2900 Tao Yu1 Yingxiang Du1,2,3 Jiaquan Chen1 Guangfu Xu1 Ke Yang1 Qi Zhang1 Jinjing Zhang1 Shuaijing Du4 Zijie Feng1 Yanjie Zhang1 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 College of Chemistry and Molecular Engineering, Peking University, Beijing, P. R. China Received June 23, 2014 Revised May 16, 2015 Accepted May 16, 2015
J. Sep. Sci. 2015, 38, 2900–2906
Research Article
Study on clarithromycin lactobionate based dual selector systems for the enantioseparation of basic drugs in capillary electrophoresis In this paper, the use of clarithromycin lactobionate, a kind of antibiotic chiral selector, in combination with four neutral cyclodextrin derivatives (glucose--cyclodextrin, hydroxyethyl--cyclodextrin, methyl--cyclodextrin and hydroxypropyl--cyclodextrin) was reported for the first time. As a result, these dual systems gave much better resolution of nefopam (the Rs increased to 3.58, 2.72, 1.49 and 1.42, respectively) compared to the single systems. The effects of buffer pH and selector concentration on the separation of nefopam were also investigated. Additionally, some other basic drugs including metoprolol, atenolol, propranolol, bisoprolol, esmolol and ritodrine were tested for the investigation and evaluation of the enantiorecognition capability of the four dual systems. As expected, the synergistic effect was observed in four systems. Different results of these dual systems were also summarized. Keywords: Capillary electrophoresis / Clarithromycin lactobionate / Cyclodextrin / Dual chiral system / Enantioseparation DOI 10.1002/jssc.201400677
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Chirality is a major concern in the modern pharmaceutical industry. It is well known that each enantiomer of a chiral drug may result in completely different pharmacokinetic, pharmacodynamic or even toxic effects in the human body, and its importance makes the study of this topic even more urgent. Thus, enantioseparation methods have been widely established as an important tool for analysis and/or theoretical studies of chiral compounds [1–4]. HPLC is a successful separation technique for the resolution of enantiomers [5,6]. Other methods used for enantiomer separation include TLC [7] and GC [8]. In the past years, the use of CE has grown rapidly,
Correspondence: Professor Yingxiang Du, Department of Analytical Chemistry, China Pharmaceutical University, No.24 Tongjiaxiang, Nanjing, Jiangsu 210009, China E-mail: [email protected] Fax: +86 25 83221790
Abbreviations: ATE, atenolol; BIS, bisoprolol fumarate; CD, cyclodextrin; CL, clarithromycin lactobionate; CS, chiral selector; ESM, esmolol hydrochloride; Glu--CD, glucose-cyclodextrin; HE--CD, hydroxyethyl--cyclodextrin; HP-CD, hydroxypropyl--cyclodextrin; Me--CD, methyl-cyclodextrin; MET, metoprolol tartrate; NEF, nefopam hydrochloride; PRO, propranolol hydrochloride; RIT, ritodrine hydrochloride; Rs, resolution
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and many new methods have been introduced into this field of chiral separation [9–13]. In fact, CE combines features of LC with high speed and simplicity of electrokinetic techniques and is nowadays considered an important technique, complimentary to HPLC and capillary GC. CE is a very versatile technique for enantioseparation because chiral selectors (CSs) can be easily altered, if necessary, to achieve the required separation. A number of chiral compounds have been enantioseparated using various kinds of CSs in CE. The most common approach in chiral CE is adding CS to the BGE to achieve enantioseparation. Cyclodextrins (CDs), antibiotics, proteins, crown ethers and polysaccharides, etc. have been used as CE CSs [14–20]. Among them, antibiotics are acquiring more and more attention for its high degree of selectivity. In some cases CE enantioseparation cannot be achieved by the addition of one single CS to running buffer. The combinations of two chiral selectors for enantioseparation have already aroused increasing attention recently because it can often lead to better enantioseparation towards chiral drugs [21–28]. The most used combinations for enantioseparation are dual CD systems [26–28]. Besides, CD/crown ethers system [22], polysaccharides/CD systems and dual polysaccharide systems [29] were also reported in a few papers. To the best of our knowledge, the combination of antibiotics with other chiral selectors to form dual system for enantioseparation has been reported in only one paper [30].
