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Study of the enantioseparation capability of chiral dual system based on chondroitin sulfate C in capillary electrophoresis
Jiaquan Chen1, Yingxiang Du1, 2, 3, *, Fenxia Zhu 1,4, Bin Chen 5, Qi Zhang1, Shuaijing Du6, Ping Li 3 1
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing, P. R. China
2
Key Laboratory of Drug Quality Control and Pharmacovigilance (Ministry of Education), China Pharmaceutical University, Nanjing, P. R. China
3
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P. R. China
4
Key Laboratory of New Drug Delivery System of Chinese Meteria Medica, Jiangsu
Provincial Academy of Chinese Medicine, Nanjing, P. R. China 5
Department of Pharmacy, Zhejiang Pharmaceutical College, Ningbo, P. R. China
6
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] (Y. Du) Tel./fax: +86 25 83221790
Received: 31-Jan-2013; Revised: 09-Nov-2014; Accepted: 14-Nov-2014 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.201300057. This article is protected by copyright. All rights reserved.
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Keywords: Capillary electrophoresis / Enantioseparation / Chiral dual system / Chondroitin sulfate C / Glycogen
Abbreviations: AML, amlodipine; ATE, atenolol; CET, cetirizine hydrochloride; CHL, chlorphenamine maleate; CIT, citalopram hydrobromide; CSA, chondroitin sulfate A; CSC, chondroitin
sulfate
C;
DUL,
duloxetine
hydrochloride;
HP-β-CD,
hydroxypropyl-β-cyclodextrin; LAU, laudanosine; NEF, nefopam hydrochloride; PRO, propranolol hydrochloride; SUL, sulconazole.
Abstract 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 capillary electrophoresis (CE) chiral separation are mostly dual cyclodextrins (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-cyclodextrin (HP-β-CD), as well as CSC/β-cyclodextrin (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 This article is protected by copyright. All rights reserved.
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Electrophoresis
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.
1 Introduction The enantiomeric separation of chiral drugs, metabolites and related substances is of great importance in the pharmaceutical field. Usually, the pharmacological activity and metabolism of the two enantiomers of one drug are different. Therefore, rapid, selective and sensitive analytical methods are required to verify the optical purity of chiral drugs. Various analytical techniques have been developed for enantioseparation, among which capillary electrophoresis (CE) has been widely used because of its simplicity, rapidity, low cost, as well as high separation efficiency [1-3]. Enantioseparation by CE is mainly achieved by a direct method in which the chiral selector is simply added to the background electrolyte (BGE). The availability of many chiral selectors makes CE an important tool for chiral analysis [4-7]. Among various selectors, cyclodextrins (CDs) and their derivatives are still the most popular ones, and the interest in antibiotics has been continuously increasing [8-14]. Besides the two kinds of selectors, polysaccharides as CE chiral selectors have attracted great attention over the past few years [15-20]. The weak UV absorption and water solubility of polysaccharides facilitates their application as chiral selectors. Additionally, their varying structures and This article is protected by copyright. All rights reserved.
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Electrophoresis
functional groups provide a wide range of enantioselectivity. Chondroitin
sulfates,
negatively
charged
polysaccharides
composed
of
N-acetylgalactosamine and glucuronic acid residues (see the Supporting Information Fig. S1 A), have been utilized successfully as chiral selectors in CE [21-24]. There are three forms of chondroitin sulfates, namely chondroitin sulfates A, B and C, all with a linear repeating sequence of β-(1,4) linkages between the disaccharide subunits and β-(1,3) linkages within the disaccharide subunits. Chondroitin sulfate C (CSC) usually displays better enantioseparation capability than chondroitin sulfate A (CSA) and B (CSB), owing to the hydroxyl group at C4 in the galactosamine residue [25]. It has been also considered as one of the most excellent polysaccharide chiral selectors so far [15, 26]. In some cases, a successful enantioseparation cannot be achieved by employing one single chiral selector in the BGE. With two different chiral selectors introduced simultaneously, chiral dual system often leads to a significant enhancement of selectivity and resolution (synergistic effect) due to differences in the complexation mechanisms of the two selectors with the analyte enantiomers [27]. The use of dual CD systems has been reported in some studies, in which different combinations of charged and neutral CDs are employed [28-35]. Taking into account the successful use of dual CD systems and the excellent enantioseparation capability of polysaccharides (such as CSC), the application of polysaccharide-based chiral dual system should be worth studying. However, the chiral dual system based on polysaccharide has been reported in only one paper by our group [36]. In this previous work, we studied three glycogen-based dual systems including glycogen/CSA, This article is protected by copyright. All rights reserved.
