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Combined use of [TBA][L-ASP] and hydroxypropyl-β-cyclodextrin as selectors for separation of Cinchona alkaloids by capillary electrophoresis
Analytical Biochemistry 462 (2014) 13–18
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Combined use of [TBA][L-ASP] and hydroxypropyl-b-cyclodextrin as selectors for separation of Cinchona alkaloids by capillary electrophoresis Yu Zhang a, Haixia Yu b, Yujiao Wu a, Wenyan Zhao a, Min Yang a, Huanwang Jing c,⇑, Anjia Chen a,⇑ a b c
College of Pharmacy, Shanxi Medical University, Taiyuan 030001, China Scientific Research Center, Shanxi Medical University, Taiyuan 030001, China College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
a r t i c l e
i n f o
Article history: Received 25 April 2014 Received in revised form 10 June 2014 Accepted 11 June 2014 Available online 20 June 2014 Keywords: Capillary electrophoresis Chiral separation Chiral ionic liquid HP-b-CD First-order derivative electropherogram
a b s t r a c t In this paper, a new capillary electrophoresis (CE) separation and detection method was developed for the chiral separation of the four major Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) using hydroxypropyl-b-cyclodextrin (HP-b-CD) and chiral ionic liquid ([TBA][L-ASP]) as selectors. Separation parameters such as buffer concentrations, pH, HP-b-CD and chiral ionic liquid concentrations, capillary temperature, and separation voltage were investigated. After optimization of separation conditions, baseline separation of the three analytes (cinchonidine, quinine, cinchonine) was achieved in fewer than 7 min in ammonium acetate background electrolyte (pH 5.0) with the addition of HPb-CD in a concentration of 40 mM and [TBA][L-ASP] of 14 mM, while the baseline separation of cinchonine and quinidine was not obtained. Therefore, the first-order derivative electropherogram was applied for resolving overlapping peaks. Regression equations revealed a good linear relationship between peak areas in first-order derivative electropherograms and concentrations of the two diastereomer pairs. The results not only indicated that the first-order derivative electropherogram was effective in determination of a low content component and of those not fully separated from adjacent ones, but also showed that the ionic liquid appeared to be a very promising chiral selector in CE. Ó 2014 Elsevier Inc. All rights reserved.
Cinchona alkaloids can be found in the bark of Cinchona and Remijia species. The bark contains a variety of alkaloids, including quinine and quinidine, and cinchonine and cinchonidine (Fig. 1A). A main structural feature of these substances is the presence of chiral centers at C8 and C9, each of which may be found in the R or S configuration. Over the past 30 years, Cinchona alkaloids have become increasingly popular in organic chemistry and are used as chiral catalysts, ligands, nuclear magnetic resonance (NMR)1 discriminating agents, and so on [1–3]. Nowadays, they were also applied in high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) stationary phases for chiral separations [4,5]. These compounds were also well known to have medicinal activity [6]. They were long cultivated as a muscle relaxant to cease
⇑ Corresponding authors. E-mail addresses: [email protected] (H. Jing), [email protected] (A. Chen). Abbreviations used: BGEs, background electrolytes; CE, capillary electrophoresis; HP-b-CD, hydroxypropyl-b-cyclodextrin; HPLC high-performance liquid chromatography; NMR, nuclear magnetic resonance. 1
http://dx.doi.org/10.1016/j.ab.2014.06.008 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
shivering due to low temperatures and as an antipyretic agent especially useful in treating malaria. Quinine is the most familiar one. Recently, some analytical methods for separation and determination of Cinchona alkaloids have been developed [7–9]. The quantitative analysis of Cinchona alkaloids has frequently been done by HPLC using C18-modified silica and ultraviolet (UV) or mass spectrometry (MS) detection systems [10–12]. In HPLC, precautions need to be taken due to the strong silanophilic interactions which can occur with these analytes and the column surface, which can lead to poor peak shape and resolution. However, CE which has the advantages of rapid analysis time, high separation efficiency, low sample, reagent consumption, high resolution, and sensitivity has been recognized as another powerful separation technique for chiral separations [12,13]. In recent decades, CE with chiral additives to the background electrolyte has been proven as the best method for separation of enantiomers. Meanwhile, chiral ionic liquids as a special chiral additive provide a new approach for the optimization of CE separations [14–17]. Chiral ionic liquids which refer to a group of organic salts are liquid at room temperature due to their unique characteristics, such as low toxicity, wide
14
Combined use of [TBA][L-ASP] and HP-b-CD as selectors for separation of Cinchona alkaloids by CE / Y. Zhang et al. / Anal. Biochem. 462 (2014) 13–18
Capillary electrophoresis instrumentation The CE experiments were carried out on a Beckman Coulter P/ ACE MDQ CE System (Beckman Coulter, Fullerton, CA, USA). Fused silica capillaries were from HeBei RuiFeng Chromatographic Instrument Company with 50.2 cm 50 lm i.d. (375 lm o.d., effective length 40 cm). New bare silica capillaries were flushed successively for 5 min with methanol, 2 min with H2O, 5 min with 0.1 M HCl, 2 min with H2O, 10 min with 0.1 M NaOH, and 5 min with H2O. Sample preparation
Fig.1. (A) chemical structure of the diastereomeric pairs of Cinchona alkaloids. (B) The chemical structure of chiral ionic liquid ([TBA][L-ASP]).
