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Determination of five anthraquinones in medicinal plants by capillary zone electrophoresis with β-cyclodextrin addition
Journal of Chromatography A, 1123 (2006) 134–137
Short communication
Determination of five anthraquinones in medicinal plants by capillary zone electrophoresis with -cyclodextrin addition Kan Tian a,b , Huige Zhang a,b , Xingguo Chen a,b,∗ , Zhide Hu a,b a
b
Department of Chemistry, Lanzhou University, Lanzhou 730000, China National Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China Received 8 February 2006; received in revised form 8 April 2006; accepted 11 April 2006 Available online 27 June 2006
Abstract A simple and rapid method for the simultaneous determination of five anthraquinone derivatives including aloe-emodin, emodin, chrysophanol, physcoin and rhein in Rheum species and Polygonum cuspidatum was established by capillary zone electrophoresis (CZE) using -cyclodextrin (CD) as modifier and urea to enhance its solubility. The apparent binding constants of these derivatives with -CD were evaluated. After an optimization study, the best conditions were selected using 35 mM phosphate buffer (pH 11.0) containing 20 mM -CD and 2 M urea, applied voltage 20 kV and detection at 254 nm. Under such conditions, all of the five anthraquinones were baseline-separated within a short analysis time of 12 min with symmetrical peaks and high theoretical plate numbers (189 000–314 000). The RSD values of the migration times and peak areas were 0.6–1.1, 1.3–1.9% (intra-day) and 0.6–1.5, 1.3–2.8% (inter-day, for a 5-day period), respectively. The limits of detection for the analytes (S/N = 3) were 0.33–0.62 g/ml. The recoveries were ranged from 93.37 to 107.69%. The proposed method was successfully applied to the determination of anthraquinones in ethanol extracts of two kinds of Rheum plants (R. palmatum and R. hotaoense) and P. cuspidatum. © 2006 Elsevier B.V. All rights reserved. Keywords: Capillary zone electrophoresis; -Cyclodextrin; Anthraquinones; Medicinal plants; Apparent binding constants
1. Introduction Rhubarb and Polygonum cuspidatum are two commonly used medicinal plants in oriental medicine. Rhubarb is also used in Europe and other places of the world [1,2]. The pharmaceutically relevant bioactive components in Rhubarb and P. cuspidatum are hydroxyanthraquinoids including physcion, emodin, rhein, aloeemodin and chrysophanol [3,4]. The structures and pKa values of these five anthraquinons are shown in Fig. 1. They are used or recommended as a laxative, antiphlogistic and hemostatic in the treatment of obstipation, gastrointestinal indigestion, diarrhea and jaundice [5,6]. Previous described methods for the determinations of these anthraquinones include TLC [7,8], HPLC [9] and capillary electrophoresis (CE) [10–16]. Qi et al. [17] reported the separation of anthraquinons in extract of Chinese herb Paedicalyx attopevensis Pierre ex Pitard. Up to now, to the best of our knowledge, there was only one report in the literature that achieved simulta∗
Corresponding author. Tel.: +86 931 8912763; fax: +86 931 8912582. E-mail address: [email protected] (X. Chen).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.04.021
neous determination of these five anthraquione derivatives using capillary zone electrophoresis (CZE) [18]. However, the analysis took as long as 40 min. Thus, it is desirable to develop a more simple and rapid method to separate these derivatives. The aim of this study was to develop an efficient and feasible CZE method to simultaneous analysis of these five anthraquinones in non-pretreated crude rhubarb and P. cuspidatum extracts. And also the apparent binding constants between anthraquinones and -CD were evaluated. 2. Experimental 2.1. Apparatus and procedures All separations were performed using a Waters Quanta 4000 capillary electrophoresis system (Milford, MA, USA), equipped with a UV detector. Data acquisition was carried out with a Maxima 820 chromatography workstation. The temperature was maintained at 23.5 ± 0.5 ◦ C with a forced-air cooling system. A fused-silica capillary of 60 cm (52.5 cm effective length) × 50 m I.D. (Yongnian Photoconductive Fiber Factory,
K. Tian et al. / J. Chromatogr. A 1123 (2006) 134–137
135
Fig. 1. The structures and pKa values of the studied anthraquinones.