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Electrodriven Separations
J. Sep. Sci. 2015, 38, 2900–2906
Recently, the macrolides, an important member of antibiotics, have been reported to be a new type of CSs [31–33]. In our recent report, clarithromycin lactobionate (CL), belonging to the group of macrolide antibiotics, was first investigated for its potential as a novel CS [32]. The structure of CL (see Supporting Information Fig. S1) is a macrocyclic lactone ring with 14 atoms and numerous functional groups. CL (with molecular mass of 1106.3) has some advantages, e.g. high solubility in acidic and neutral solution, high stability and low viscosity in the solvent and very weak UV absorption. CDs are the most extensive and successful selectors in the enantioseparation of chiral compounds until now. In this paper, we first reported the enantioselectivity of four CL-based dual selector systems towards some chiral drugs, including CL/hydroxyethyl--cyclodextrin (HE--CD), CL/methyl-cyclodextrin (Me--CD), CL/glucose--cyclodextrin (Glu-CD) and CL/hydroxypropyl--cyclodextrin (HP--CD) systems. In the course of the work, a synergistic effect was observed in all of the four dual chiral selector systems. We present details of these CL-based dual systems for CE enantioseparation in this paper.
2 Materials and methods 2.1 Chemicals and reagents CL (purity > 99%) and esmolol hydrochloride (ESM, pKa 9.7) were purchased from Shandong Keyuan Pharmaceutical (Jinan, China). Propranolol hydrochloride (PRO, pKa 9.4) was purchased from Sigma (St. Louis, MO, USA). Metoprolol tartrate (MET, pKa 9.7), atenolol (ATE, pKa 9.6), ritodrine hydrochloride (RIT, pKa 9.8) and nefopam hydrochloride (NEF, pKa 9.0) were supplied by Jiangsu Institute for Food and Drug Control. Bisoprolol fumarate (BIS, pKa 9.7) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products. All these drug samples were racemic mixtures. HE--CD (Mw approx. 1443), Me--CD (Mw approx. 1310), Glu--CD (Mw approx. 1320) and HP-CD (Mw approx. 1540) were purchased from Zibo Qianhui Fine Chemical (Shandong, China). Methanol (HPLC grade) was purchased from Jiangsu Hanbon Sci. & Tech. (Nanjing, China). Sodium hydroxide (NaOH), hydrochloric acid (HCl) and sodium tetraborate decahydrate, all of analytical grade, were purchased from Nanjing Chemical Reagent (Nanjing, China). Double distilled water was used throughout all the experiments.
2.2 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 sys C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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tem control, data collection and analysis. It was equipped with a 33 cm (24.5 cm effective length) × 50 m id uncoated fused-silica capillary (Hebei Yongnian County Reafine Chromatography, Hebei, China). A new capillary was flushed for 30 min with 0.2 M NaOH and 30 min with water, respectively. At the beginning of each day, the capillary was flushed with 0.2 M NaOH (10 min) and water (10 min). At the end of each day it was flushed successively with 0.2 M HCl (10 min) and water (10 min). Between consecutive injections the capillary was rinsed with 0.2 M HCl, water, 0.2 M NaOH, water and running buffer for 3 min each. Sample injections were performed by pressure (50 mbar, 4 s). Enantioseparations were performed at a constant voltage of 20 kV, and the temperature of capillary was controlled at 25⬚C using an air-cooling system.
2.3 Procedures Buffer solution was a 25 mM borax buffer adjusted to a specified pH value with 1 M HCl, mixed with methanol (1:1). The running buffer was freshly prepared by dissolving appropriate amounts of CL and -CD derivatives (HE--CD, Me--CD, Glu--CD, HP--CD) in the buffer solution, and then adjusting pH exactly to a desired value by adding a small volume of HCl or NaOH using a microsyringe. Acetone was used as a neutral marker to determine the EOF. The racemic samples (0.5 mg/mL) were dissolved in methanol (ATE) and distilled water (others). Running buffers and samples were filtered with a 0.45 m pore membrane filter and degassed by sonication before use.
3 Results and discussion 3.1 Enantioseparation of NEF in four dual systems In the previous study [32], CL exhibited a very poor selectivity towards NEF compared to other tested racemic drugs when it was used alone. Thus, in this paper, NEF was first selected as the model drug to evaluate the enantioseparation performance of CL-based dual systems. As expected, the combined use of CL and some neutral CD derivatives (HE--CD, Me-CD, HP--CD and Glu--CD) in the running buffer to form dual systems (CL/HE--CD, CL/Me--CD, CL/HP--CD and CL/Glu--CD systems) could achieve significantly improved separations for NEF enantiomers (see Fig. 1). It is worth noting that when these CDs were used alone, the NEF enantiomers were only poorly separated under the same experiment conditions. This clearly showed that CL and the neutral CD derivatives may cooperate during the enantiorecognition process. Although these four neutral CD derivatives all exhibited synergistic effect with CL, the optimal separation was achieved at different experiment conditions for these dual systems. This may be attributed to the different characteristics of the substituent group on the CDs. It seems that the modification of the native CDs leads to significant changes in www.jss-journal.com
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J. Sep. Sci. 2015, 38, 2900–2906
Figure 1. The electropherogram of NEF in four dual chiral systems and single systems. Conditions: (A) CL concentration, 50 mM, Glu--CD concentration, 40 mM, buffer pH, 5.5 (B) CL concentration, 50 mM, HE--CD concentration, 20 mM, buffer pH, 4.5 (C) CL concentration, 40 mM, Me--CD concentration, 20 mM, buffer pH, 6.5 (D) CL concentration, 50 mM, HP--CD concentration, 15 mM, buffer pH, 7.0; fused-silica capillary, 33 cm (24.5 cm effective length) ×50 m id; BGE, 12.5 mM borax buffer with methanol (50% v/v); applied voltage, 20 kV; capillary temperature, 25⬚C.
their physicochemical properties and their chiral recognition ability.
tem and pH 7.0 in CL/HP--CD system, respectively. Below their optimum pH value, the Rs of NEF all tended to decrease.
