Anal Bioanal Chem (2014) 406:1557–1566 DOI 10.1007/s00216-013-7544-3
RESEARCH PAPER
Investigation of chondroitin sulfate D and chondroitin sulfate E as novel chiral selectors in capillary electrophoresis Qi Zhang & Yingxiang Du & Jiaquan Chen & Guangfu Xu & Tao Yu & Xiaoyi Hua & Jinjing Zhang
Received: 29 April 2013 / Revised: 3 October 2013 / Accepted: 29 November 2013 / Published online: 21 December 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Various chiral selectors have been utilized successfully in capillary electrophoresis (CE); however, the number of polysaccharides used as chiral selectors is still small and the mechanism of enantiorecognition has not been fully elucidated. Chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE), belonging to the group of glycosaminoglycans, are linear, sulfated polysaccharides with large mass. In this paper, they were investigated for the first time for their potential as chiral selectors by CE. The effect of buffer composition and pH, chiral selector concentration, and applied voltage were systematically examined and optimized. A variety of drug enantiomers were resolved in the buffer pH range of 2.8–3.4 using 20 mM Tris/H3PO4 buffer with 5.0 % CSD or CSE and 20 kVapplied voltage. A central composite design was used to validate the optimized separation parameters and satisfactory uniformity was obtained. As observed, CSE allowed satisfactory separation of the enantiomers of amlodipine, laudanosine, nefopam, sulconazole, and tryptophan methyl ester, as well as partial resolution of citalopram, duloxetine, and propranolol under the optimized conditions. CSD allowed partial or nearly baseline separation of amlodipine, laudanosine, nefopam, and sulconazole. The results indicated that CSE has a better enantiorecognition capability than CSD toward the tested drugs. Q. Zhang : Y. Du (*) : J. Chen : G. Xu : T. Yu : X. Hua : J. Zhang Department of Analytical Chemistry, China Pharmaceutical University, No. 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China e-mail: [email protected] Y. Du Key Laboratory of Drug Quality Control and Pharmacovigilance (Ministry of Education), China Pharmaceutical University, Nanjing 210009, China Y. Du State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
Keywords Capillary electrophoresis . Enantiomeric separation . Chondroitin sulfate D . Chondroitin sulfate E . Chiral selector
Introduction Enantiomeric separation is one of the important issues in pharmaceutical analysis where a wide number of drugs with asymmetric centers exist as pairs of enantiomers [1]. Very often, the pharmacological activity and metabolism of two enantiomers of a drug may be different [2]. For this reason, chiral separation is getting more and more important for drug quality control, pharmacodynamic, and pharmacokinetic studies as well as toxicological investigations. Various analytical techniques have been developed for chiral separation, such as high-performance liquid chromatography (HPLC) [3], gas chromatography [4], supercritical fluid chromatography [5], etc. Among them, HPLC is still the dominant method for its several advantages such as low limit of detection, good reproducibility, etc. However, the column efficiency of HPLC is relatively low and it requires relatively large volumes of samples and chiral reagents. The use of chiral HPLC column is also very expensive while these chiral columns always have limited enantiorecognition capability toward various drug enantiomers. 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 [6–12]. The most common approach for enantiomeric separation in CE involves the addition of one or more chiral selectors into the running buffer. Thus, various kinds of chiral selectors have been developed, including cyclodextrins [13, 14] and their derivatives [14, 15], macrocyclic antibiotics [16, 17], and
1558
many other small molecules [18, 19]. Linear oligo- and polysaccharides have attracted much interest over the past several years [20–24]. Varying structures and functional groups of polysaccharides provide a range of enantioselectivity in CE and many of them are water-soluble. Additionally, the weak UVabsorption of polysaccharides facilitates the application of these materials as CE chiral additives. Despite some enantiomeric separations have been achieved with neutral polysaccharides such as dextrin and dextran [25–29], more attention has been focused on the use of anionic polysaccharides. Among them, heparin, dextran sulfate, and pentosan polysulfate were successfully used for the chiral separation of basic and neutral drugs by CE [30–33]. Chondroitin sulfates, which are negatively charged polysaccharides, composed of the N-acetylgalactosamine and glucuronic acid residues, are widely existed in different animal tissues [34]. They showed wide enantioselectivity toward basic drugs, and in previous studies, authors noted the function of sulfate group in chiral recognition and at the same time pointed out the significance of polymer network from the comparison with neutral polysaccharides. The first paper on enantioseparation with chondroitin sulfate C as chiral selector was published by Nishi et al. [35] in 1995. Besides, chondroitin sulfate A, dermatan sulfate (chondroitin sulfate B), and some related glycosaminoglycans were also successfully used for the chiral separation of basic and neutral drugs by CE in the next few years [28, 36–38]. However, after these papers, few literatures focused on the use of new chondroitin sulfates and derivatives for chiral separation. In general, the uses of polysaccharides as chiral selectors are still less and the mechanism of enantiorecognition has not been fully elucidated. In this study, chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE), as shown in Fig. 1, belonging to the group of anionic glycosaminoglycans, were first investigated for their potential as chiral selectors. We presented details of enantioseparation of the studied drugs using CSD and CSE as buffer additives by CE.