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glycogen/β-CD and glycogen/HP-β-CD. Among them, significant synergistic effect was observed in the glycogen/CSA system. However, no synergistic effect was found in glycogen/β-CD and glycogen/HP-β-CD system. This paper is a follow up study of our previous research [36]. The enantioseparation performance of four CSC-based dual systems, including CSC/CSA, CSC/glycogen and CSC/natural CDs (β-CD or HP-β-CD), was investigated for the first time. As observed, the CSC/glycogen and CSC/HP-β-CD systems gave significant synergistic effect to some drugs enantiomers, suggesting their value for practical use. The details and results in the present work are different from our previous paper, and a number of advances exist. It also provides an amount of useful information to the fundamental research of polysaccharide-based chiral dual system.
2 Materials and methods 2.1 Chemicals and reagents CSC and CSA were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Glycogen (from Mytilus edulis; mean molecular weigth: 2, 560, 000 Dalton) was purchased from Takeda Pure Chemicals, Ltd. (Osaka, Japan). β-CD was purchased from Yunan Forever Bright Monosodium Glutamate Co. Ltd (Guangdong, China). Hydroxypropyl-β-cyclodextrin (HP-β-CD) was purchased from Xi’an Deli Biochemical Company (Xi’an, China). Citalopram hydrobromide (CIT, pKa 9.78) were obtained from Chongqing Lummy Pharm Techn Co., Ltd (Chongqing, China). Propranolol hydrochloride (PRO, pKa 9.67), chlorphenamine maleate (CHL, pKa 9.47), atenolol (ATE, pKa 9.67), sulconazole (SUL, pKa This article is protected by copyright. All rights reserved.
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6.69) and laudanosine (LAU, pKa 8.3) were purchased from Sigma (St. Louis, MO, USA). Cetirizine hydrochloride (CET, pKa 2.19, 2.93, and 8.00), nefopam hydrochloride (NEF, pKa 8.98), duloxetine hydrochloride (DUL, pKa 9.70), amlodipine (AML, pKa 9.45) and its R-isomer were kind gifts from Jiangsu Institute For Drug Control (Nanjing, China). The structures of all these chiral drugs are shown in Supporting Information Fig. S1 B. All these compounds were racemates besides R-AML enantiomer. Nylon filters (0.45 µm) and HPLC grade methanol were purchased from Jiangsu Hanbon Sci.&Tech. Co., Ltd (Nanjing, China). Sodium dihydrogen phosphate, phosphoric acid and sodium hydroxide were of analytical grade from Nanjing Chemical Reagent CO., Ltd (Nanjing, China). The water used to prepare the buffer and the sample solution was distilled and doubly deionized.