Hebei Province, China) was used. Samples were introduced from anodic end of the capillary by hydrodynamic injection where the samples vial was raised by 10 cm for 5 s and the detection wavelength was operated at 254 nm. Acetonitrile was the marker of electroosmotic flow (EOF). A PHS-10A acidity meter (Xiaoshan Science Instrumentation Factory, Zhejiang, China) was used for the pH measurement. The pH was adjusted with 0.5 M NaOH or HCl. The measurements were run at least in triplicate for each different composition of the running buffer to ensure repeatability. Before their first use, capillaries were washed successively with 1 M NaOH for 5 min, water for 3 min, 0.1 M NaOH for 5 min and water for 3 min. The capillary rinsed between the runs as follows: 2 min with water, 3 min with 0.1 M NaOH, 2 min with water and 3 min with background electrolyte (BGE).
0.45 m membrane filter (Shanghai Xinya Purification Apparatus Factory, Shanghai, China) prior to use. A 0.10 g sample of P. cuspidatum powder (100 mesh) was immersed in 10 ml ethanol at room temperature for 12 h, after that, it was extracted for 20 min in an ultrasonic bath and followed by centrifugation at 1500 × g for 5 min. This procedure was repeated three times, then the extracts were combined, evaporated to near-dryness on a water bath and the residue was dissolved in 5 ml acetonitrile as the stock solution; a 0.05 g powder of R. palmatum and R. hotaoense were extracted, respectively, using the same procedure as mentioned above. Before injection into the capillary electrophoresis system, the solutions were appropriately diluted and passed through a 0.45 m filter.
2.2. Materials
The viscosity adjusted electrophoretic mobility was determined by multiplying the experimental mobility value by the ratio of the current obtained without -CD in the buffer over the current measured at the selector concentration of interest [20].
Raw rhubarb (R. palmatum and R. hotaoense) and P. cuspidatum were purchased from Z 1hongyou drugstore (Lanzhou, China). Standards of anthraquinone derivatives aloe-emodin, emodin, chrysophanol, physcoin and rhein were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). -CD was purchased from China Medicine Group, Shanghai Chemical Reagent Company (Shanghai, China). Urea, phosphate and borate were purchased from Tianjin Chemical Reagent Factory (Tianjin, China). All chemicals and solvents were of analytical reagent grade and were used without further purification. 2.3. Solutions and sample preparation The stock solutions of standards were prepared by dissolving precisely weighed anthraquinones in 5 ml acetonitrile and stored at −4 ◦ C. The running buffers were prepared daily by adding variable amounts of -CD to the phosphate/urea BGE to give final concentrations of -CD ranging from 0 to 35 mM. The reason for using urea was the limited solubility of -CD in water (16 mM) [19]. All solutions were filtered through a
2.4. Viscosity correction
3. Results and discussion 3.1. CZE separation with β-CD addition Preliminary experiments showed that the studied compounds could not be completely separated by ordinary CZE because the little difference in pKa values between chrysophanol and aloeemodin. Thus, -CD was added to the buffer to enhance the separation [21,22]. The effects on the separation by parameters such as -CD and urea concentration, background electrolyte pH and concentration, applied voltage, were evaluated. After an appropriate optimization, the best conditions were obtained with running electrolyte containing 35 mM phosphate, 20 mM -CD and 2 M urea at pH 11.0. Under the optimum conditions, all the five analytes were well separated within 12 min with symmetrical peaks. The numbers of theoretical plates per meter measured were in the range of 189 000–314 000, which were not reported in Ref. [18]. The typical electropherogram for a standard mix-
136
K. Tian et al. / J. Chromatogr. A 1123 (2006) 134–137
Fig. 2. The typical electropherograms of the standards mixture solution and the real samples under the optimum conditions: (A) the standards mixture; (B) R. hotaoense; (C) R. palmatum and (D) P. cuspidatum. The concentrations of the standards were 45 g/ml for chrysophanol, physcionand and emodin; 40 g/ml for aloe-emodin; 65 g/ml for rhein, respectively; Conditions: 35 mM phosphate containing 20 mM -CD and 2 M urea; pH 11.0; I = 49 A; uncoated fused-silica capillary, total length 60 cm (52.5 cm effective length) × 50 m I.D.; applied voltage, 20 kV; temperature, 23.5 ± 0.5 ◦ C; detection, 254 nm; the peaks were numbered according to the analyte notation in Fig. 1.