3.1.1 Effect of buffer pH on enantioseparation of NEF
3.1.2 Effect of CS concentration on enantioseparation of NEF
As same as the single chiral system, pH of the running buffer remains to be a very important experimental parameter to be controlled in dual chiral separation systems. The charge of both selector and analytes can be modulated with buffer pH. Moreover, EOF, as well as the interactions between the CSs and analytes can also be affected. In this study, the effects of buffer pH on Rs of NEF were investigated by changing the pH value from 7.5 to 4.0, using a 12.5 mM borax buffer (containing 50% v/v methanol). As shown in Fig. 2, the electro-osmotic mobility (eof ) went down when pH decreased in all four CL/CDs dual systems. No separation was obtained for NEF at pH 7.5 in four dual systems. However, the decreased pH resulted in a general increased Rs for NEF, and the optimum separation in terms of Rs was obtained at pH 4.5 in CL/HE--CD system, pH 5.5 in CL/Glu--CD system, pH 6.5 in CL/Me--CD sys C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
To obtain the optimum concentration of CL and the CDs, we varied the concentration of one selector while keeping the other concentration fixed for each dual chiral system. In the CL/Glu--CD system, the effect of Glu--CD concentration on separation was carried out by varying Glu--CD concentration in the range of 10–50 mM while holding the CL concentration at 50 mM (the results are listed in Table 1). It could be found that the Rs and ␣ of NEF increased significantly as the Glu--CD concentration increased from 10 to 40 mM and then decreased. Therefore, we selected 40 mM as the optimum concentration of Glu--CD. The effect of CL concentration (30–60 mM) on separation was then examined with the concentration of Glu--CD kept constant at 40 mM. It can be seen from Fig. 3, the Rs and ␣ of NEF increased with the rise of CL concentration (30–50 mM), then decreased from www.jss-journal.com
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Figure 2. Effect of the buffer pH on EOF (A) and the enantioseparation of NEF (B) in four dual chiral systems. Conditions: CL/Me--CD system: CL concentration, 40 mM, Me--CD concentration, 15 mM; CL/Glu--CD system: CL concentration, 50 mM, Glu--CD concentration, 10 mM; CL/HE--CD system: CL concentration, 50 mM, HE--CD concentration, 10 mM; CL/HP--CD system: CL concentration, 40 mM, HP--CD concentration, 10 mM; all other conditions, as in Fig. 1.
Figure 3. Electropherograms of the enantioseparation of NEF at different CL concentration in CL/Glu--CD system. Conditions: CL concentration, 30 mM (A), 40 mM (B), 50 mM (C), 60 mM (D), Glu--CD concentration, 40 mM; buffer pH, 5.5; all other conditions, as in Fig. 1.
50 to 60 mM. Consequently, a CL concentration of 50 mM was selected. At low concentration, the Rs and ␣ increased gradually naturally owing to the intensive CS-enantiomer interactions. When the concentration was higher than its optimum value, the Rs and ␣ of NEF generally tended to decrease due to saturated complexation and peak broadening. For other three dual systems, CL/HE--CD system, CL/HP--CD system and CL/Me--CD system, the similar approach was applied for the investigation of the effect of different CS concentrations on the enantioseparation of NEF. The final optimized separation conditions, as well as the Rs and ␣ of NEF in different dual systems are summarized in Table 2. We have also optimized the enantioseparation of NEF with only CD as chiral selectors. The results showed that even C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
at the optimized separation condition of single CD systems, much worse separations were observed compared to dual selector systems (complete separation of NEF still cannot be achieved, Rs < 1.35, data not shown).
3.2 CL/Me--CD dual system for other drugs In addition to NEF, other six basic drugs including of MET, ATE, PRO, BIS, ESM and RIT were also tested for the evaluation of the enantioseparation ability of CL/Me--CD system. As illustrated in Table 3, in this system, the separation of all of the tested drugs were improved compared to the single system under the tested conditions. Supporting Information Fig. S2 shows the representative comparison www.jss-journal.com
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Table 1. Effect of the concentration of Glu--CD on the enantioseparation of NEF in CL/ Glu--CD system
NEF Glu--CD (mM)
t1
t2
Rs
␣
10 20 30 40 50
5.957 6.808 7.295 8.101 9.460
6.118 7.029 7.552 8.413 9.789
1.34 2.16 2.70 3.58 2.97
1.027 1.032 1.035 1.039 1.035
Conditions: CL concentration, 50 mM; Glu--CD concentration, 10, 20, 30, 40, 50 mM; buffer pH, 5.5; all other conditions, as in Fig. 1. t1 and t2 , the migration time of the enantiomers.
