chiral selector in an electrophoretic or a chromatographic system was reported in only three papers. In this study,
we
designed
a
lactobionic
acid
(LA)-based
ionic
liquid,
tetramethylammonium-lactobionate (TMA-LA), and it is very interesting to find that the chiral separation capability can be remarkably improved when a conventional saccharide chiral selector evolved into an ionic liquid chiral selector. A comparative study of the enantiorecognition capability of three separation systems (Single LA system, LA + tetramethylammonium-chloride (TMA-Cl) system, and TMA-LA ionic liquid system) was also conducted, and the results turned out that the use of TMA-LA ionic liquid as the sole chiral selector exhibited a remarkable superiority. A series of parameters affecting the enantioseparation, such as the type and proportion of organic modifier, buffer composition and pH, chiral selector concentration, as well as applied voltage were systematically investigated. The best enantioseparation was obtained at pH 7.6 using a 40 mM borax buffer with 40% v/v methanol, 200 mM TMA-LA, and 20 kV applied voltage. It is the first time that a saccharide-based ionic liquid is evaluated as a sole chiral selector in CE, and we hope this study would provide a new direction for the development of novel ILs chiral selectors based on conventional chiral selectors. Keywords: Capillary electrophoresis; Chiral selector; Enantioseparation; Ionic liquids; Tetramethylammonium-lactobionate This article is protected by copyright. All rights reserved.
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Electrophoresis
1. Introduction The development of methods for enantiomer separation is attracting increasing interest [1]. This interest stems from the fact that enantiomers of a racemic drug usually display markedly
different
physiological
activities,
as
well
as
different
even
opposite
pharmacokinetic and pharmacodynamic effects [2, 3]. Various analytical techniques have been developed for chiral separation. Among them, Capillary electrophoresis (CE) has been regarded as an attractive technique over the last few decades due to its several advantages such as high separation efficiency, short analysis time, convenient change in separation condition and extremely small volume requirements for sample and separation media [4-7]. The most common approach for CE enantioseparation involves the addition of different chiral selectors to the running buffer. Thus, various kinds of chiral selectors have been developed, including cyclodextrins and their derivatives [8-10], antibiotics [11, 12], proteins [13], saccharides [14], etc. Even though a majority of enantiomeric separations could be achieved with these different kinds of conventional chiral additives, the development of novel chiral selector remains an urgent priority for heavy enantioseparation tasks. Ionic liquids (ILs), which are a group of organic salts with melting point close to or below room temperature, have been studied extensively in recent years. They have many unique physicochemical characters, such as negligible vapor pressure, good thermal stability, relatively high ionic conductivity and, especially, the designable properties [15-18]. ILs have successfully been applied to various areas, such as replacing conventional organic solvent in organic or inorganic synthesis, electrochemical reactions, stationary phases in GC and mobile This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 4
Electrophoresis
phases additive in HPLC [19-24]. In CE, ILs have already been used as electrolyte [25], running buffer modifiers [26], or supported coatings on the capillary wall [27]. Chiral ILs, which have either a chiral cation or a chiral anion, or both, are particularly attractive for their potential applications to chiral discrimination. The designable properties of chiral ILs open a way of developing new chiral recognition material for enantiomeric separation. However, only a few papers have reported the application of chiral ILs in CE for enantioseparation [28-38], and the use of chiral ILs as the sole chiral selector in an electrophoretic or a chromatographic system was reported in only three papers [36-38]. Recently, we have reported the use of clarithromycin lactobionate (CL) as a novel chiral selector for enantioseparation [39]. In the present study, we first found that when lactobionic acid (LA) was used alone, it also showed some enantiorecongnition capability. In order to investigate what would happen when a conventional saccharide chiral selector evolved into an ionic liquid chiral selector, we further designed a LA-based ionic liquid, tetramethylammonium-lactobionate
(TMA-LA).
Interestingly,
the
TMA-LA showed
markedly improved enantioseparation capability as an ionic liquid chiral selector compared to LA. It is the first time that a saccharide-based ionic liquid is evaluated as a sole chiral selector in CE, and we hope this study would provide a new direction for the development of novel ionic liquid chiral selectors based on conventional chiral selectors.
This article is protected by copyright. All rights reserved.