ture of five analytes is shown in Fig. 2A. The recorded current was lower than 50 A. 3.2. Evaluation of apparent binding constants For a better understanding of the interactions of anthraquinones with -CD, the apparent binding constants of anthraquinones to -CD were evaluated based on the dependence of the effective electrophoretic mobility of anthraquinones on the concentrations of -CD. The model that described a 1:1 guest–host interaction had been derived elsewhere [23,24]. The experiment was performed with buffers containing 35 mM phosphate (pH 11.0), 2 M urea and 0–35 mM -CD. Table 1 lists the apparent binding constants simultaneously determined by non-linear regression fitting method and three different linearization plots methods (named double reciprocal, x-reciprocal and y-reciprocal), and the results were adjusted by the viscosity correction factor. Comparing the binding constants of anthraquinone derivatives revealed that the substituent group(s) in the R position(s) of aromatic ring significantly affected the inclusion behaviors of these analytes. The sta-
bility of the inclusion complex of anthraquinones follows the order physcion > chrysophanol > aloe-emodin, emodin > rhein. Accordingly, it was the decreasing hydrophobicity of analytes introduced by different substituent group(s) in the R position(s) of analytes. The hydrophobic parameter of log POW values for these compounds were 3.85 for physcion, 3.63 for chrysophanol, 2.68 for aloe-emodin, 2.91 for emodin and 2.42 for rhein [11]. Though emodin and rhein substituted with polar groups in R position(s) of benzene ring, they displayed strong interaction with -CD. The reason for this maybe that hydroxyl and carboxylic acid group attached to the R position(s) of aromatic ring were involved in the host–guest complex formation enabling hydrogen bonds. In this study, the baseline resolution of chrysophanol and aloe-emodin was attributed to the fact that the interactions of chrysophanol with -CD were stronger than those with aloe-emodin and migrated quickly and resolved. 3.3. Method validation Under optimal conditions, the linear relationship between the concentration of the five anthraquinones 1–5 and the corresponding peak areas were investigated. The data of regression equations of curves, their correlation coefficients, linearity range and detection limits are shown in Table 2. The repeatability of the method was determined by measuring six replicate injection of the standard mixture solution at the level of 35 g/ml for each analytes, respectively. The RSD values of the migration times and peak areas were 0.6–1.1, 1.3–1.9% (intra-day), and 0.6–1.5, 1.3–2.8% (inter-day, for a 5-day period), respectively. Ensure accuracy of the method, the recoveries of the method were determined with the standard addition method for anthraquinones 1–5 in the extracts of R. palmatum, R. hotaoens and P. cuspidatum with results of 93.37, 98.19 and 95.98% for 1, 105.57, 104.29 and 95.46% for 2, 98.23, 107.31 and 104.23% for 3, 102.34, 105.47 and 104.83% for 4, 94.51, 98.48 and 107.69% for 5, respectively. These results were similar to that obtained by HPLC [25]. 3.4. Analysis of real samples Under the optimized conditions, two kinds of Rhubarb plants, R. palmatum and R. hotaoens, and P. cuspidatum were analyzed. The peaks were identified by spiking a small amount of each analyte into the sample solutions, and also by comparing their
Table 1 The apparent binding constants (at 23.5 ◦ C) obtained by the different calculation methods with the viscosity correction Compound
Apparent binding constants (×102 M−1 )a Non-linear fitting (r2 )
1 2 3 4 5
1.70 1.07 0.78 1.38 0.74 a
Error 1.96σ.