Table 2. The final optimized separation conditions, Rs and ␣ of NEF in four dual systems
Dual systems
Concentration (mM)CL/CDs
Buffer pH
Rs/␣
CL/Glu--CD CL/HE--CD CL/Me--CD CL/HP--CD
50/40 60/20 40/20 50/15
5.5 4.5 6.5 7.0
3.58/1.039 2.72/1.035 1.49/1.016 1.42/1.014
Conditions: all other conditions, as in Fig. 1.
electropherograms of MET and ATE in CL/Me--CD system and single CL system. All the results illustrated above demonstrated the existence of synergistic effect between CL and Me--CD. These two CSs can supplement with each other during the enantiorecognition process, and thus provide better separation capability towards tested drugs. The mixtures of CL and Me--CD, forming dual chiral systems, may enhance the interactions between the selectors and the chiral compounds. We further investigated the effect of buffer pH, CSs combination (including the CS concentration ratio and the total CS concentration) on the enantioseparation in the CL/Me-CD system.
3.2.1 Effect of buffer pH in CL/Me--CD system Generally, higher buffer pH increased the EOF and accordingly gave shorter migration times. However, it was observed that the migration times prolonged as the buffer pH increased in our experiment (Table 4). This may be attributed to a decrease of the positive charge of analytes (pKa from 9.4 to 9.8) which resulted in a decrease in electrophoretic mobilities and longer migration times. Simultaneously, with the buffer pH increasing from 6.8 to 7.5, both Rs and ␣ of all tested enantiomers increased due to the increasing complexation between analytes and CSs. When the buffer pH was over 7.5, CL exhibited low solubility and the precipitation of CL could be observed during the runs. Thus, a buffer pH of 7.5 was chosen as optimal for the CL/Me--CD system. 3.2.2 Effect of the CS concentrations in CL/Me--CD system The influence of the CL to Me--CD ratio was first studied by varying the CL-to-Me--CD ratio while keeping the total CS concentration constant. Experiments showed that the optimum chiral separations occurred at a CL-to-Me--CD ratio of 8:3 for all of the tested drugs (see Fig. 4A). We further optimized the total concentrations of the selectors. The CL-to-Me--CD ratio was set at 8:3, a series of total concentrations (45, 55, 65 and 75 mM) were studied. As shown in Fig. 4B, low concentration (45 mM) only gave partial separations. The Rs of all tested drugs increased as the total concentration of CL and Me--CD rose, and reached maximum value at 55 mM. At higher total concentrations, decreased Rs of studied drug enantiomers were observed. Therefore a total concentration of 55 mM was determined for the CL/Me--CD system. 3.3 CL/Glu--CD, CL/HE--CD and CL/HP--CD systems for other drugs The synergistic effect of three other CL-based dual chiral systems (CL/Glu--CD, CL/HE--CD and CL/HP--CD system) towards the above mentioned other drugs were also studied.
Table 3. The comparison of the results obtained in the single systems and CL/ Me--CD system
CL
CL+ Me--CD
Me--CD
Drugs
t2 /t1 (min)
Rs/␣
t2 /t1 (min)
Rs/␣
t2 /t1 (min)
Rs/␣
MET ATE PRO BIS ESM RIT
5.462/5.304 5.645/5.476 5.292/5.139 5.533/5.379 5.553/5.392 6.600/6.489
1.16/1.030 1.15/1.031 1.35/1.030 1.21/1.029 1.28/1.030 0.99/1.017
8.409/8.102 7.739/7.463 6.795/6.581 7.345/7.120 7.444/7.234 8.603/8.460
2.10/1.038 1.77/1.037 1.70/1.033 1.88/1.032 1.75/1.029 1.33/1.017
2.666 2.545 2.602 3.045 2.745 3.064
NS NS NS NS NS NS
Conditions: CL concentration, 40 mM, Me--CD concentration, 15 mM; buffer pH, 7.5; all other conditions, as in Fig. 1. NS, not separated.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 4. Effects of the buffer pH on the enantioseparation of MET, ATE, PRO, BIS, ESM and RIT in CL/ Me--CD system
6.8
7.0
7.3
7.5
Drugs
t2 /t1 (min)
Rs/␣
t2 /t1 (min)
Rs/␣
t2 /t1 (min)
Rs/␣
t2 /t1 (min)
Rs/␣
MET ATE PRO BIS ESM RIT
6.358 6.215 6.012 6.763/6.727 6.643 7.208/7.174
NS NS NS 0.41/1.005 NS
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 College of Chemistry and Molecular Engineering, Peking University, Beijing, P. R. China Received June 23, 2014 Revised May 16, 2015 Accepted May 16, 2015
J. Sep. Sci. 2015, 38, 2900–2906
Research Article
Study on clarithromycin lactobionate based dual selector systems for the enantioseparation of basic drugs in capillary electrophoresis In this paper, the use of clarithromycin lactobionate, a kind of antibiotic chiral selector, in combination with four neutral cyclodextrin derivatives (glucose--cyclodextrin, hydroxyethyl--cyclodextrin, methyl--cyclodextrin and hydroxypropyl--cyclodextrin) was reported for the first time. As a result, these dual systems gave much better resolution of nefopam (the Rs increased to 3.58, 2.72, 1.49 and 1.42, respectively) compared to the single systems. The effects of buffer pH and selector concentration on the separation of nefopam were also investigated. Additionally, some other basic drugs including metoprolol, atenolol, propranolol, bisoprolol, esmolol and ritodrine were tested for the investigation and evaluation of the enantiorecognition capability of the four dual systems. As expected, the synergistic effect was observed in four systems. Different results of these dual systems were also summarized. Keywords: Capillary electrophoresis / Clarithromycin lactobionate / Cyclodextrin / Dual chiral system / Enantioseparation DOI 10.1002/jssc.201400677