liquid range, high chemical stability, wide electrochemical window, and nonvolatility, and thus have attracted much research interest. They have been considered as an alternative to conventional molecular solvents and were applied widely. A good example was the increasing use of chiral ionic liquids in modern analytical chemistry specially for chiral separation, in which they have played a variety of roles in facilitating the chromatographic separation either as mobile phase additives or as stationary phases. In CE separation processes, chiral ionic liquids have manifested some fascinating strengths when used as background electrolytes (BGEs) additives or capillary-wall modifiers in a variety of CE applications, such as improving resolution, controlling electroosmotic flow (EOF), and suppressing wall adsorption problems due to the repulsion forces between the analyte and the capillary surface. In this paper, a chiral ionic liquid ([TBA][L-ASP]; see Fig. 1B), synthesized by our research group, combined with hydroxypropyl-b-cyclodextrin (HP-b-CD), was used as chiral selectors for separation and detection of Cinchona alkaloids by CE. Two diastereomeric pairs of Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) were used as model analytes. Compared with conventional separation and detection methods, this method shows many advantages including lower analyte wall adsorption, faster separation, better resolution, and shorter migration time. The separation condition, the migration order, and the separation mechanisms in this system are discussed in detail. Experimental Chemicals and reagent Cinchona alkaloids (quinine, quinidine, cinchonine, and cinchonidine) were purchased from Sinopharm Chemical Reagent Co., Ltd. Chiral ionic liquid ([TBA][L-ASP]) was synthesized by our research group and HP-b-CD was from Tianjin Guangfu Fine Chemical Company. Each was prepared by dissolving in 40 mM ammonium acetate background electrolyte with sodium hydroxide and acetic acid to the required pH (3.5–5.5). Other chemicals used for chiral separation were of analytical grade. The real samples (Compound Quinine Injection was from Wuhan Fusion Biological Pharmaceutical Co., Ltd, Watsons Tonic Water was from Watsons Food & Beverage Co., Ltd, and Cinchona tree bark was from Guangdong) were purchased.
The stock solutions were prepared by dissolving the four Cinchona alkaloids in methanol at a concentration of 500 lg/mL. All solutions were filtered through 0.45 lm membranes (Shanghai Milipore Co., Ltd), and degassed by ultrasonication (Kunshan Ultrasonic Instruments CO., Ltd) before use. Water was purified by a distillation apparatus. The extracts of Cinchona barks were prepared by grinding to a particle size smaller than 0.8 mm, treated with methanol in an ultrasonic bath for 30 min, and filtered through a paper filter. Other test mixtures were prepared and diluted with the BGEs in a 1:1 ratio before injection. Method Between each injection, the capillary was conditioned by flushing with the BGEs for 5 min. When the electrolyte composition was changed, the capillary was washed and equilibrated with new electrolytes. Injections were made in hydrodynamic mode for a period of 5 s. The electrophoresis was performed at a voltage of 15 kV for the separation. UV detection was set at the cathodic end of capillary and wavelength at 235 nm for Cinchona alkaloids. All experiments were carried out at room temperature (25 °C). Electroosmotic flow was determined with the equation
leof ¼
1L teof V
by using methanol as a neutral marker. Results and discussion Effect of pH on separation and EOF As an important characteristic, the pH value of the background electrolyte not only significantly influenced the chiral separation behaviors, but also had impact on the EOF. From a practical viewpoint, pH was examined by gradually increasing its value from 3.5 to 5.5 by the use of HP-b-CD and ammonium acetate as test samples. Under such pH conditions, the mobility of analytes decreased with the increase of pH, while the EOF increased. As shown in Fig. 2A, pH 5.0 was chosen as the optimal acidity of BGEs for the subsequent study. The corresponding resolution data and EOF are also given in Fig. 2B. These behaviors could be explained by the fact that under acidic conditions, the vast majority of compounds were ionized and bore positive charges, as inclusion complex decreased the mobility and slowed the migration of the analytes, providing more time for the separation of the diastereomers. Meanwhile, these positively charged complexes probably were associated with the negatively charged silanol groups on the inner surface of the capillary, which resulted in the increase of EOF. Effect of buffer concentration on separation and EOF The effects of the ammonium acetate buffer solution concentration were investigated on the separation and EOF. Buffers were usually added to BGEs in CE in order to ionize chiral selectors/
Combined use of [TBA][L-ASP] and HP-b-CD as selectors for separation of Cinchona alkaloids by CE / Y. Zhang et al. / Anal. Biochem. 462 (2014) 13–18
Fig.2. (A) Effect of the pH on the separation of the diastereomers. (A) pH 3.5, (B) pH 4.0, (C) pH 4.5, (D) pH 5.0, (E) pH 5.5. Conditions: 25 mM ammonium acetate buffer with 40 mM HP-b-CD, applied voltage 15 kV, temperature 25 °C. Peaks: 1, cinchonidine; 2, quinine; 3, cinchonine; 4, quinidine. (B) Effect of the pH on the resolutions (C1 cinchonidine/quinine, D1 quinine/cinchonine, E1 cinchonine/quinidine) and (B) leof of the diastereomers. CE conditions were same as those in panel A.
analytes and improve peak shapes. In some cases, the addition of buffers can also provide better enantioselectivity. Ammonium acetate was usually used as a buffer as it had fair solubility in most organic solvents. By increasing the buffer concentration from 10 to 30 mM, it was demonstrated that the four Cinchona alkaloids can be well resolved by using the running electrolyte containing 25 mM ammonium acetate buffer at pH 5.0. It is shown in Fig. 3A, and the corresponding separation data and EOF are also given in Fig. 3B. It is seen that migration time increased with an increase of the buffer concentration. This phenomenon was different from that in EOF which may partially be attributable to the enhanced ionic strength and possible capillary wall adsorption. Effect of HP-b-CD and chiral ionic liquid concentration on separation and EOF The chiral recognition depended on the complex stability constants of the formation of complexes with the analytes. Many factors affected the formation of complexes, and the variables were interdependent. In this paper, HP-b-CD and chiral ionic liquid worked as the chiral selector additive, and both of them were optimized for this requirement, respectively. The concentration of the HP-b-CD was changed from 10 to 50 mM. The results are shown in Fig. 4A, and the corresponding separation data and EOF are also given in Fig. 4B. The resolutions increased with the increase of the HP-b-CD concentration, while longer migration time and lower EOF were obtained. It should be noted that the best separation can be achieved at a concentration of 40 mM. This trend is in agreement with the prediction concerning the existence of a
15
Fig.3. (A) Effect of the buffer concentration on the separation of the diastereomers. (A) 10 mM, (B) 15 mM, (C) 20 mM, (D) 25 mM, (E) 30 mM. Conditions: 40 mM HP-bCD with pH 5.0, applied voltage 15 kV, temperature 25 °C. The serial numbers of peak were the same as those in Fig. 2A. (B) Effect of the buffer concentration on the resolutions (C1 cinchonidine/quinine, D1 quinine/cinchonine, E1 cinchonine/quinidine) and (B) leof of the diastereomers. CE conditions were same as those in panel A.