± ± ± ± ±
0.08 (0.9968) 0.05 (0.9974) 0.05 (0.9960) 0.05 (0.9980) 0.05 (0.9954)
Double reciprocal (r2 ) 1.69 1.04 0.71 1.34 0.71
± ± ± ± ±
0.05 (0.9982) 0.03 (0.9987) 0.03 (0.9978) 0.03 (0.9989) 0.03 (0.9982)
y-reciprocal (r2 ) 1.70 1.04 0.77 1.39 0.73
± ± ± ± ±
0.08 (0.9995) 0.05 (0.9991) 0.05 (0.9974) 0.06 (0.9995) 0.05 (0.9968)
x-reciprocal (r2 ) 1.69 1.05 0.73 1.36 0.72
± ± ± ± ±
0.06 (0.9937) 0.04 (0.9928) 0.04 (0.9834) 0.04 (0.9956) 0.04 (0.9850)
K. Tian et al. / J. Chromatogr. A 1123 (2006) 134–137
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Table 2 The results of regression analysis on calibration curves and the detection limits Compound
Calibration curves y = a + bxa
Correlation coefficient
Linear range (g/ml)
Detection limit (g/ml)b
1 2 3 4 5
y = −185.6 + 109.7x y = −38.4 + 141.2x y = 144.9 + 88.7x y = −35.3 + 75.7x y = 71.4 + 132.2x
0.9995 0.9998 0.9986 0.9988 0.9992
1.41–90 1.41–90 1.25–120 0.71–135 1.56–87
0.42 0.33 0.52 0.62 0.36
a b
y and x stand for the peak area and the concentration (g/ml)of the analytes, respectively. The detection limits corresponding to concentrations giving signal-to-noise ratio of 3.
Table 3 Contents of the five anthraquinones in real samples and RSD (n = 5) Sample 1 hotaoensa
R. R. palmatum P. cuspidatum a b c
2 (0.62)b
0.20 0.41 (0.81) 0.57 (0.79)
0.71 (2.18) 0.76 (1.13) –c
3
4
5
0.089 (1.37) 0.13 (1.54) –
0.33 (1.74) 1.32 (2.21) 1.46 (1.53)
0.086 (2.63) 0.072 (3.04) –
Percentage of dry mass. The data in parentheses refer to the RSD. Not found.
migration times with the migration time of the standards. The typical electropherograms are shown in Fig. 2B–D. The contents of the five anthraquinones together with RSDs (n = 5) are listed in Table 3. The reason that the content of rhein in R. palmatum was low compared to that obtained by HPLC probably due to the different collection time or cultivation region of the plants [26]. 4. Conclusions In this study, the effective separation of five anthraquinones in Rhubarb species and P. cuspidatum were achieved by CD-CZE. The binding constants were evaluated by non-linear regression and three different linear plots methods. The developed method, for their rapidity, simple and the high selectivity, was very suitable for the quality control of medicinal plants containing the studied anthraquinones. Acknowledgement The authors are grateful for the financial support provided by the Natural Science Foundation of China (grant no. 20275014). References [1] British Pharmacopoeia, 17th ed., Stationary Office, Department of Health, London, 1999, p. 1251. [2] European Pharmacopoeia, 3rd ed., European Department for the Quality of Medicines, Council of Europe, Strasbourg, 1997, p. 1441.
[3] P.G. Xiao, L.Y. He, L.W. Wang, J. Ethnopharmacol. 10 (1984) 275. [4] D.X. Wang, G.L. Yang, X.R. Song, Electrophoresis 22 (2001) 464. [5] W. Tang, G. Eisenbrand, Chinese Drugs of Plant Origin, Springer, Berlin, Heidelberg, 1992, p. 855. [6] C.A. Newall, L.A. Anderson, J.D. Phillipson, Herbal Medicines, The Pharmaceutical Press, London, 1996, p. 228. [7] X. Ma, Y. Chen, R. Hui, Chromatographia 27 (1989) 465. [8] S. Nyiredy, T. Dallenbach, G.C. Zogg, O. Sticher, J. Chromatogr. 499 (1990) 453. [9] L.W. Shi, J.H. Zheng, J. Beijing Med. Univ. 25 (1993) 15. [10] C.H. Kuo, S.W. Sun, Anal. Chim. Acta 482 (2003) 47. [11] X.Y. Shang, Z.B. Yuan, Bioorg. Med. Chem. Lett. 13 (2003) 617. [12] S.W. Sun, P.C. Yeh, J. Pharm. Biomed. Anal. 36 (2005) 995. [13] G.B. Li, X.G. Chen, M.C. Liu, Z.D. Hu, Analyst 123 (1998) 1501. [14] Y. Li, H. Liu, X. Ji, J. Li, Electrophoresis 21 (2000) 3109. [15] W.C. Weng, S.J. Sheu, J. High Resolut. Chromatogr. 