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Chirality is a major concern in the modern pharmaceutical industry. It is well known that each enantiomer of a chiral drug may result in completely different pharmacokinetic, pharmacodynamic or even toxic effects in the human body, and its importance makes the study of this topic even more urgent. Thus, enantioseparation methods have been widely established as an important tool for analysis and/or theoretical studies of chiral compounds [1–4]. HPLC is a successful separation technique for the resolution of enantiomers [5,6]. Other methods used for enantiomer separation include TLC [7] and GC [8]. In the past years, the use of CE has grown rapidly,
Correspondence: Professor Yingxiang Du, Department of Analytical Chemistry, China Pharmaceutical University, No.24 Tongjiaxiang, Nanjing, Jiangsu 210009, China E-mail: [email protected] Fax: +86 25 83221790
Abbreviations: ATE, atenolol; BIS, bisoprolol fumarate; CD, cyclodextrin; CL, clarithromycin lactobionate; CS, chiral selector; ESM, esmolol hydrochloride; Glu--CD, glucose-cyclodextrin; HE--CD, hydroxyethyl--cyclodextrin; HP-CD, hydroxypropyl--cyclodextrin; Me--CD, methyl-cyclodextrin; MET, metoprolol tartrate; NEF, nefopam hydrochloride; PRO, propranolol hydrochloride; RIT, ritodrine hydrochloride; Rs, resolution
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and many new methods have been introduced into this field of chiral separation [9–13]. In fact, CE combines features of LC with high speed and simplicity of electrokinetic techniques and is nowadays considered an important technique, complimentary to HPLC and capillary GC. CE is a very versatile technique for enantioseparation because chiral selectors (CSs) can be easily altered, if necessary, to achieve the required separation. A number of chiral compounds have been enantioseparated using various kinds of CSs in CE. The most common approach in chiral CE is adding CS to the BGE to achieve enantioseparation. Cyclodextrins (CDs), antibiotics, proteins, crown ethers and polysaccharides, etc. have been used as CE CSs [14–20]. Among them, antibiotics are acquiring more and more attention for its high degree of selectivity. In some cases CE enantioseparation cannot be achieved by the addition of one single CS to running buffer. The combinations of two chiral selectors for enantioseparation have already aroused increasing attention recently because it can often lead to better enantioseparation towards chiral drugs [21–28]. The most used combinations for enantioseparation are dual CD systems [26–28]. Besides, CD/crown ethers system [22], polysaccharides/CD systems and dual polysaccharide systems [29] were also reported in a few papers. To the best of our knowledge, the combination of antibiotics with other chiral selectors to form dual system for enantioseparation has been reported in only one paper [30].
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Electrodriven Separations
J. Sep. Sci. 2015, 38, 2900–2906
Recently, the macrolides, an important member of antibiotics, have been reported to be a new type of CSs [31–33]. In our recent report, clarithromycin lactobionate (CL), belonging to the group of macrolide antibiotics, was first investigated for its potential as a novel CS [32]. The structure of CL (see Supporting Information Fig. S1) is a macrocyclic lactone ring with 14 atoms and numerous functional groups. CL (with molecular mass of 1106.3) has some advantages, e.g. high solubility in acidic and neutral solution, high stability and low viscosity in the solvent and very weak UV absorption. CDs are the most extensive and successful selectors in the enantioseparation of chiral compounds until now. In this paper, we first reported the enantioselectivity of four CL-based dual selector systems towards some chiral drugs, including CL/hydroxyethyl--cyclodextrin (HE--CD), CL/methyl-cyclodextrin (Me--CD), CL/glucose--cyclodextrin (Glu-CD) and CL/hydroxypropyl--cyclodextrin (HP--CD) systems. In the course of the work, a synergistic effect was observed in all of the four dual chiral selector systems. We present details of these CL-based dual systems for CE enantioseparation in this paper.