Experimental Chemicals and reagents Chondroitin sulfate D (90 %) was purchased from Hefei Bomei Biotechnology Co., Ltd (Hefei, China); chondroitin sulfate E (90 %) was purchased from Wuhan Yuancheng Gong-chuang Technology Co., Ltd (Wuhan, China). The molecular mass distributions of CSD and CSE were 15,000–30,000 and 20,000–30,000, respectively. Laudanosine (LAU), propranolol hydrochloride (PRO), and tryptophan methyl ester (TME) were purchased from Sigma (St. Louis, MO, USA). Amlodipine besylate (AML), citalopram hydrobromide (CIT), duloxetine
Q. Zhang et al.
Fig. 1 Unit structure of: a chondroitin sulfate D; b chondroitin sulfate E
hydrochloride (DUL), nefopam hydrochloride (NEF), and sulconazole (SUL) were supplied by Jiangsu Institute for Food and Drug Control (Nanjing, China). All these drug samples were racemic mixtures. Tris (hydroxymethyl) aminomethane (Tris) was purchased from Shanghai Huixing Biochemistry Reagent (Shanghai, China). Nylon filters (0.45 μm) were purchased from Jiangsu Hanbon Science and Technology (Nanjing, China). Phosphoric acid and sodium hydrogen were of analytical reagent from Nanjing Chemical Reagent (Nanjing, China). Double distilled water was used throughout all the experiments. Apparatus Electrophoretic experiments were performed with an Agilent 3D CE system (Agilent Technologies, Waldbronn, Germany), which consisted of 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 ChemStation software (Revision B.02.01) for system control, data collection, and analysis. It was equipped with a 50 cm (41.5 cm effective length)×50 μm id uncoated fused-silica capillary (Hebei Yongnian County Reafine Chromatography Ltd., Hebei, China). The samples were introduced hydrodynamically for 5 s (injection pressure 50 mbar). All separations were carried out at 15 °C using a voltage in a range of 10–20 kV. The wavelength for detection was 237 nm (AML, CIT), 230 nm (DUL, LAU, and SUL), 215 nm (NEF), or 225 nm (PRO, TME). 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 (20 min), followed by the 0.1 M NaOH and water (10 min), respectively. At the beginning of each day, the
Chondroitin sulfate D and E as novel chiral selectors
1559
Fig. 2 Structures of the racemic drugs studied in this paper
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.
Running buffers and samples were filtered with a 0.45-μm pore membrane filter and degassed by sonication prior to use.
Procedures
The resolution (R s) and selectivity factor (α) of the enantiomers were calculated from R s =1.18(t 2 −t 1)/(w 1 +w 2) and α =t 2/t 1, where t 1 and t 2 are the migration times of the two enantiomers, and w 1 and w 2 are the peak widths at half-height of the first and second eluting enantiomer. The electroosmotic flow (EOF) and apparent mobilities (μ app) were expressed by the equation, μ eof =(L ×l)/(V ×t 0), and μ app =(L ×l)/(V ×t m ), where L, l, V, t 0, and t m are total capillary length, effective capillary length, applied voltage, migration time of acetone (a neutral marker), and migration time of the first migrating enantiomer in the
The background electrolyte (BGE) consisted of 20 mM Tris solution, adjusted to specified pH value with H3PO4 (10 % v/v). The running buffer solutions were freshly prepared by dissolving appropriate amounts of chondroitin sulfate D or chondroitin sulfate E in BGE, and then adjusting pH exactly to a desired value by adding a small volume of H3PO4 (10 % v/v) using a microsyringe. The racemic samples (0.5 mg/ml) were dissolved in methanol (LAU) or distilled water (others).