23 (2000) 143. [16] Y.F. Chai, S.G. Ji, Y.T. Wu, D.S. Liang, Z.M. Xu, Biomed. Chromatogr. 12 (1998) 193. [17] S.D. Qi, S.Y. Cui, X.G. Chen, Z.D. Hu, J. Chromatogr. A 1059 (2004) 191. [18] Y.X. Gong, S.P. Li, Y.T. Wang, P. Li, F.Q. Yang, Electrophoresis 26 (2005) 1778. [19] D.Y. Pharr, Z.S. Fu, T.K. Smith, W.L. Hinze, Anal. Chem. 61 (1989) 275. [20] S.A.C. Wren, R.C. Rowe, J. Chromatogr. 603 (1992) 235. [21] M.H. Chen, W.H. Ding, J. Chromatogr. A 1033 (2004) 167. [22] W. Li, S.X. Gong, D.W. Wen, B.Q. Che, Y.P. Liao, H.W. Liu, X.F. Feng, S.L. Hu, J. Chromatogr. A 1049 (2004) 211. [23] K.L. Rundlett, D.W. Armstrong, J. Chromatogr. A 721 (1996) 173. [24] M.T. Bowser, D.D.Y. Chen, Anal. Chem. 70 (1998) 3261. [25] C. Yu, H.M. Lan, Y. Wang, Primary J. Zhongcaoyao 16 (2002) 8. [26] D.M. Lin, L. Li, H.S. Cheng, F.M. Li, J. Zhongcaoyao 18 (2005) 197.
Short communication
Determination of five anthraquinones in medicinal plants by capillary zone electrophoresis with -cyclodextrin addition Kan Tian a,b , Huige Zhang a,b , Xingguo Chen a,b,∗ , Zhide Hu a,b a
b
Department of Chemistry, Lanzhou University, Lanzhou 730000, China National Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China Received 8 February 2006; received in revised form 8 April 2006; accepted 11 April 2006 Available online 27 June 2006
Abstract A simple and rapid method for the simultaneous determination of five anthraquinone derivatives including aloe-emodin, emodin, chrysophanol, physcoin and rhein in Rheum species and Polygonum cuspidatum was established by capillary zone electrophoresis (CZE) using -cyclodextrin (CD) as modifier and urea to enhance its solubility. The apparent binding constants of these derivatives with -CD were evaluated. After an optimization study, the best conditions were selected using 35 mM phosphate buffer (pH 11.0) containing 20 mM -CD and 2 M urea, applied voltage 20 kV and detection at 254 nm. Under such conditions, all of the five anthraquinones were baseline-separated within a short analysis time of 12 min with symmetrical peaks and high theoretical plate numbers (189 000–314 000). The RSD values of the migration times and peak areas were 0.6–1.1, 1.3–1.9% (intra-day) and 0.6–1.5, 1.3–2.8% (inter-day, for a 5-day period), respectively. The limits of detection for the analytes (S/N = 3) were 0.33–0.62 g/ml. The recoveries were ranged from 93.37 to 107.69%. The proposed method was successfully applied to the determination of anthraquinones in ethanol extracts of two kinds of Rheum plants (R. palmatum and R. hotaoense) and P. cuspidatum. © 2006 Elsevier B.V. All rights reserved. Keywords: Capillary zone electrophoresis; -Cyclodextrin; Anthraquinones; Medicinal plants; Apparent binding constants
1. Introduction Rhubarb and Polygonum cuspidatum are two commonly used medicinal plants in oriental medicine. Rhubarb is also used in Europe and other places of the world [1,2]. The pharmaceutically relevant bioactive components in Rhubarb and P. cuspidatum are hydroxyanthraquinoids including physcion, emodin, rhein, aloeemodin and chrysophanol [3,4]. The structures and pKa values of these five anthraquinons are shown in Fig. 1. They are used or recommended as a laxative, antiphlogistic and hemostatic in the treatment of obstipation, gastrointestinal indigestion, diarrhea and jaundice [5,6]. Previous described methods for the determinations of these anthraquinones include TLC [7,8], HPLC [9] and capillary electrophoresis (CE) [10–16]. Qi et al. [17] reported the separation of anthraquinons in extract of Chinese herb Paedicalyx attopevensis Pierre ex Pitard. Up to now, to the best of our knowledge, there was only one report in the literature that achieved simulta∗