maximum resolution at a certain concentration of chiral selector, when the apparent electrophoretic mobility difference of two pairs of enantiomers reached their maximum. We have also investigated chiral ionic liquids as other influencing factors in the mobile phase. Chiral ionic liquids have manifested some fascinating strengths when being used as BGEs additives or capillary wall modifiers in a variety of CE applications, such as improving resolution, reducing wall adsorption, and controlling EOF. First, the chiral ionic liquid was tested as a single influencing factor of chiral selectors from 1 to 20 mM. It was noted that the migration time and the resolution changed with the increase of [TBA][L-ASP], as shown in Fig. 5. For migration time, it increased from 3.4 to 4.3 s at concentrations of 1 to 16 mM. The resolution of the analytes remained unchanged until the concentration of [TBA][L-ASP] reached to 8 mM, gradually increased in the range of 12–16 mM, and then decreased with the further increase of the concentration of [TBA][L-ASP] from 16 to 20 mM. This phenomenon might be related to the higher viscosity of the electrophoretic system and the adsorption of the cations on the capillary which could lead to a decrease EOF. It can be seen that the good efficiency of the separation was gained with [TBA][L-ASP] in the range from 8 to 16 mM. As discussed above, we maintained the BGEs due to ammonium acetate background electrolyte pH 5.0 with the addition of HP-bCD at a concentration of 40 mM, and [TBA][L-ASP] was investigated respectively using concentrations of 8, 12, 14, and 16 mM. It indicated that both the mobility and the separation of analytes increased with increasing [TBA][L-ASP] concentration ranging from 8 to 14 mM (except D1, 12 M; shown in Fig. 6A), and then they
16
Combined use of [TBA][L-ASP] and HP-b-CD as selectors for separation of Cinchona alkaloids by CE / Y. Zhang et al. / Anal. Biochem. 462 (2014) 13–18
Fig.4. (A) Effect of the HP-b-CD concentration on the separation of the diastereomers. (A) 10 mM, (B) 20 mM, (C) 30 mM, (D) 40 mM, (E) 50 mM. Conditions: 25 mM ammonium acetate buffer with pH 5.0, voltage 15 kV, temperature 25 °C. The serial numbers of peak were the same as those in Fig. 2A. (B) Effect of the HP-b-CD concentration on the resolutions (C1 cinchonidine/quinine, D1 quinine/cinchonine, E1 cinchonine/quinidine) and (B) leof of the diastereomers. CE conditions were same as those in panel A.
Fig.5. Effect of the [TBA][L-ASP] concentration on the separation of the diastereomers. (A) 1 mM, (B) 4 mM, (C) 8 mM, (D) 16 mM, (E) 20 mM. Conditions: 25 mM ammonium acetate buffer with pH 5.0, voltage 15 kV, temperature 25 °C. The serial numbers of peak were the same as those in Fig. 2A.
decreased with the further increase of [TBA][L-ASP]. The corresponding separation data and EOF are also given in Fig. 6B. A possible explanation for this separation performance was the integration of the decreased EOF and the interaction between the chiral selector and the analytes. Effect of voltage and temperature on separation and EOF The effect of voltage on the chiral separation of analytes was investigated respectively using voltages of 10, 15, and 20 kV. The
Fig.6. (A) Effect of the mixture of [TBA][L-ASP] and HP-b-CD on the separation of the diastereomers. (A) 8 mM, (B) 12 mM, (C) 14 mM, (D) 16 mM. Conditions: 25 mM ammonium acetate buffer with 40 mM HP-b-CD, pH 5.0, voltage 15 kV, temperature 25 °C. The serial numbers of peak were the same as those in Fig. 2A. (B) Effect of the [TBA][L-ASP] concentration on the resolutions (C1 cinchonidine/ quinine, D1 quinine/cinchonine, E1 cinchonine/quinidine) and (B) leof of the diastereomers. CE conditions were same the same as those in panel A.
Fig.7. The electropherograms of standard solution. (A) Standard solution, (B) firstorder derivative electropherogram of standard solution. Peaks: 1, cinchonidine; 2, quinine; 3, cinchonine; 4, quinidine. Conditions: ammonium acetate buffer, 25 mM; HP-b-CD, 40 mM; [TBA] [L-ASP], 14 mM; pH, 5.0; voltage, 15 kV; temperature, 25 °C.
results showed that the migration times of the four analytes were shortened with increased applied voltage, but a slight decrease in resolution of the four analytes was also found. Therefore, 15 kV was selected for relatively good separation of the four compounds with shorter analysis time. The influence of the capillary temperature was also studied with optimized electrophoretic medium. As the temperature
17
Combined use of [TBA][L-ASP] and HP-b-CD as selectors for separation of Cinchona alkaloids by CE / Y. Zhang et al. / Anal. Biochem. 462 (2014) 13–18 Table 1 Calibration range, regression equation, correlation coefficients, and RSDs.
a
Analytes
Linear range (lg/mL)
Regression equationa
Correlation coefficient
Intraday, RSD (%, n = 5)
Interday, RSD (%, n = 5)
Quinine Quinidine Cinchonine Cinchonidine
1–500 1–500 1–500 1–500
Y = 771.5x 834.8 Y = 820.7x 693.2 Y = 881.0x + 797.8 Y = 821.6x 886.6
0.9975 0.9994 0.9993 0.9986
4.0 3.5 2.6 2.9
4.1 3.4 3.3 3.5
Y and x stand for the first-order derivative peak area and the concentration (lg/mL) of the analytes, respectively.
quinine, and cinchonine) was repeatedly achieved in fewer than 7 min. The following migration times were obtained and shown in Fig. 7: cinchonidine 5.621 min; quinine 6.075 min; cinchonine 6.417 min; quinidine 6.779 min. The numbers of theoretical plates were >970,000 plates/m. Quantitative analysis
Fig.8. The electropherograms of Cinchona tree bark. (A) Cinchona tree bark, (B) first-order derivative electropherograms of Cinchona tree bark. Peaks: 1, cinchonidine; 2, quinine; 3, cinchonine; 4, quinidine. CE conditions were same as those in Fig. 7.