2.2 Apparatus and procedures Separations were performed on an Aglient
3D
CE automated capillary electrophoresis
system (Waldbronn, Germany) equipped with a diode array detector (190 to 600 nm). Agilent ChemStation software (Revision B.02.01) was used for instrumental control and data analysis. The CE system was operated in the conventional mode with the anode at the injector end of the capillary. Uncoated capillaries (Hebei Yongnian County Reafine Chromatography, Hebei, China) of 50 cm length (41.5cm to the detector) ×50 µm id were used, with the temperature keeping at 20 ℃ throughout the experiments. A new capillary was initially washed with methanol (10 min), followed by water (5 min), 1 M HCl (10 min), water (5 min), 1 M NaOH (10 min), and water (5 min) successively. As a daily routine procedure, the capillary was rinsed with 1 M NaOH (10 min) followed by a 10-min rinse with water, and then flushed This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
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Electrophoresis
with running buffer (5 min) before sample injections. Before every run, the capillary was washed with 1 M NaOH, water, and running buffer for 2 min in sequence. The samples were introduced hydrodynamically for 4 s (injection pressure 50 mbar). Running buffer solutions were freshly prepared by dissolving appropriate amounts of chiral selectors in the BGE consisting of 20 or 40 mM NaH2PO4 solution, and adjusted to the desired pH value with H3PO4 (10 %, w/v). All stock solutions of either racemic or pure enantiomeric samples were prepared in methanol at a concentration of ca. 1.0 mg/mL. The sample solutions were obtained at a concentration of ca. 0.1 mg/mL by diluting above stock solutions with water. All running buffers and sample solutions were filtered with a 0.45 µm nylon filter and degassed by sonication prior to use. All the measurements were performed three times if not stated otherwise.
3 Results and discussion 3.1 Dual system consisting of CSC and glycogen To investigate the enantioseparation performance of the dual system consisting of CSC (an anionic polysaccharide) and glycogen (a neutral polysaccharide), comparison experiments, namely, the enantioseparation using single system (CSC or glycogen) and dual system (CSC and glycogen) were performed, respectively. According to the references [20, 21, 26], the single system was carried out using a 40 mM Na2HPO4 (pH 3.0) running buffer containing 3.0 % w/v of glycogen or 2.0 % w/v of CSC alone. As illustrated in Table 1, the resolutions (Rs) of CET, NEF, DUL, ATE, SUL and LAU increased when the dual system was employed, especially for NEF, SUL and DUL, with their Rs increasing from 1.64 to 2.85,
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Electrophoresis
Study of the enantioseparation capability of chiral dual system based on chondroitin sulfate C in capillary electrophoresis
Jiaquan Chen1, Yingxiang Du1, 2, 3, *, Fenxia Zhu 1,4, Bin Chen 5, Qi Zhang1, Shuaijing Du6, Ping Li 3 1
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing, P. R. China
2
Key Laboratory of Drug Quality Control and Pharmacovigilance (Ministry of Education), China Pharmaceutical University, Nanjing, P. R. China
3
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P. R. China
4
Key Laboratory of New Drug Delivery System of Chinese Meteria Medica, Jiangsu
Provincial Academy of Chinese Medicine, Nanjing, P. R. China 5
Department of Pharmacy, Zhejiang Pharmaceutical College, Ningbo, P. R. China
6
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] (Y. Du) Tel./fax: +86 25 83221790
Received: 31-Jan-2013; Revised: 09-Nov-2014; Accepted: 14-Nov-2014 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.201300057. This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 2
Electrophoresis
Keywords: Capillary electrophoresis / Enantioseparation / Chiral dual system / Chondroitin sulfate C / Glycogen
Abbreviations: AML, amlodipine; ATE, atenolol; CET, cetirizine hydrochloride; CHL, chlorphenamine maleate; CIT, citalopram hydrobromide; CSA, chondroitin sulfate A; CSC, chondroitin
sulfate
C;
DUL,
duloxetine
hydrochloride;
HP-β-CD,
hydroxypropyl-β-cyclodextrin; LAU, laudanosine; NEF, nefopam hydrochloride; PRO, propranolol hydrochloride; SUL, sulconazole.
Abstract 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 capillary electrophoresis (CE) chiral separation are mostly dual cyclodextrins (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-cyclodextrin (HP-β-CD), as well as CSC/β-cyclodextrin (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 This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 3
Electrophoresis
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.