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Page 5
Electrophoresis
2. Experimental 2.1. Chemicals and reagents TMA-LA ionic liquid (purity>99%) was custom made by Shanghai Cheng Jie Chemical Co., Ltd. (Shanghai, China). LA (purity>99%, the LA used in this work was from the same batch used for the synthesis of TMA-LA ionic liquid) was purchased from Shanghai Cheng Jie Chemical Co., Ltd. (Shanghai, China), their structures are shown in Fig. 1. Atenolol hydrochloride (ATE, pKa 9.6), metoprolol tartrate (MET, pKa 9.6), nefopam hydrochloride (NEF, pKa 9.0) and duloxetine hydrochloride (DUL, pKa 9.7) were supplied by Jiangsu Institute for Food and Drug Control (Nanjing, China). Bisoprolol fumarate (BIS, pKa 9.5) was purchased from National Institute for the Control of Pharmaceutical and Biological Products. Propranolol hydrochloride (PRO, pKa 9.5) was purchased from Sigma (St. Louis, MO, USA). All these drug samples are racemic mixtures. Nylon filters (0.45 mm), methanol, ethanol and acetonitrile (ACN), all of HPLC grade, were purchased from Jiangsu Hanbon Sci. & Tech. Co., Ltd. (Nanjing, China). Sodium hydroxide, hydrochloric acid and sodium tetraborate decahydrate, all of analytical grade, were purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Double distilled water was used throughout all the experiments. Figure 1
This article is protected by copyright. All rights reserved.
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Page 6
Electrophoresis
2.2. Apparatus Electrophoretic experiments were performed with a Beckman P/ACE MDQ capillary electrophoresis system with UV detection (Beckman Instruments, Fullerton, CA, USA). The whole system was driven by 32 karat software (Version 7.0) for instrument control, data collection and analysis. It was equipped with a 48.5 cm (38.5 cm effective length) ×50 µm id uncoated fused-silica capillary (Hebei Yongnian County Reafine Chromatography Ltd., Hebei, China). Samples were introduced into the capillary by a 5 s, 0.7 psi pressure injection. All separations were carried out at 15℃ using a voltage in the range of 15–25 kV. The wavelength for detection was 220 nm (ATE, BIS, MET and PRO), 230 nm (DUL), or 215 nm (NEF). The CE system was operated in the conventional mode with the anode at the injector end of the capillary. A new capillary was first rinsed with 1.0 M NaOH (30 min), followed by 0.1 M NaOH (20 min) and water (20 min), respectively. At the beginning of each day, the capillary was flushed with 0.1 M NaOH (10 min) followed by water (10 min). Between consecutive injections, the capillary was rinsed with 0.1 M NaOH, water and running buffer for 3 min each.
2.3. Procedures A methanol aqueous solution (40% v/v, if not stated otherwise) was used for preparation of the 40 mM borax buffer solution. The running background electrolyte (BGE) containing chiral additives, was freshly prepared by dissolving appropriate amounts of TMA-LA or other additives in the borax buffer solution having a specified apparent pH, and then adjusting the apparent pH exactly to a desired value by adding a small volume of hydrochloric acid or sodium hydroxide solution using a microsyringe. Thiourea was used as a neutral marker to detect the electroosmotic flow. The racemic samples (0.6 mg/ml) were dissolved in distilled water. Running buffers and samples were filtered with a 0.45 µm pore membrane filter and degassed by sonication prior to use.
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2.4. Calculations The resolution (Rs) and selectivity factor (α) of the enantiomers were calculated from Rs 2(t 2 t1 ) /( w1 w2 ) and t 2 / t1 , where t1 and t2 are the migration times of the two
enantiomers, and w1 and w2 are the widths of their peaks at the baseline.
3. Results and discussion 3.1. Separation performance of three chiral systems To demonstrate the superiority of the TMA-LA ionic liquid chiral selector, we investigated the enantioseparation performance of three chiral systems, including LA system, TMA-LA ionic liquid system, and LA + TMA-Cl system. Several racemic drugs (ATE, BIS, DUL, MET, NEF and PRO) were selected as model analytes. As seen in Table 1, the enantiomers of ATE, BIS, MET and PRO were baseline or partially resolved in the LA system with the resolution values in the range of 1.31 to 1.89. However, poor separation was obtained for DUL (Rs