increased from 15 to 25 °C, the migration times and the resolutions of the analytes remained almost unchanged. Thus the room temperature of 25 °C was selected as the optimum. Based on comprehensive considerations of CE requirements, such as high resolution, low electrolyte concentration, low electric current, and stable baseline, 25 mM ammonium acetate buffer was selected with the addition of 40 mM HP-b-CD and 14 mM [TBA][LASP] at pH 5.0, 15 kV applied voltage across the capillary, and 25 °C as the optimal conditions. According to the above conditions, baseline resolution was obtained among cinchonidine, quinine, and cinchonine. Yet, complete baseline separations between analytes and sample matrices in real samples were not obtained in the determination of cinchonine and quinidine. Therefore, the first-order derivative electropherogram was introduced for quantitative analysis and the results were satisfactory. Under the optimal conditions, baseline separation of the three analytes (cinchonidine,
The linear relationships between the concentration of four analytes and the corresponding peak area were investigated under the optimum separation conditions. Regression equations revealed a good linear relationship over the concentration range of 1– 500 lg/mL for the four analytes. The results are shown in Table 1. The reproducibility test of four analytes in the experiment was determined by repeated five injections of a standard mixture solution intraday and interday, under the optimum conditions. It is also shown in Table 1. Then, the limit of detection (LOD, S/N = 3) was 0.24 lg/mL for quinine, 0.21 lg/mL for quinidine, 0.30 lg/mL for cinchonine, and 0.17 lg/mL for cinchonidine respectively; the limit of quantitation (LOQ, S/N = 10) was 0.87 lg/mL for quinine, 0.9 lg/mL for quinidine, 0.94 lg/mL for cinchonine, and 0.78 lg/mL for cinchonidine respectively. Application Under the optimal conditions, the real samples were examined, including Cinchona tree bark, Compound Quinine Injection, and Watsons Tonic Water to demonstrate the potential of the method. The sample electropherogram and the corresponding first- order derivative electropherogram of Cinchona tree bark are shown in Fig. 8. The contents and recovery of the four Cinchona alkaloids in the samples were determined with the standard addition method under exactly the same conditions, and the results are also given in Table 2, Table 3.1, and Table 3.2.
Table 2 The contents of the four Cinchona alkaloids in the samples. Samples
Quinine
RSD (%, n = 3)
Quinidine
RSD (%, n = 3)
Cinchonine
RSD (%, n = 3)
Cinchonidine
RSD (%, n = 3)
Cinchona tree bark (mg/g) Compound quinine injection (mg/mL) Watsons tonic water (mg/mL)
0.0924 0.7742 0.8550
3.8 2.4 3.0
0.2624 – –
2.1 – –
0.1148 – –
4.6 – –
0.3086 – –
2.9 – –
Table 3.1 The average recoveries of quinine and quinidine in different samples (n = 5). Samples
Cinchona tree bark Compound Quinine Injection Watsons Tonic Water
Quinine
Quinidine
Content (mg)
Added (mg)
Found (mg)
Recovery (%)
RSD (%)
Content (mg)
Added (mg)
Found (mg)
Recovery (%)
RSD (%)
0.0462 0.3871
0.0400 0.3840
0.0933 0.7927
107.0 105.6
7.8 5.2
0.1312 0
0.1200 0.3840
0.2488 0.3685
98.0 95.9
3.2 4.4
0.4275
0.4000
0.8196
98.2
5.1
0
0.4000
0.3894
97.35
2.5
18
Combined use of [TBA][L-ASP] and HP-b-CD as selectors for separation of Cinchona alkaloids by CE / Y. Zhang et al. / Anal. Biochem. 462 (2014) 13–18
Table 3.2 The average recoveries of cinchonine and cinchonidine in different samples (n = 5). Samples
Cinchona tree bark Compound Quinine Injection Watsons Tonic Water
Cinchonine
Cinchonidine
Content (mg)
Added (mg)
Found (mg)
Recovery (%)
RSD (%)
Content (mg)
Added (mg)
Found (mg)
Recovery (%)
RSD (%)
0.0574 0
0.0500 0.3840
0.1018 0.4018
90.2 104.6
4.3 3.7
0.1543 0
0.1520 0.3840
0.3011 0.3953
96.6 102.9
1.9 6.2
0
0.4000
0.3894
97.3
3.9
0
0.4000
0.4012
100.3
5.8
Discussion From the above results, we realized that HP-b-CD and [TBA][LASP] were good alternatives as chiral selectors for the chiral separations. In CE, HP-b-CD was endowed with high aqueous solubility, excellent chiral discriminating ability, and reproducibility toward chiral analytes. As HP-b-CD in the BGEs, analytes shifted the complex formation equilibrium toward the formation of HP-b-CD analytes inclusion complex, which led to slower migration due to improvement of the resolution. Meanwhile, the stability of inclusion complexes was coupled to hydrogen bonding interactions between the chiral selector HP-b-CD and the analytes. From a CE point of view, chiral separation can only be achieved by the addition of another chiral anion or chiral neutral compound, when separation results were not satisfied. In this paper, [TBA][L-ASP], as another chiral selector, had a key role. Hydrogen bonding and steric hindrance of chiral ionic liquid were supposed to afford a supplementary intermolecular interaction for stereoselectivity. The special chemical structure directly connected to the chiral ionic liquid and the analytes, exhibiting stereoselectivity. Meanwhile, chiral ionic liquid also played an important role in reducing the adsorption of the cations on the capillary wall. In this experiment, it has been demonstrated that the special chemical structure of [TBA][L-ASP] and HP-b-CD is collaboratively related to the chiral separation of Cinchona alkaloids.