1 Introduction The enantiomeric separation of chiral drugs, metabolites and related substances is of great importance in the pharmaceutical field. Usually, the pharmacological activity and metabolism of the two enantiomers of one drug are different. Therefore, rapid, selective and sensitive analytical methods are required to verify the optical purity of chiral drugs. Various analytical techniques have been developed for enantioseparation, among which capillary electrophoresis (CE) has been widely used because of its simplicity, rapidity, low cost, as well as high separation efficiency [1-3]. Enantioseparation by CE is mainly achieved by a direct method in which the chiral selector is simply added to the background electrolyte (BGE). The availability of many chiral selectors makes CE an important tool for chiral analysis [4-7]. Among various selectors, cyclodextrins (CDs) and their derivatives are still the most popular ones, and the interest in antibiotics has been continuously increasing [8-14]. Besides the two kinds of selectors, polysaccharides as CE chiral selectors have attracted great attention over the past few years [15-20]. The weak UV absorption and water solubility of polysaccharides facilitates their application as chiral selectors. Additionally, their varying structures and This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 4
Electrophoresis
functional groups provide a wide range of enantioselectivity. Chondroitin
sulfates,
negatively
charged
polysaccharides
composed
of
N-acetylgalactosamine and glucuronic acid residues (see the Supporting Information Fig. S1 A), have been utilized successfully as chiral selectors in CE [21-24]. There are three forms of chondroitin sulfates, namely chondroitin sulfates A, B and C, all with a linear repeating sequence of β-(1,4) linkages between the disaccharide subunits and β-(1,3) linkages within the disaccharide subunits. Chondroitin sulfate C (CSC) usually displays better enantioseparation capability than chondroitin sulfate A (CSA) and B (CSB), owing to the hydroxyl group at C4 in the galactosamine residue [25]. It has been also considered as one of the most excellent polysaccharide chiral selectors so far [15, 26]. In some cases, a successful enantioseparation cannot be achieved by employing one single chiral selector in the BGE. With two different chiral selectors introduced simultaneously, chiral dual system often leads to a significant enhancement of selectivity and resolution (synergistic effect) due to differences in the complexation mechanisms of the two selectors with the analyte enantiomers [27]. The use of dual CD systems has been reported in some studies, in which different combinations of charged and neutral CDs are employed [28-35]. Taking into account the successful use of dual CD systems and the excellent enantioseparation capability of polysaccharides (such as CSC), the application of polysaccharide-based chiral dual system should be worth studying. However, the chiral dual system based on polysaccharide has been reported in only one paper by our group [36]. In this previous work, we studied three glycogen-based dual systems including glycogen/CSA, This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 5
Electrophoresis
glycogen/β-CD and glycogen/HP-β-CD. Among them, significant synergistic effect was observed in the glycogen/CSA system. However, no synergistic effect was found in glycogen/β-CD and glycogen/HP-β-CD system. This paper is a follow up study of our previous research [36]. The enantioseparation performance of four CSC-based dual systems, including CSC/CSA, CSC/glycogen and CSC/natural CDs (β-CD or HP-β-CD), was investigated for the first time. As observed, the CSC/glycogen and CSC/HP-β-CD systems gave significant synergistic effect to some drugs enantiomers, suggesting their value for practical use. The details and results in the present work are different from our previous paper, and a number of advances exist. It also provides an amount of useful information to the fundamental research of polysaccharide-based chiral dual system.