2 Materials and methods 2.1 Chemicals and reagents CL (purity > 99%) and esmolol hydrochloride (ESM, pKa 9.7) were purchased from Shandong Keyuan Pharmaceutical (Jinan, China). Propranolol hydrochloride (PRO, pKa 9.4) was purchased from Sigma (St. Louis, MO, USA). Metoprolol tartrate (MET, pKa 9.7), atenolol (ATE, pKa 9.6), ritodrine hydrochloride (RIT, pKa 9.8) and nefopam hydrochloride (NEF, pKa 9.0) were supplied by Jiangsu Institute for Food and Drug Control. Bisoprolol fumarate (BIS, pKa 9.7) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products. All these drug samples were racemic mixtures. HE--CD (Mw approx. 1443), Me--CD (Mw approx. 1310), Glu--CD (Mw approx. 1320) and HP-CD (Mw approx. 1540) were purchased from Zibo Qianhui Fine Chemical (Shandong, China). Methanol (HPLC grade) was purchased from Jiangsu Hanbon Sci. & Tech. (Nanjing, China). Sodium hydroxide (NaOH), hydrochloric acid (HCl) and sodium tetraborate decahydrate, all of analytical grade, were purchased from Nanjing Chemical Reagent (Nanjing, China). Double distilled water was used throughout all the experiments.
2.2 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 sys C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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tem control, data collection and analysis. It was equipped with a 33 cm (24.5 cm effective length) × 50 m id uncoated fused-silica capillary (Hebei Yongnian County Reafine Chromatography, Hebei, China). A new capillary was flushed for 30 min with 0.2 M NaOH and 30 min with water, respectively. At the beginning of each day, the capillary was flushed with 0.2 M NaOH (10 min) and water (10 min). At the end of each day it was flushed successively with 0.2 M HCl (10 min) and water (10 min). Between consecutive injections the capillary was rinsed with 0.2 M HCl, water, 0.2 M NaOH, water and running buffer for 3 min each. Sample injections were performed by pressure (50 mbar, 4 s). Enantioseparations were performed at a constant voltage of 20 kV, and the temperature of capillary was controlled at 25⬚C using an air-cooling system.
2.3 Procedures Buffer solution was a 25 mM borax buffer adjusted to a specified pH value with 1 M HCl, mixed with methanol (1:1). The running buffer was freshly prepared by dissolving appropriate amounts of CL and -CD derivatives (HE--CD, Me--CD, Glu--CD, HP--CD) in the buffer solution, and then adjusting pH exactly to a desired value by adding a small volume of HCl or NaOH using a microsyringe. Acetone was used as a neutral marker to determine the EOF. The racemic samples (0.5 mg/mL) were dissolved in methanol (ATE) and distilled water (others). Running buffers and samples were filtered with a 0.45 m pore membrane filter and degassed by sonication before use.
3 Results and discussion 3.1 Enantioseparation of NEF in four dual systems In the previous study [32], CL exhibited a very poor selectivity towards NEF compared to other tested racemic drugs when it was used alone. Thus, in this paper, NEF was first selected as the model drug to evaluate the enantioseparation performance of CL-based dual systems. As expected, the combined use of CL and some neutral CD derivatives (HE--CD, Me-CD, HP--CD and Glu--CD) in the running buffer to form dual systems (CL/HE--CD, CL/Me--CD, CL/HP--CD and CL/Glu--CD systems) could achieve significantly improved separations for NEF enantiomers (see Fig. 1). It is worth noting that when these CDs were used alone, the NEF enantiomers were only poorly separated under the same experiment conditions. This clearly showed that CL and the neutral CD derivatives may cooperate during the enantiorecognition process. Although these four neutral CD derivatives all exhibited synergistic effect with CL, the optimal separation was achieved at different experiment conditions for these dual systems. This may be attributed to the different characteristics of the substituent group on the CDs. It seems that the modification of the native CDs leads to significant changes in www.jss-journal.com
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Figure 1. The electropherogram of NEF in four dual chiral systems and single systems. Conditions: (A) CL concentration, 50 mM, Glu--CD concentration, 40 mM, buffer pH, 5.5 (B) CL concentration, 50 mM, HE--CD concentration, 20 mM, buffer pH, 4.5 (C) CL concentration, 40 mM, Me--CD concentration, 20 mM, buffer pH, 6.5 (D) CL concentration, 50 mM, HP--CD concentration, 15 mM, buffer pH, 7.0; fused-silica capillary, 33 cm (24.5 cm effective length) ×50 m id; BGE, 12.5 mM borax buffer with methanol (50% v/v); applied voltage, 20 kV; capillary temperature, 25⬚C.
their physicochemical properties and their chiral recognition ability.
tem and pH 7.0 in CL/HP--CD system, respectively. Below their optimum pH value, the Rs of NEF all tended to decrease.