Calculations
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Q. Zhang et al.
Fig. 3 Typical electropherograms of the chiral separations of studied drug enantiomers under the optimal conditions. Conditions: fused-silica capillary, 50 cm (41.5 cm effective length) × 50 μm id; 20 mM
Tris/H3PO4 buffer solution containing 5.0 % CSE (a) or CSD (b); buffer pH, 2.8 (3.4 for NEF) for CSE system, 3.4 (2.8 for AML) for CSD system; applied voltage, 15 kV; capillary temperature, 15 °C
presence of chiral additives, respectively. Effective mobilities (μ eff) were calculated from μ eff =μ app −μ eof .
composition and pH, chiral selector concentration, and applied voltage because the enantiorecognition process could be facilitated by these parameters among others. In the course of operation, one of the aforementioned parameters was varied while the others were kept constant.
Results and discussion A variety of racemic drugs were selected as the model analytes (their structures are shown in Fig. 2). We carried out CE experiments to investigate the optimal chiral separation of these drugs under various electrophoretic conditions such as buffer
Structures and properties of CSD and CSE Chondroitin sulfates are natural-occuring substances found in connective tissue or mast cell granules and are soluble in water.
Chondroitin sulfate D and E as novel chiral selectors
1561
of chondroitin sulfate A, B, and C in sites or the number of sulfate groups. CSD and CSE both have a sulfate group at C6 position in the galactosamine residue. Besides, CSD has an extra sulfate group at C2 position in the glucuronic acid residue, while CSE has an extra sulfate group at C4 position in the galactosamine residue (see Fig. 1). These anionic characters of chondroitins also enhance their aqueous solubility while offering the potential for considerable electrophoretic mobility. For these reasons, CSD and CSE are expected that their chiral recognition abilities could be different from the chondroitin sulfates that reported in previous studies. Enantioseparation of racemic drugs by CSD and CSE Fig. 4 Effects of buffer pH on the effective mobilities (μ eff, in square centimeter per second per volt·10−5) of selected enantiomers. Conditions: 20 mM Tris/H3PO4 buffer solution containing 5.0 % CSE; applied voltage, 15 kV; other conditions as in “Experimental”
CSD and CSE are both sulfated polysaccharides composed of unit structures of β-(1→4)-linked disaccharide subunits within which hexosamine and uronic acid are connected through β-(1→3) linkages. Related ionic galactosaminogylcans (chondroitin sulfate A, B, and C) have been employed as chiral selectors for the enantiomeric separation of several pharmaceutical compounds by CE. The ionic characters of these galactosaminogylcans are very important features that affect the enantioseparation capability in the separation environment. As for the anionic character, chondroitin sulfate A, B, and C have about one ionic group (carboxyl group or sulfate group) per monosaccharide unit, with one sulfate group distributed per disaccharide unit. CSD and CSE unit structures differ from that
On the basis of discussed considerations, CSD and CSE should be useful chiral selectors preferably under acidic conditions as described for other galactosaminogylcans because researchers have noted the contribution of ionic interaction to the enantioseparation [33, 35, 37]. Since the pK a values of most analytes used in this study were in the range of 8–10, the enantiomers should be significantly protonated during the separation process. The protonated nitrogens can interact electrostatically with sulfate groups in the residues of CSD and CSE. Combined with possible hydrogen bond, hydrophobic, and steric interactions, large mobility difference between the free analyte and its complexed form would occur, and thus result in a successful enantioseparation. Through the study of a series of racemic drugs, we obtained the best enantioresolution in strong acidic background electrolyte (pH 2.8–3.4) for most analytes. As observed, CSE allowed satisfactory separation (R s >1.5) of AML, LAU, NEF, SUL, and TME, as well as partial resolution of CIT,
Table 1 Effects of running buffer pH on the enantioseparation of 8 drugs with CSE Drugs Buffer pH 2.0 t 2/t 1 (min)
2.4 R s/α
t 2/t 1 (min)
2.8 R s/α
t 2/t 1 (min)
3.4 R s/α
t 2/t 1 (min)
4.0 R s/α
t 2/t 1 (min)
R s/α
AML 16.217/15.828 1.57/1.025 23.132/22.197 2.25/1.042 21.607/20.876 2.65/1.035 15.639/15.303 1.57/1.022 13.823/13.746 0.54/1.006 CIT DUL LAU NEF PRO SUL TME
16.185/16.060 18.327/18.176 18.997/18.481 14.289/14.060 16.896/16.778 17.432/16.976 19.760/19.411
RESEARCH PAPER
Investigation of chondroitin sulfate D and chondroitin sulfate E as novel chiral selectors in capillary electrophoresis Qi Zhang & Yingxiang Du & Jiaquan Chen & Guangfu Xu & Tao Yu & Xiaoyi Hua & Jinjing Zhang