Conclusions In the present paper, the chiral selector of [TBA][L-ASP] was synthesized and successfully used for the enantioseparation of four main Cinchona alkaloids. Though baseline separation of cinchonine and quinidine was not obtained under optimum experimental conditions, the use of a first-order derivative electropherogram made the simultaneous determination of the four compounds possible. Compared with previously developed CE and HPLC methods, the method was superior in terms of short time, high separation efficiency, and easy operation. The study not only showed that chiral ionic liquid, as the selector, had a huge potential for chiral separation techniques, but also proved that the first-order derivative electropherogram was effective in determination of the analytes which was sometimes hard to obtain with the baseline separation. Acknowledgments This work was financially supported by National Natural Science Foundation of China (81241137), Shanxi Scholarship Council
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Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Combined use of [TBA][L-ASP] and hydroxypropyl-b-cyclodextrin as selectors for separation of Cinchona alkaloids by capillary electrophoresis Yu Zhang a, Haixia Yu b, Yujiao Wu a, Wenyan Zhao a, Min Yang a, Huanwang Jing c,⇑, Anjia Chen a,⇑ a b c
College of Pharmacy, Shanxi Medical University, Taiyuan 030001, China Scientific Research Center, Shanxi Medical University, Taiyuan 030001, China College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
a r t i c l e
i n f o
Article history: Received 25 April 2014 Received in revised form 10 June 2014 Accepted 11 June 2014 Available online 20 June 2014 Keywords: Capillary electrophoresis Chiral separation Chiral ionic liquid HP-b-CD First-order derivative electropherogram
a b s t r a c t In this paper, a new capillary electrophoresis (CE) separation and detection method was developed for the chiral separation of the four major Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) using hydroxypropyl-b-cyclodextrin (HP-b-CD) and chiral ionic liquid ([TBA][L-ASP]) as selectors. Separation parameters such as buffer concentrations, pH, HP-b-CD and chiral ionic liquid concentrations, capillary temperature, and separation voltage were investigated. After optimization of separation conditions, baseline separation of the three analytes (cinchonidine, quinine, cinchonine) was achieved in fewer than 7 min in ammonium acetate background electrolyte (pH 5.0) with the addition of HPb-CD in a concentration of 40 mM and [TBA][L-ASP] of 14 mM, while the baseline separation of cinchonine and quinidine was not obtained. Therefore, the first-order derivative electropherogram was applied for resolving overlapping peaks. Regression equations revealed a good linear relationship between peak areas in first-order derivative electropherograms and concentrations of the two diastereomer pairs. The results not only indicated that the first-order derivative electropherogram was effective in determination of a low content component and of those not fully separated from adjacent ones, but also showed that the ionic liquid appeared to be a very promising chiral selector in CE. Ó 2014 Elsevier Inc. All rights reserved.
Cinchona alkaloids can be found in the bark of Cinchona and Remijia species. The bark contains a variety of alkaloids, including quinine and quinidine, and cinchonine and cinchonidine (Fig. 1A). A main structural feature of these substances is the presence of chiral centers at C8 and C9, each of which may be found in the R or S configuration. Over the past 30 years, Cinchona alkaloids have become increasingly popular in organic chemistry and are used as chiral catalysts, ligands, nuclear magnetic resonance (NMR)1 discriminating agents, and so on [1–3]. Nowadays, they were also applied in high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) stationary phases for chiral separations [4,5]. These compounds were also well known to have medicinal activity [6]. They were long cultivated as a muscle relaxant to cease
⇑ Corresponding authors. E-mail addresses: [email protected] (H. Jing), [email protected] (A. Chen). Abbreviations used: BGEs, background electrolytes; CE, capillary electrophoresis; HP-b-CD, hydroxypropyl-b-cyclodextrin; HPLC high-performance liquid chromatography; NMR, nuclear magnetic resonance. 1
http://dx.doi.org/10.1016/j.ab.2014.06.008 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
shivering due to low temperatures and as an antipyretic agent especially useful in treating malaria. Quinine is the most familiar one. Recently, some analytical methods for separation and determination of Cinchona alkaloids have been developed [7–9]. The quantitative analysis of Cinchona alkaloids has frequently been done by HPLC using C18-modified silica and ultraviolet (UV) or mass spectrometry (MS) detection systems [10–12]. In HPLC, precautions need to be taken due to the strong silanophilic interactions which can occur with these analytes and the column surface, which can lead to poor peak shape and resolution. However, CE which has the advantages of rapid analysis time, high separation efficiency, low sample, reagent consumption, high resolution, and sensitivity has been recognized as another powerful separation technique for chiral separations [12,13]. In recent decades, CE with chiral additives to the background electrolyte has been proven as the best method for separation of enantiomers. Meanwhile, chiral ionic liquids as a special chiral additive provide a new approach for the optimization of CE separations [14–17]. Chiral ionic liquids which refer to a group of organic salts are liquid at room temperature due to their unique characteristics, such as low toxicity, wide
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Combined use of [TBA][L-ASP] and HP-b-CD as selectors for separation of Cinchona alkaloids by CE / Y. Zhang et al. / Anal. Biochem. 462 (2014) 13–18
Capillary electrophoresis instrumentation The CE experiments were carried out on a Beckman Coulter P/ ACE MDQ CE System (Beckman Coulter, Fullerton, CA, USA). Fused silica capillaries were from HeBei RuiFeng Chromatographic Instrument Company with 50.2 cm 50 lm i.d. (375 lm o.d., effective length 40 cm). New bare silica capillaries were flushed successively for 5 min with methanol, 2 min with H2O, 5 min with 0.1 M HCl, 2 min with H2O, 10 min with 0.1 M NaOH, and 5 min with H2O. Sample preparation
Fig.1. (A) chemical structure of the diastereomeric pairs of Cinchona alkaloids. (B) The chemical structure of chiral ionic liquid ([TBA][L-ASP]).