Corresponding author. Tel.: +86 931 8912763; fax: +86 931 8912582. E-mail address: [email protected] (X. Chen).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.04.021
neous determination of these five anthraquione derivatives using capillary zone electrophoresis (CZE) [18]. However, the analysis took as long as 40 min. Thus, it is desirable to develop a more simple and rapid method to separate these derivatives. The aim of this study was to develop an efficient and feasible CZE method to simultaneous analysis of these five anthraquinones in non-pretreated crude rhubarb and P. cuspidatum extracts. And also the apparent binding constants between anthraquinones and -CD were evaluated. 2. Experimental 2.1. Apparatus and procedures All separations were performed using a Waters Quanta 4000 capillary electrophoresis system (Milford, MA, USA), equipped with a UV detector. Data acquisition was carried out with a Maxima 820 chromatography workstation. The temperature was maintained at 23.5 ± 0.5 ◦ C with a forced-air cooling system. A fused-silica capillary of 60 cm (52.5 cm effective length) × 50 m I.D. (Yongnian Photoconductive Fiber Factory,
K. Tian et al. / J. Chromatogr. A 1123 (2006) 134–137
135
Fig. 1. The structures and pKa values of the studied anthraquinones.
Hebei Province, China) was used. Samples were introduced from anodic end of the capillary by hydrodynamic injection where the samples vial was raised by 10 cm for 5 s and the detection wavelength was operated at 254 nm. Acetonitrile was the marker of electroosmotic flow (EOF). A PHS-10A acidity meter (Xiaoshan Science Instrumentation Factory, Zhejiang, China) was used for the pH measurement. The pH was adjusted with 0.5 M NaOH or HCl. The measurements were run at least in triplicate for each different composition of the running buffer to ensure repeatability. Before their first use, capillaries were washed successively with 1 M NaOH for 5 min, water for 3 min, 0.1 M NaOH for 5 min and water for 3 min. The capillary rinsed between the runs as follows: 2 min with water, 3 min with 0.1 M NaOH, 2 min with water and 3 min with background electrolyte (BGE).
0.45 m membrane filter (Shanghai Xinya Purification Apparatus Factory, Shanghai, China) prior to use. A 0.10 g sample of P. cuspidatum powder (100 mesh) was immersed in 10 ml ethanol at room temperature for 12 h, after that, it was extracted for 20 min in an ultrasonic bath and followed by centrifugation at 1500 × g for 5 min. This procedure was repeated three times, then the extracts were combined, evaporated to near-dryness on a water bath and the residue was dissolved in 5 ml acetonitrile as the stock solution; a 0.05 g powder of R. palmatum and R. hotaoense were extracted, respectively, using the same procedure as mentioned above. Before injection into the capillary electrophoresis system, the solutions were appropriately diluted and passed through a 0.45 m filter.
2.2. Materials
The viscosity adjusted electrophoretic mobility was determined by multiplying the experimental mobility value by the ratio of the current obtained without -CD in the buffer over the current measured at the selector concentration of interest [20].