3.1.1 Effect of buffer pH on enantioseparation of NEF
3.1.2 Effect of CS concentration on enantioseparation of NEF
As same as the single chiral system, pH of the running buffer remains to be a very important experimental parameter to be controlled in dual chiral separation systems. The charge of both selector and analytes can be modulated with buffer pH. Moreover, EOF, as well as the interactions between the CSs and analytes can also be affected. In this study, the effects of buffer pH on Rs of NEF were investigated by changing the pH value from 7.5 to 4.0, using a 12.5 mM borax buffer (containing 50% v/v methanol). As shown in Fig. 2, the electro-osmotic mobility (eof ) went down when pH decreased in all four CL/CDs dual systems. No separation was obtained for NEF at pH 7.5 in four dual systems. However, the decreased pH resulted in a general increased Rs for NEF, and the optimum separation in terms of Rs was obtained at pH 4.5 in CL/HE--CD system, pH 5.5 in CL/Glu--CD system, pH 6.5 in CL/Me--CD sys C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
To obtain the optimum concentration of CL and the CDs, we varied the concentration of one selector while keeping the other concentration fixed for each dual chiral system. In the CL/Glu--CD system, the effect of Glu--CD concentration on separation was carried out by varying Glu--CD concentration in the range of 10–50 mM while holding the CL concentration at 50 mM (the results are listed in Table 1). It could be found that the Rs and ␣ of NEF increased significantly as the Glu--CD concentration increased from 10 to 40 mM and then decreased. Therefore, we selected 40 mM as the optimum concentration of Glu--CD. The effect of CL concentration (30–60 mM) on separation was then examined with the concentration of Glu--CD kept constant at 40 mM. It can be seen from Fig. 3, the Rs and ␣ of NEF increased with the rise of CL concentration (30–50 mM), then decreased from www.jss-journal.com
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Figure 2. Effect of the buffer pH on EOF (A) and the enantioseparation of NEF (B) in four dual chiral systems. Conditions: CL/Me--CD system: CL concentration, 40 mM, Me--CD concentration, 15 mM; CL/Glu--CD system: CL concentration, 50 mM, Glu--CD concentration, 10 mM; CL/HE--CD system: CL concentration, 50 mM, HE--CD concentration, 10 mM; CL/HP--CD system: CL concentration, 40 mM, HP--CD concentration, 10 mM; all other conditions, as in Fig. 1.
Figure 3. Electropherograms of the enantioseparation of NEF at different CL concentration in CL/Glu--CD system. Conditions: CL concentration, 30 mM (A), 40 mM (B), 50 mM (C), 60 mM (D), Glu--CD concentration, 40 mM; buffer pH, 5.5; all other conditions, as in Fig. 1.
50 to 60 mM. Consequently, a CL concentration of 50 mM was selected. At low concentration, the Rs and ␣ increased gradually naturally owing to the intensive CS-enantiomer interactions. When the concentration was higher than its optimum value, the Rs and ␣ of NEF generally tended to decrease due to saturated complexation and peak broadening. For other three dual systems, CL/HE--CD system, CL/HP--CD system and CL/Me--CD system, the similar approach was applied for the investigation of the effect of different CS concentrations on the enantioseparation of NEF. The final optimized separation conditions, as well as the Rs and ␣ of NEF in different dual systems are summarized in Table 2. We have also optimized the enantioseparation of NEF with only CD as chiral selectors. The results showed that even C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
at the optimized separation condition of single CD systems, much worse separations were observed compared to dual selector systems (complete separation of NEF still cannot be achieved, Rs < 1.35, data not shown).
3.2 CL/Me--CD dual system for other drugs In addition to NEF, other six basic drugs including of MET, ATE, PRO, BIS, ESM and RIT were also tested for the evaluation of the enantioseparation ability of CL/Me--CD system. As illustrated in Table 3, in this system, the separation of all of the tested drugs were improved compared to the single system under the tested conditions. Supporting Information Fig. S2 shows the representative comparison www.jss-journal.com
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Table 1. Effect of the concentration of Glu--CD on the enantioseparation of NEF in CL/ Glu--CD system
NEF Glu--CD (mM)
t1
t2
Rs
␣
10 20 30 40 50
5.957 6.808 7.295 8.101 9.460
6.118 7.029 7.552 8.413 9.789
1.34 2.16 2.70 3.58 2.97
1.027 1.032 1.035 1.039 1.035
Conditions: CL concentration, 50 mM; Glu--CD concentration, 10, 20, 30, 40, 50 mM; buffer pH, 5.5; all other conditions, as in Fig. 1. t1 and t2 , the migration time of the enantiomers.