liquid range, high chemical stability, wide electrochemical window, and nonvolatility, and thus have attracted much research interest. They have been considered as an alternative to conventional molecular solvents and were applied widely. A good example was the increasing use of chiral ionic liquids in modern analytical chemistry specially for chiral separation, in which they have played a variety of roles in facilitating the chromatographic separation either as mobile phase additives or as stationary phases. In CE separation processes, chiral ionic liquids have manifested some fascinating strengths when used as background electrolytes (BGEs) additives or capillary-wall modifiers in a variety of CE applications, such as improving resolution, controlling electroosmotic flow (EOF), and suppressing wall adsorption problems due to the repulsion forces between the analyte and the capillary surface. In this paper, a chiral ionic liquid ([TBA][L-ASP]; see Fig. 1B), synthesized by our research group, combined with hydroxypropyl-b-cyclodextrin (HP-b-CD), was used as chiral selectors for separation and detection of Cinchona alkaloids by CE. Two diastereomeric pairs of Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) were used as model analytes. Compared with conventional separation and detection methods, this method shows many advantages including lower analyte wall adsorption, faster separation, better resolution, and shorter migration time. The separation condition, the migration order, and the separation mechanisms in this system are discussed in detail. Experimental Chemicals and reagent Cinchona alkaloids (quinine, quinidine, cinchonine, and cinchonidine) were purchased from Sinopharm Chemical Reagent Co., Ltd. Chiral ionic liquid ([TBA][L-ASP]) was synthesized by our research group and HP-b-CD was from Tianjin Guangfu Fine Chemical Company. Each was prepared by dissolving in 40 mM ammonium acetate background electrolyte with sodium hydroxide and acetic acid to the required pH (3.5–5.5). Other chemicals used for chiral separation were of analytical grade. The real samples (Compound Quinine Injection was from Wuhan Fusion Biological Pharmaceutical Co., Ltd, Watsons Tonic Water was from Watsons Food & Beverage Co., Ltd, and Cinchona tree bark was from Guangdong) were purchased.
The stock solutions were prepared by dissolving the four Cinchona alkaloids in methanol at a concentration of 500 lg/mL. All solutions were filtered through 0.45 lm membranes (Shanghai Milipore Co., Ltd), and degassed by ultrasonication (Kunshan Ultrasonic Instruments CO., Ltd) before use. Water was purified by a distillation apparatus. The extracts of Cinchona barks were prepared by grinding to a particle size smaller than 0.8 mm, treated with methanol in an ultrasonic bath for 30 min, and filtered through a paper filter. Other test mixtures were prepared and diluted with the BGEs in a 1:1 ratio before injection. Method Between each injection, the capillary was conditioned by flushing with the BGEs for 5 min. When the electrolyte composition was changed, the capillary was washed and equilibrated with new electrolytes. Injections were made in hydrodynamic mode for a period of 5 s. The electrophoresis was performed at a voltage of 15 kV for the separation. UV detection was set at the cathodic end of capillary and wavelength at 235 nm for Cinchona alkaloids. All experiments were carried out at room temperature (25 °C). Electroosmotic flow was determined with the equation
leof ¼
1L teof V
by using methanol as a neutral marker. Results and discussion Effect of pH on separation and EOF As an important characteristic, the pH value of the background electrolyte not only significantly influenced the chiral separation behaviors, but also had impact on the EOF. From a practical viewpoint, pH was examined by gradually increasing its value from 3.5 to 5.5 by the use of HP-b-CD and ammonium acetate as test samples. Under such pH conditions, the mobility of analytes decreased with the increase of pH, while the EOF increased. As shown in Fig. 2A, pH 5.0 was chosen as the optimal acidity of BGEs for the subsequent study. The corresponding resolution data and EOF are also given in Fig. 2B. These behaviors could be explained by the fact that under acidic conditions, the vast majority of compounds were ionized and bore positive charges, as inclusion complex decreased the mobility and slowed the migration of the analytes, providing more time for the separation of the diastereomers. Meanwhile, these positively charged complexes probably were associated with the negatively charged silanol groups on the inner surface of the capillary, which resulted in the increase of EOF. Effect of buffer concentration on separation and EOF The effects of the ammonium acetate buffer solution concentration were investigated on the separation and EOF. Buffers were usually added to BGEs in CE in order to ionize chiral selectors/
Combined use of [TBA][L-ASP] and HP-b-CD as selectors for separation of Cinchona alkaloids by CE / Y. Zhang et al. / Anal. Biochem. 462 (2014) 13–18
Fig.2. (A) Effect of the pH on the separation of the diastereomers. (A) pH 3.5, (B) pH 4.0, (C) pH 4.5, (D) pH 5.0, (E) pH 5.5. Conditions: 25 mM ammonium acetate buffer with 40 mM HP-b-CD, applied voltage 15 kV, temperature 25 °C. Peaks: 1, cinchonidine; 2, quinine; 3, cinchonine; 4, quinidine. (B) Effect of the pH on the resolutions (C1 cinchonidine/quinine, D1 quinine/cinchonine, E1 cinchonine/quinidine) and (B) leof of the diastereomers. CE conditions were same as those in panel A.
analytes and improve peak shapes. In some cases, the addition of buffers can also provide better enantioselectivity. Ammonium acetate was usually used as a buffer as it had fair solubility in most organic solvents. By increasing the buffer concentration from 10 to 30 mM, it was demonstrated that the four Cinchona alkaloids can be well resolved by using the running electrolyte containing 25 mM ammonium acetate buffer at pH 5.0. It is shown in Fig. 3A, and the corresponding separation data and EOF are also given in Fig. 3B. It is seen that migration time increased with an increase of the buffer concentration. This phenomenon was different from that in EOF which may partially be attributable to the enhanced ionic strength and possible capillary wall adsorption. Effect of HP-b-CD and chiral ionic liquid concentration on separation and EOF The chiral recognition depended on the complex stability constants of the formation of complexes with the analytes. Many factors affected the formation of complexes, and the variables were interdependent. In this paper, HP-b-CD and chiral ionic liquid worked as the chiral selector additive, and both of them were optimized for this requirement, respectively. The concentration of the HP-b-CD was changed from 10 to 50 mM. The results are shown in Fig. 4A, and the corresponding separation data and EOF are also given in Fig. 4B. The resolutions increased with the increase of the HP-b-CD concentration, while longer migration time and lower EOF were obtained. It should be noted that the best separation can be achieved at a concentration of 40 mM. This trend is in agreement with the prediction concerning the existence of a
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Fig.3. (A) Effect of the buffer concentration on the separation of the diastereomers. (A) 10 mM, (B) 15 mM, (C) 20 mM, (D) 25 mM, (E) 30 mM. Conditions: 40 mM HP-bCD with pH 5.0, applied voltage 15 kV, temperature 25 °C. The serial numbers of peak were the same as those in Fig. 2A. (B) Effect of the buffer concentration on the resolutions (C1 cinchonidine/quinine, D1 quinine/cinchonine, E1 cinchonine/quinidine) and (B) leof of the diastereomers. CE conditions were same as those in panel A.