Received: 29 April 2013 / Revised: 3 October 2013 / Accepted: 29 November 2013 / Published online: 21 December 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Various chiral selectors have been utilized successfully in capillary electrophoresis (CE); however, the number of polysaccharides used as chiral selectors is still small and the mechanism of enantiorecognition has not been fully elucidated. Chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE), belonging to the group of glycosaminoglycans, are linear, sulfated polysaccharides with large mass. In this paper, they were investigated for the first time for their potential as chiral selectors by CE. The effect of buffer composition and pH, chiral selector concentration, and applied voltage were systematically examined and optimized. A variety of drug enantiomers were resolved in the buffer pH range of 2.8–3.4 using 20 mM Tris/H3PO4 buffer with 5.0 % CSD or CSE and 20 kVapplied voltage. A central composite design was used to validate the optimized separation parameters and satisfactory uniformity was obtained. As observed, CSE allowed satisfactory separation of the enantiomers of amlodipine, laudanosine, nefopam, sulconazole, and tryptophan methyl ester, as well as partial resolution of citalopram, duloxetine, and propranolol under the optimized conditions. CSD allowed partial or nearly baseline separation of amlodipine, laudanosine, nefopam, and sulconazole. The results indicated that CSE has a better enantiorecognition capability than CSD toward the tested drugs. Q. Zhang : Y. Du (*) : J. Chen : G. Xu : T. Yu : X. Hua : J. Zhang Department of Analytical Chemistry, China Pharmaceutical University, No. 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China e-mail: [email protected] Y. Du Key Laboratory of Drug Quality Control and Pharmacovigilance (Ministry of Education), China Pharmaceutical University, Nanjing 210009, China Y. Du State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
Keywords Capillary electrophoresis . Enantiomeric separation . Chondroitin sulfate D . Chondroitin sulfate E . Chiral selector
Introduction Enantiomeric separation is one of the important issues in pharmaceutical analysis where a wide number of drugs with asymmetric centers exist as pairs of enantiomers [1]. Very often, the pharmacological activity and metabolism of two enantiomers of a drug may be different [2]. For this reason, chiral separation is getting more and more important for drug quality control, pharmacodynamic, and pharmacokinetic studies as well as toxicological investigations. Various analytical techniques have been developed for chiral separation, such as high-performance liquid chromatography (HPLC) [3], gas chromatography [4], supercritical fluid chromatography [5], etc. Among them, HPLC is still the dominant method for its several advantages such as low limit of detection, good reproducibility, etc. However, the column efficiency of HPLC is relatively low and it requires relatively large volumes of samples and chiral reagents. The use of chiral HPLC column is also very expensive while these chiral columns always have limited enantiorecognition capability toward various drug enantiomers. 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 [6–12]. The most common approach for enantiomeric separation in CE involves the addition of one or more chiral selectors into the running buffer. Thus, various kinds of chiral selectors have been developed, including cyclodextrins [13, 14] and their derivatives [14, 15], macrocyclic antibiotics [16, 17], and
1558
many other small molecules [18, 19]. Linear oligo- and polysaccharides have attracted much interest over the past several years [20–24]. Varying structures and functional groups of polysaccharides provide a range of enantioselectivity in CE and many of them are water-soluble. Additionally, the weak UVabsorption of polysaccharides facilitates the application of these materials as CE chiral additives. Despite some enantiomeric separations have been achieved with neutral polysaccharides such as dextrin and dextran [25–29], more attention has been focused on the use of anionic polysaccharides. Among them, heparin, dextran sulfate, and pentosan polysulfate were successfully used for the chiral separation of basic and neutral drugs by CE [30–33]. Chondroitin sulfates, which are negatively charged polysaccharides, composed of the N-acetylgalactosamine and glucuronic acid residues, are widely existed in different animal tissues [34]. They showed wide enantioselectivity toward basic drugs, and in previous studies, authors noted the function of sulfate group in chiral recognition and at the same time pointed out the significance of polymer network from the comparison with neutral polysaccharides. The first paper on enantioseparation with chondroitin sulfate C as chiral selector was published by Nishi et al. [35] in 1995. Besides, chondroitin sulfate A, dermatan sulfate (chondroitin sulfate B), and some related glycosaminoglycans were also successfully used for the chiral separation of basic and neutral drugs by CE in the next few years [28, 36–38]. However, after these papers, few literatures focused on the use of new chondroitin sulfates and derivatives for chiral separation. In general, the uses of polysaccharides as chiral selectors are still less and the mechanism of enantiorecognition has not been fully elucidated. In this study, chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE), as shown in Fig. 1, belonging to the group of anionic glycosaminoglycans, were first investigated for their potential as chiral selectors. We presented details of enantioseparation of the studied drugs using CSD and CSE as buffer additives by CE.