Table 2. The final optimized separation conditions, Rs and ␣ of NEF in four dual systems
Dual systems
Concentration (mM)CL/CDs
Buffer pH
Rs/␣
CL/Glu--CD CL/HE--CD CL/Me--CD CL/HP--CD
50/40 60/20 40/20 50/15
5.5 4.5 6.5 7.0
3.58/1.039 2.72/1.035 1.49/1.016 1.42/1.014
Conditions: all other conditions, as in Fig. 1.
electropherograms of MET and ATE in CL/Me--CD system and single CL system. All the results illustrated above demonstrated the existence of synergistic effect between CL and Me--CD. These two CSs can supplement with each other during the enantiorecognition process, and thus provide better separation capability towards tested drugs. The mixtures of CL and Me--CD, forming dual chiral systems, may enhance the interactions between the selectors and the chiral compounds. We further investigated the effect of buffer pH, CSs combination (including the CS concentration ratio and the total CS concentration) on the enantioseparation in the CL/Me-CD system.
3.2.1 Effect of buffer pH in CL/Me--CD system Generally, higher buffer pH increased the EOF and accordingly gave shorter migration times. However, it was observed that the migration times prolonged as the buffer pH increased in our experiment (Table 4). This may be attributed to a decrease of the positive charge of analytes (pKa from 9.4 to 9.8) which resulted in a decrease in electrophoretic mobilities and longer migration times. Simultaneously, with the buffer pH increasing from 6.8 to 7.5, both Rs and ␣ of all tested enantiomers increased due to the increasing complexation between analytes and CSs. When the buffer pH was over 7.5, CL exhibited low solubility and the precipitation of CL could be observed during the runs. Thus, a buffer pH of 7.5 was chosen as optimal for the CL/Me--CD system. 3.2.2 Effect of the CS concentrations in CL/Me--CD system The influence of the CL to Me--CD ratio was first studied by varying the CL-to-Me--CD ratio while keeping the total CS concentration constant. Experiments showed that the optimum chiral separations occurred at a CL-to-Me--CD ratio of 8:3 for all of the tested drugs (see Fig. 4A). We further optimized the total concentrations of the selectors. The CL-to-Me--CD ratio was set at 8:3, a series of total concentrations (45, 55, 65 and 75 mM) were studied. As shown in Fig. 4B, low concentration (45 mM) only gave partial separations. The Rs of all tested drugs increased as the total concentration of CL and Me--CD rose, and reached maximum value at 55 mM. At higher total concentrations, decreased Rs of studied drug enantiomers were observed. Therefore a total concentration of 55 mM was determined for the CL/Me--CD system. 3.3 CL/Glu--CD, CL/HE--CD and CL/HP--CD systems for other drugs The synergistic effect of three other CL-based dual chiral systems (CL/Glu--CD, CL/HE--CD and CL/HP--CD system) towards the above mentioned other drugs were also studied.
Table 3. The comparison of the results obtained in the single systems and CL/ Me--CD system
CL
CL+ Me--CD
Me--CD
Drugs
t2 /t1 (min)
Rs/␣
t2 /t1 (min)
Rs/␣
t2 /t1 (min)
Rs/␣
MET ATE PRO BIS ESM RIT
5.462/5.304 5.645/5.476 5.292/5.139 5.533/5.379 5.553/5.392 6.600/6.489
1.16/1.030 1.15/1.031 1.35/1.030 1.21/1.029 1.28/1.030 0.99/1.017
8.409/8.102 7.739/7.463 6.795/6.581 7.345/7.120 7.444/7.234 8.603/8.460
2.10/1.038 1.77/1.037 1.70/1.033 1.88/1.032 1.75/1.029 1.33/1.017
2.666 2.545 2.602 3.045 2.745 3.064
NS NS NS NS NS NS
Conditions: CL concentration, 40 mM, Me--CD concentration, 15 mM; buffer pH, 7.5; all other conditions, as in Fig. 1. NS, not separated.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 4. Effects of the buffer pH on the enantioseparation of MET, ATE, PRO, BIS, ESM and RIT in CL/ Me--CD system
6.8
7.0
7.3
7.5
Drugs
t2 /t1 (min)
Rs/␣
t2 /t1 (min)
Rs/␣
t2 /t1 (min)
Rs/␣
t2 /t1 (min)
Rs/␣
MET ATE PRO BIS ESM RIT
6.358 6.215 6.012 6.763/6.727 6.643 7.208/7.174
NS NS NS 0.41/1.005 NS