maximum resolution at a certain concentration of chiral selector, when the apparent electrophoretic mobility difference of two pairs of enantiomers reached their maximum. We have also investigated chiral ionic liquids as other influencing factors in the mobile phase. Chiral ionic liquids have manifested some fascinating strengths when being used as BGEs additives or capillary wall modifiers in a variety of CE applications, such as improving resolution, reducing wall adsorption, and controlling EOF. First, the chiral ionic liquid was tested as a single influencing factor of chiral selectors from 1 to 20 mM. It was noted that the migration time and the resolution changed with the increase of [TBA][L-ASP], as shown in Fig. 5. For migration time, it increased from 3.4 to 4.3 s at concentrations of 1 to 16 mM. The resolution of the analytes remained unchanged until the concentration of [TBA][L-ASP] reached to 8 mM, gradually increased in the range of 12–16 mM, and then decreased with the further increase of the concentration of [TBA][L-ASP] from 16 to 20 mM. This phenomenon might be related to the higher viscosity of the electrophoretic system and the adsorption of the cations on the capillary which could lead to a decrease EOF. It can be seen that the good efficiency of the separation was gained with [TBA][L-ASP] in the range from 8 to 16 mM. As discussed above, we maintained the BGEs due to ammonium acetate background electrolyte pH 5.0 with the addition of HP-bCD at a concentration of 40 mM, and [TBA][L-ASP] was investigated respectively using concentrations of 8, 12, 14, and 16 mM. It indicated that both the mobility and the separation of analytes increased with increasing [TBA][L-ASP] concentration ranging from 8 to 14 mM (except D1, 12 M; shown in Fig. 6A), and then they
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Combined use of [TBA][L-ASP] and HP-b-CD as selectors for separation of Cinchona alkaloids by CE / Y. Zhang et al. / Anal. Biochem. 462 (2014) 13–18
Fig.4. (A) Effect of the HP-b-CD concentration on the separation of the diastereomers. (A) 10 mM, (B) 20 mM, (C) 30 mM, (D) 40 mM, (E) 50 mM. Conditions: 25 mM ammonium acetate buffer with pH 5.0, voltage 15 kV, temperature 25 °C. The serial numbers of peak were the same as those in Fig. 2A. (B) Effect of the HP-b-CD concentration on the resolutions (C1 cinchonidine/quinine, D1 quinine/cinchonine, E1 cinchonine/quinidine) and (B) leof of the diastereomers. CE conditions were same as those in panel A.
Fig.5. Effect of the [TBA][L-ASP] concentration on the separation of the diastereomers. (A) 1 mM, (B) 4 mM, (C) 8 mM, (D) 16 mM, (E) 20 mM. Conditions: 25 mM ammonium acetate buffer with pH 5.0, voltage 15 kV, temperature 25 °C. The serial numbers of peak were the same as those in Fig. 2A.
decreased with the further increase of [TBA][L-ASP]. The corresponding separation data and EOF are also given in Fig. 6B. A possible explanation for this separation performance was the integration of the decreased EOF and the interaction between the chiral selector and the analytes. Effect of voltage and temperature on separation and EOF The effect of voltage on the chiral separation of analytes was investigated respectively using voltages of 10, 15, and 20 kV. The
Fig.6. (A) Effect of the mixture of [TBA][L-ASP] and HP-b-CD on the separation of the diastereomers. (A) 8 mM, (B) 12 mM, (C) 14 mM, (D) 16 mM. Conditions: 25 mM ammonium acetate buffer with 40 mM HP-b-CD, pH 5.0, voltage 15 kV, temperature 25 °C. The serial numbers of peak were the same as those in Fig. 2A. (B) Effect of the [TBA][L-ASP] concentration on the resolutions (C1 cinchonidine/ quinine, D1 quinine/cinchonine, E1 cinchonine/quinidine) and (B) leof of the diastereomers. CE conditions were same the same as those in panel A.
Fig.7. The electropherograms of standard solution. (A) Standard solution, (B) firstorder derivative electropherogram of standard solution. Peaks: 1, cinchonidine; 2, quinine; 3, cinchonine; 4, quinidine. Conditions: ammonium acetate buffer, 25 mM; HP-b-CD, 40 mM; [TBA] [L-ASP], 14 mM; pH, 5.0; voltage, 15 kV; temperature, 25 °C.
results showed that the migration times of the four analytes were shortened with increased applied voltage, but a slight decrease in resolution of the four analytes was also found. Therefore, 15 kV was selected for relatively good separation of the four compounds with shorter analysis time. The influence of the capillary temperature was also studied with optimized electrophoretic medium. As the temperature
17
Combined use of [TBA][L-ASP] and HP-b-CD as selectors for separation of Cinchona alkaloids by CE / Y. Zhang et al. / Anal. Biochem. 462 (2014) 13–18 Table 1 Calibration range, regression equation, correlation coefficients, and RSDs.
a
Analytes
Linear range (lg/mL)
Regression equationa
Correlation coefficient
Intraday, RSD (%, n = 5)
Interday, RSD (%, n = 5)
Quinine Quinidine Cinchonine Cinchonidine
1–500 1–500 1–500 1–500
Y = 771.5x 834.8 Y = 820.7x 693.2 Y = 881.0x + 797.8 Y = 821.6x 886.6
0.9975 0.9994 0.9993 0.9986
4.0 3.5 2.6 2.9
4.1 3.4 3.3 3.5
Y and x stand for the first-order derivative peak area and the concentration (lg/mL) of the analytes, respectively.
quinine, and cinchonine) was repeatedly achieved in fewer than 7 min. The following migration times were obtained and shown in Fig. 7: cinchonidine 5.621 min; quinine 6.075 min; cinchonine 6.417 min; quinidine 6.779 min. The numbers of theoretical plates were >970,000 plates/m. Quantitative analysis
Fig.8. The electropherograms of Cinchona tree bark. (A) Cinchona tree bark, (B) first-order derivative electropherograms of Cinchona tree bark. Peaks: 1, cinchonidine; 2, quinine; 3, cinchonine; 4, quinidine. CE conditions were same as those in Fig. 7.