Experimental Chemicals and reagents Chondroitin sulfate D (90 %) was purchased from Hefei Bomei Biotechnology Co., Ltd (Hefei, China); chondroitin sulfate E (90 %) was purchased from Wuhan Yuancheng Gong-chuang Technology Co., Ltd (Wuhan, China). The molecular mass distributions of CSD and CSE were 15,000–30,000 and 20,000–30,000, respectively. Laudanosine (LAU), propranolol hydrochloride (PRO), and tryptophan methyl ester (TME) were purchased from Sigma (St. Louis, MO, USA). Amlodipine besylate (AML), citalopram hydrobromide (CIT), duloxetine
Q. Zhang et al.
Fig. 1 Unit structure of: a chondroitin sulfate D; b chondroitin sulfate E
hydrochloride (DUL), nefopam hydrochloride (NEF), and sulconazole (SUL) were supplied by Jiangsu Institute for Food and Drug Control (Nanjing, China). All these drug samples were racemic mixtures. Tris (hydroxymethyl) aminomethane (Tris) was purchased from Shanghai Huixing Biochemistry Reagent (Shanghai, China). Nylon filters (0.45 μm) were purchased from Jiangsu Hanbon Science and Technology (Nanjing, China). Phosphoric acid and sodium hydrogen were of analytical reagent from Nanjing Chemical Reagent (Nanjing, China). Double distilled water was used throughout all the experiments. Apparatus Electrophoretic experiments were performed with an Agilent 3D CE system (Agilent Technologies, Waldbronn, Germany), which consisted of 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 ChemStation software (Revision B.02.01) for system control, data collection, and analysis. It was equipped with a 50 cm (41.5 cm effective length)×50 μm id uncoated fused-silica capillary (Hebei Yongnian County Reafine Chromatography Ltd., Hebei, China). The samples were introduced hydrodynamically for 5 s (injection pressure 50 mbar). All separations were carried out at 15 °C using a voltage in a range of 10–20 kV. The wavelength for detection was 237 nm (AML, CIT), 230 nm (DUL, LAU, and SUL), 215 nm (NEF), or 225 nm (PRO, TME). 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 (20 min), followed by the 0.1 M NaOH and water (10 min), respectively. At the beginning of each day, the
Chondroitin sulfate D and E as novel chiral selectors
1559
Fig. 2 Structures of the racemic drugs studied in this paper
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.
Running buffers and samples were filtered with a 0.45-μm pore membrane filter and degassed by sonication prior to use.
Procedures
The resolution (R s) and selectivity factor (α) of the enantiomers were calculated from R s =1.18(t 2 −t 1)/(w 1 +w 2) and α =t 2/t 1, where t 1 and t 2 are the migration times of the two enantiomers, and w 1 and w 2 are the peak widths at half-height of the first and second eluting enantiomer. The electroosmotic flow (EOF) and apparent mobilities (μ app) were expressed by the equation, μ eof =(L ×l)/(V ×t 0), and μ app =(L ×l)/(V ×t m ), where L, l, V, t 0, and t m are total capillary length, effective capillary length, applied voltage, migration time of acetone (a neutral marker), and migration time of the first migrating enantiomer in the
The background electrolyte (BGE) consisted of 20 mM Tris solution, adjusted to specified pH value with H3PO4 (10 % v/v). The running buffer solutions were freshly prepared by dissolving appropriate amounts of chondroitin sulfate D or chondroitin sulfate E in BGE, and then adjusting pH exactly to a desired value by adding a small volume of H3PO4 (10 % v/v) using a microsyringe. The racemic samples (0.5 mg/ml) were dissolved in methanol (LAU) or distilled water (others).
Calculations
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Q. Zhang et al.