Raw rhubarb (R. palmatum and R. hotaoense) and P. cuspidatum were purchased from Z 1hongyou drugstore (Lanzhou, China). Standards of anthraquinone derivatives aloe-emodin, emodin, chrysophanol, physcoin and rhein were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). -CD was purchased from China Medicine Group, Shanghai Chemical Reagent Company (Shanghai, China). Urea, phosphate and borate were purchased from Tianjin Chemical Reagent Factory (Tianjin, China). All chemicals and solvents were of analytical reagent grade and were used without further purification. 2.3. Solutions and sample preparation The stock solutions of standards were prepared by dissolving precisely weighed anthraquinones in 5 ml acetonitrile and stored at −4 ◦ C. The running buffers were prepared daily by adding variable amounts of -CD to the phosphate/urea BGE to give final concentrations of -CD ranging from 0 to 35 mM. The reason for using urea was the limited solubility of -CD in water (16 mM) [19]. All solutions were filtered through a
2.4. Viscosity correction
3. Results and discussion 3.1. CZE separation with β-CD addition Preliminary experiments showed that the studied compounds could not be completely separated by ordinary CZE because the little difference in pKa values between chrysophanol and aloeemodin. Thus, -CD was added to the buffer to enhance the separation [21,22]. The effects on the separation by parameters such as -CD and urea concentration, background electrolyte pH and concentration, applied voltage, were evaluated. After an appropriate optimization, the best conditions were obtained with running electrolyte containing 35 mM phosphate, 20 mM -CD and 2 M urea at pH 11.0. Under the optimum conditions, all the five analytes were well separated within 12 min with symmetrical peaks. The numbers of theoretical plates per meter measured were in the range of 189 000–314 000, which were not reported in Ref. [18]. The typical electropherogram for a standard mix-
136
K. Tian et al. / J. Chromatogr. A 1123 (2006) 134–137
Fig. 2. The typical electropherograms of the standards mixture solution and the real samples under the optimum conditions: (A) the standards mixture; (B) R. hotaoense; (C) R. palmatum and (D) P. cuspidatum. The concentrations of the standards were 45 g/ml for chrysophanol, physcionand and emodin; 40 g/ml for aloe-emodin; 65 g/ml for rhein, respectively; Conditions: 35 mM phosphate containing 20 mM -CD and 2 M urea; pH 11.0; I = 49 A; uncoated fused-silica capillary, total length 60 cm (52.5 cm effective length) × 50 m I.D.; applied voltage, 20 kV; temperature, 23.5 ± 0.5 ◦ C; detection, 254 nm; the peaks were numbered according to the analyte notation in Fig. 1.
ture of five analytes is shown in Fig. 2A. The recorded current was lower than 50 A. 3.2. Evaluation of apparent binding constants For a better understanding of the interactions of anthraquinones with -CD, the apparent binding constants of anthraquinones to -CD were evaluated based on the dependence of the effective electrophoretic mobility of anthraquinones on the concentrations of -CD. The model that described a 1:1 guest–host interaction had been derived elsewhere [23,24]. The experiment was performed with buffers containing 35 mM phosphate (pH 11.0), 2 M urea and 0–35 mM -CD. Table 1 lists the apparent binding constants simultaneously determined by non-linear regression fitting method and three different linearization plots methods (named double reciprocal, x-reciprocal and y-reciprocal), and the results were adjusted by the viscosity correction factor. Comparing the binding constants of anthraquinone derivatives revealed that the substituent group(s) in the R position(s) of aromatic ring significantly affected the inclusion behaviors of these analytes. The sta-
bility of the inclusion complex of anthraquinones follows the order physcion > chrysophanol > aloe-emodin, emodin > rhein. Accordingly, it was the decreasing hydrophobicity of analytes introduced by different substituent group(s) in the R position(s) of analytes. The hydrophobic parameter of log POW values for these compounds were 3.85 for physcion, 3.63 for chrysophanol, 2.68 for aloe-emodin, 2.91 for emodin and 2.42 for rhein [11]. Though emodin and rhein substituted with polar groups in R position(s) of benzene ring, they displayed strong interaction with -CD. The reason for this maybe that hydroxyl and carboxylic acid group attached to the R position(s) of aromatic ring were involved in the host–guest complex formation enabling hydrogen bonds. In this study, the baseline resolution of chrysophanol and aloe-emodin was attributed to the fact that the interactions of chrysophanol with -CD were stronger than those with aloe-emodin and migrated quickly and resolved. 3.3. Method validation Under optimal conditions, the linear relationship between the concentration of the five anthraquinones 1–5 and the corresponding peak areas were investigated. The data of regression equations of curves, their correlation coefficients, linearity range and detection limits are shown in Table 2. The repeatability of the method was determined by measuring six replicate injection of the standard mixture solution at the level of 35 g/ml for each analytes, respectively. The RSD values of the migration times and peak areas were 0.6–1.1, 1.3–1.9% (intra-day), and 0.6–1.5, 1.3–2.8% (inter-day, for a 5-day period), respectively. Ensure accuracy of the method, the recoveries of the method were determined with the standard addition method for anthraquinones 1–5 in the extracts of R. palmatum, R. hotaoens and P. cuspidatum with results of 93.37, 98.19 and 95.98% for 1, 105.57, 104.29 and 95.46% for 2, 98.23, 107.31 and 104.23% for 3, 102.34, 105.47 and 104.83% for 4, 94.51, 98.48 and 107.69% for 5, respectively. These results were similar to that obtained by HPLC [25]. 3.4. Analysis of real samples Under the optimized conditions, two kinds of Rhubarb plants, R. palmatum and R. hotaoens, and P. cuspidatum were analyzed. The peaks were identified by spiking a small amount of each analyte into the sample solutions, and also by comparing their
Table 1 The apparent binding constants (at 23.5 ◦ C) obtained by the different calculation methods with the viscosity correction Compound
Apparent binding constants (×102 M−1 )a Non-linear fitting (r2 )
1 2 3 4 5
1.70 1.07 0.78 1.38 0.74 a
Error 1.96σ.