2 Materials and methods 2.1 Chemicals and reagents CSC and CSA were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Glycogen (from Mytilus edulis; mean molecular weigth: 2, 560, 000 Dalton) was purchased from Takeda Pure Chemicals, Ltd. (Osaka, Japan). β-CD was purchased from Yunan Forever Bright Monosodium Glutamate Co. Ltd (Guangdong, China). Hydroxypropyl-β-cyclodextrin (HP-β-CD) was purchased from Xi’an Deli Biochemical Company (Xi’an, China). Citalopram hydrobromide (CIT, pKa 9.78) were obtained from Chongqing Lummy Pharm Techn Co., Ltd (Chongqing, China). Propranolol hydrochloride (PRO, pKa 9.67), chlorphenamine maleate (CHL, pKa 9.47), atenolol (ATE, pKa 9.67), sulconazole (SUL, pKa This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
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Electrophoresis
6.69) and laudanosine (LAU, pKa 8.3) were purchased from Sigma (St. Louis, MO, USA). Cetirizine hydrochloride (CET, pKa 2.19, 2.93, and 8.00), nefopam hydrochloride (NEF, pKa 8.98), duloxetine hydrochloride (DUL, pKa 9.70), amlodipine (AML, pKa 9.45) and its R-isomer were kind gifts from Jiangsu Institute For Drug Control (Nanjing, China). The structures of all these chiral drugs are shown in Supporting Information Fig. S1 B. All these compounds were racemates besides R-AML enantiomer. Nylon filters (0.45 µm) and HPLC grade methanol were purchased from Jiangsu Hanbon Sci.&Tech. Co., Ltd (Nanjing, China). Sodium dihydrogen phosphate, phosphoric acid and sodium hydroxide were of analytical grade from Nanjing Chemical Reagent CO., Ltd (Nanjing, China). The water used to prepare the buffer and the sample solution was distilled and doubly deionized.
2.2 Apparatus and procedures Separations were performed on an Aglient
3D
CE automated capillary electrophoresis
system (Waldbronn, Germany) equipped with a diode array detector (190 to 600 nm). Agilent ChemStation software (Revision B.02.01) was used for instrumental control and data analysis. The CE system was operated in the conventional mode with the anode at the injector end of the capillary. Uncoated capillaries (Hebei Yongnian County Reafine Chromatography, Hebei, China) of 50 cm length (41.5cm to the detector) ×50 µm id were used, with the temperature keeping at 20 ℃ throughout the experiments. A new capillary was initially washed with methanol (10 min), followed by water (5 min), 1 M HCl (10 min), water (5 min), 1 M NaOH (10 min), and water (5 min) successively. As a daily routine procedure, the capillary was rinsed with 1 M NaOH (10 min) followed by a 10-min rinse with water, and then flushed This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
Page 7
Electrophoresis
with running buffer (5 min) before sample injections. Before every run, the capillary was washed with 1 M NaOH, water, and running buffer for 2 min in sequence. The samples were introduced hydrodynamically for 4 s (injection pressure 50 mbar). Running buffer solutions were freshly prepared by dissolving appropriate amounts of chiral selectors in the BGE consisting of 20 or 40 mM NaH2PO4 solution, and adjusted to the desired pH value with H3PO4 (10 %, w/v). All stock solutions of either racemic or pure enantiomeric samples were prepared in methanol at a concentration of ca. 1.0 mg/mL. The sample solutions were obtained at a concentration of ca. 0.1 mg/mL by diluting above stock solutions with water. All running buffers and sample solutions were filtered with a 0.45 µm nylon filter and degassed by sonication prior to use. All the measurements were performed three times if not stated otherwise.
3 Results and discussion 3.1 Dual system consisting of CSC and glycogen To investigate the enantioseparation performance of the dual system consisting of CSC (an anionic polysaccharide) and glycogen (a neutral polysaccharide), comparison experiments, namely, the enantioseparation using single system (CSC or glycogen) and dual system (CSC and glycogen) were performed, respectively. According to the references [20, 21, 26], the single system was carried out using a 40 mM Na2HPO4 (pH 3.0) running buffer containing 3.0 % w/v of glycogen or 2.0 % w/v of CSC alone. As illustrated in Table 1, the resolutions (Rs) of CET, NEF, DUL, ATE, SUL and LAU increased when the dual system was employed, especially for NEF, SUL and DUL, with their Rs increasing from 1.64 to 2.85,