Fig. 3 Typical electropherograms of the chiral separations of studied drug enantiomers under the optimal conditions. Conditions: fused-silica capillary, 50 cm (41.5 cm effective length) × 50 μm id; 20 mM
Tris/H3PO4 buffer solution containing 5.0 % CSE (a) or CSD (b); buffer pH, 2.8 (3.4 for NEF) for CSE system, 3.4 (2.8 for AML) for CSD system; applied voltage, 15 kV; capillary temperature, 15 °C
presence of chiral additives, respectively. Effective mobilities (μ eff) were calculated from μ eff =μ app −μ eof .
composition and pH, chiral selector concentration, and applied voltage because the enantiorecognition process could be facilitated by these parameters among others. In the course of operation, one of the aforementioned parameters was varied while the others were kept constant.
Results and discussion A variety of racemic drugs were selected as the model analytes (their structures are shown in Fig. 2). We carried out CE experiments to investigate the optimal chiral separation of these drugs under various electrophoretic conditions such as buffer
Structures and properties of CSD and CSE Chondroitin sulfates are natural-occuring substances found in connective tissue or mast cell granules and are soluble in water.
Chondroitin sulfate D and E as novel chiral selectors
1561
of chondroitin sulfate A, B, and C in sites or the number of sulfate groups. CSD and CSE both have a sulfate group at C6 position in the galactosamine residue. Besides, CSD has an extra sulfate group at C2 position in the glucuronic acid residue, while CSE has an extra sulfate group at C4 position in the galactosamine residue (see Fig. 1). These anionic characters of chondroitins also enhance their aqueous solubility while offering the potential for considerable electrophoretic mobility. For these reasons, CSD and CSE are expected that their chiral recognition abilities could be different from the chondroitin sulfates that reported in previous studies. Enantioseparation of racemic drugs by CSD and CSE Fig. 4 Effects of buffer pH on the effective mobilities (μ eff, in square centimeter per second per volt·10−5) of selected enantiomers. Conditions: 20 mM Tris/H3PO4 buffer solution containing 5.0 % CSE; applied voltage, 15 kV; other conditions as in “Experimental”
CSD and CSE are both sulfated polysaccharides composed of unit structures of β-(1→4)-linked disaccharide subunits within which hexosamine and uronic acid are connected through β-(1→3) linkages. Related ionic galactosaminogylcans (chondroitin sulfate A, B, and C) have been employed as chiral selectors for the enantiomeric separation of several pharmaceutical compounds by CE. The ionic characters of these galactosaminogylcans are very important features that affect the enantioseparation capability in the separation environment. As for the anionic character, chondroitin sulfate A, B, and C have about one ionic group (carboxyl group or sulfate group) per monosaccharide unit, with one sulfate group distributed per disaccharide unit. CSD and CSE unit structures differ from that
On the basis of discussed considerations, CSD and CSE should be useful chiral selectors preferably under acidic conditions as described for other galactosaminogylcans because researchers have noted the contribution of ionic interaction to the enantioseparation [33, 35, 37]. Since the pK a values of most analytes used in this study were in the range of 8–10, the enantiomers should be significantly protonated during the separation process. The protonated nitrogens can interact electrostatically with sulfate groups in the residues of CSD and CSE. Combined with possible hydrogen bond, hydrophobic, and steric interactions, large mobility difference between the free analyte and its complexed form would occur, and thus result in a successful enantioseparation. Through the study of a series of racemic drugs, we obtained the best enantioresolution in strong acidic background electrolyte (pH 2.8–3.4) for most analytes. As observed, CSE allowed satisfactory separation (R s >1.5) of AML, LAU, NEF, SUL, and TME, as well as partial resolution of CIT,
Table 1 Effects of running buffer pH on the enantioseparation of 8 drugs with CSE Drugs Buffer pH 2.0 t 2/t 1 (min)
2.4 R s/α
t 2/t 1 (min)
2.8 R s/α
t 2/t 1 (min)
3.4 R s/α
t 2/t 1 (min)
4.0 R s/α
t 2/t 1 (min)
R s/α
AML 16.217/15.828 1.57/1.025 23.132/22.197 2.25/1.042 21.607/20.876 2.65/1.035 15.639/15.303 1.57/1.022 13.823/13.746 0.54/1.006 CIT DUL LAU NEF PRO SUL TME
16.185/16.060 18.327/18.176 18.997/18.481 14.289/14.060 16.896/16.778 17.432/16.976 19.760/19.411