± ± ± ± ±
0.08 (0.9968) 0.05 (0.9974) 0.05 (0.9960) 0.05 (0.9980) 0.05 (0.9954)
Double reciprocal (r2 ) 1.69 1.04 0.71 1.34 0.71
± ± ± ± ±
0.05 (0.9982) 0.03 (0.9987) 0.03 (0.9978) 0.03 (0.9989) 0.03 (0.9982)
y-reciprocal (r2 ) 1.70 1.04 0.77 1.39 0.73
± ± ± ± ±
0.08 (0.9995) 0.05 (0.9991) 0.05 (0.9974) 0.06 (0.9995) 0.05 (0.9968)
x-reciprocal (r2 ) 1.69 1.05 0.73 1.36 0.72
± ± ± ± ±
0.06 (0.9937) 0.04 (0.9928) 0.04 (0.9834) 0.04 (0.9956) 0.04 (0.9850)
K. Tian et al. / J. Chromatogr. A 1123 (2006) 134–137
137
Table 2 The results of regression analysis on calibration curves and the detection limits Compound
Calibration curves y = a + bxa
Correlation coefficient
Linear range (g/ml)
Detection limit (g/ml)b
1 2 3 4 5
y = −185.6 + 109.7x y = −38.4 + 141.2x y = 144.9 + 88.7x y = −35.3 + 75.7x y = 71.4 + 132.2x
0.9995 0.9998 0.9986 0.9988 0.9992
1.41–90 1.41–90 1.25–120 0.71–135 1.56–87
0.42 0.33 0.52 0.62 0.36
a b
y and x stand for the peak area and the concentration (g/ml)of the analytes, respectively. The detection limits corresponding to concentrations giving signal-to-noise ratio of 3.
Table 3 Contents of the five anthraquinones in real samples and RSD (n = 5) Sample 1 hotaoensa
R. R. palmatum P. cuspidatum a b c
2 (0.62)b
0.20 0.41 (0.81) 0.57 (0.79)
0.71 (2.18) 0.76 (1.13) –c
3
4
5
0.089 (1.37) 0.13 (1.54) –
0.33 (1.74) 1.32 (2.21) 1.46 (1.53)
0.086 (2.63) 0.072 (3.04) –
Percentage of dry mass. The data in parentheses refer to the RSD. Not found.
migration times with the migration time of the standards. The typical electropherograms are shown in Fig. 2B–D. The contents of the five anthraquinones together with RSDs (n = 5) are listed in Table 3. The reason that the content of rhein in R. palmatum was low compared to that obtained by HPLC probably due to the different collection time or cultivation region of the plants [26]. 4. Conclusions In this study, the effective separation of five anthraquinones in Rhubarb species and P. cuspidatum were achieved by CD-CZE. The binding constants were evaluated by non-linear regression and three different linear plots methods. The developed method, for their rapidity, simple and the high selectivity, was very suitable for the quality control of medicinal plants containing the studied anthraquinones. Acknowledgement The authors are grateful for the financial support provided by the Natural Science Foundation of China (grant no. 20275014). References [1] British Pharmacopoeia, 17th ed., Stationary Office, Department of Health, London, 1999, p. 1251. [2] European Pharmacopoeia, 3rd ed., European Department for the Quality of Medicines, Council of Europe, Strasbourg, 1997, p. 1441.
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