increased from 15 to 25 °C, the migration times and the resolutions of the analytes remained almost unchanged. Thus the room temperature of 25 °C was selected as the optimum. Based on comprehensive considerations of CE requirements, such as high resolution, low electrolyte concentration, low electric current, and stable baseline, 25 mM ammonium acetate buffer was selected with the addition of 40 mM HP-b-CD and 14 mM [TBA][LASP] at pH 5.0, 15 kV applied voltage across the capillary, and 25 °C as the optimal conditions. According to the above conditions, baseline resolution was obtained among cinchonidine, quinine, and cinchonine. Yet, complete baseline separations between analytes and sample matrices in real samples were not obtained in the determination of cinchonine and quinidine. Therefore, the first-order derivative electropherogram was introduced for quantitative analysis and the results were satisfactory. Under the optimal conditions, baseline separation of the three analytes (cinchonidine,
The linear relationships between the concentration of four analytes and the corresponding peak area were investigated under the optimum separation conditions. Regression equations revealed a good linear relationship over the concentration range of 1– 500 lg/mL for the four analytes. The results are shown in Table 1. The reproducibility test of four analytes in the experiment was determined by repeated five injections of a standard mixture solution intraday and interday, under the optimum conditions. It is also shown in Table 1. Then, the limit of detection (LOD, S/N = 3) was 0.24 lg/mL for quinine, 0.21 lg/mL for quinidine, 0.30 lg/mL for cinchonine, and 0.17 lg/mL for cinchonidine respectively; the limit of quantitation (LOQ, S/N = 10) was 0.87 lg/mL for quinine, 0.9 lg/mL for quinidine, 0.94 lg/mL for cinchonine, and 0.78 lg/mL for cinchonidine respectively. Application Under the optimal conditions, the real samples were examined, including Cinchona tree bark, Compound Quinine Injection, and Watsons Tonic Water to demonstrate the potential of the method. The sample electropherogram and the corresponding first- order derivative electropherogram of Cinchona tree bark are shown in Fig. 8. The contents and recovery of the four Cinchona alkaloids in the samples were determined with the standard addition method under exactly the same conditions, and the results are also given in Table 2, Table 3.1, and Table 3.2.
Table 2 The contents of the four Cinchona alkaloids in the samples. Samples
Quinine
RSD (%, n = 3)
Quinidine
RSD (%, n = 3)
Cinchonine
RSD (%, n = 3)
Cinchonidine
RSD (%, n = 3)
Cinchona tree bark (mg/g) Compound quinine injection (mg/mL) Watsons tonic water (mg/mL)
0.0924 0.7742 0.8550
3.8 2.4 3.0
0.2624 – –
2.1 – –
0.1148 – –
4.6 – –
0.3086 – –
2.9 – –
Table 3.1 The average recoveries of quinine and quinidine in different samples (n = 5). Samples
Cinchona tree bark Compound Quinine Injection Watsons Tonic Water
Quinine
Quinidine
Content (mg)
Added (mg)
Found (mg)
Recovery (%)
RSD (%)
Content (mg)
Added (mg)
Found (mg)
Recovery (%)
RSD (%)
0.0462 0.3871
0.0400 0.3840
0.0933 0.7927
107.0 105.6
7.8 5.2
0.1312 0
0.1200 0.3840
0.2488 0.3685
98.0 95.9
3.2 4.4
0.4275
0.4000
0.8196
98.2
5.1
0
0.4000
0.3894
97.35
2.5
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Combined use of [TBA][L-ASP] and HP-b-CD as selectors for separation of Cinchona alkaloids by CE / Y. Zhang et al. / Anal. Biochem. 462 (2014) 13–18
Table 3.2 The average recoveries of cinchonine and cinchonidine in different samples (n = 5). Samples
Cinchona tree bark Compound Quinine Injection Watsons Tonic Water
Cinchonine
Cinchonidine
Content (mg)
Added (mg)
Found (mg)
Recovery (%)
RSD (%)
Content (mg)
Added (mg)
Found (mg)
Recovery (%)
RSD (%)
0.0574 0
0.0500 0.3840
0.1018 0.4018
90.2 104.6
4.3 3.7
0.1543 0
0.1520 0.3840
0.3011 0.3953
96.6 102.9
1.9 6.2
0
0.4000
0.3894
97.3
3.9
0
0.4000
0.4012
100.3
5.8
Discussion From the above results, we realized that HP-b-CD and [TBA][LASP] were good alternatives as chiral selectors for the chiral separations. In CE, HP-b-CD was endowed with high aqueous solubility, excellent chiral discriminating ability, and reproducibility toward chiral analytes. As HP-b-CD in the BGEs, analytes shifted the complex formation equilibrium toward the formation of HP-b-CD analytes inclusion complex, which led to slower migration due to improvement of the resolution. Meanwhile, the stability of inclusion complexes was coupled to hydrogen bonding interactions between the chiral selector HP-b-CD and the analytes. From a CE point of view, chiral separation can only be achieved by the addition of another chiral anion or chiral neutral compound, when separation results were not satisfied. In this paper, [TBA][L-ASP], as another chiral selector, had a key role. Hydrogen bonding and steric hindrance of chiral ionic liquid were supposed to afford a supplementary intermolecular interaction for stereoselectivity. The special chemical structure directly connected to the chiral ionic liquid and the analytes, exhibiting stereoselectivity. Meanwhile, chiral ionic liquid also played an important role in reducing the adsorption of the cations on the capillary wall. In this experiment, it has been demonstrated that the special chemical structure of [TBA][L-ASP] and HP-b-CD is collaboratively related to the chiral separation of Cinchona alkaloids.
Conclusions In the present paper, the chiral selector of [TBA][L-ASP] was synthesized and successfully used for the enantioseparation of four main Cinchona alkaloids. Though baseline separation of cinchonine and quinidine was not obtained under optimum experimental conditions, the use of a first-order derivative electropherogram made the simultaneous determination of the four compounds possible. Compared with previously developed CE and HPLC methods, the method was superior in terms of short time, high separation efficiency, and easy operation. The study not only showed that chiral ionic liquid, as the selector, had a huge potential for chiral separation techniques, but also proved that the first-order derivative electropherogram was effective in determination of the analytes which was sometimes hard to obtain with the baseline separation. Acknowledgments This work was financially supported by National Natural Science Foundation of China (81241137), Shanxi Scholarship Council
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