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Determination of sinomenine in Sinomenium acutum by capillary electrophoresis with electrochemiluminescence detection
Analytica Chimica Acta 587 (2007) 104–109
Determination of sinomenine in Sinomenium acutum by capillary electrophoresis with electrochemiluminescence detection Min Zhou, Yong-Jun Ma, Xiao-Na Ren, Xiu-Ying Zhou, Li Li, Hui Chen ∗ Gansu Key Laboratory of Polymer Materials, College of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou 730070, China Received 23 October 2006; received in revised form 21 December 2006; accepted 10 January 2007 Available online 16 January 2007
Abstract A Ru(bpy)3 2+ -based electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE) has been established for the determination of sinomenine for the first time. Optimum separation was achieved with a fused-silica capillary column (50 cm × 25 m i.d.) and a background electrolyte of 50 mM sodium phosphate (pH 5.0) at a separation voltage of 15 kV. The content of sinomenine was detected by ECL at the detection voltage of 1.15 V (versus Ag/AgCl) with 5 mM Ru(bpy)3 2+ in 75 mM phosphate solution (pH 8.0) when a chemically modified platinum electrode by europium(III)-doped prussian blue analogue (Eu-PB) was used as a working electrode. Under the optimized conditions, the ECL intensity was in proportion to sinomenine concentration in the range from 0.01 to 1.0 g mL−1 with a detection limit of 2.0 ng mL−1 (3σ). The relative standard derivations of migration time and ECL intensity were 0.93 and 1.11%, respectively. The level of sinomenine in Sinomenium acutum Rehd. et Wils was easily determined with recoveries between 98.6 and 102.7%. © 2007 Elsevier B.V. All rights reserved. Keywords: Electrochemiluminescence; Capillary electrophoresis; Sinomenine
1. Introduction Sinomenine (7,8-didehydro-4-hydroxyl-3,7-dimethoxy-17methylmorphinan-6-one; SIN) is a principal alkaloid isolated from the stem and root of Chinese medical plant Sinomenium acutum Rehd. et Wils, and its chemical structure is shown in Fig. 1. Due to its analgesic and anti-inflammatory effects, sinomenine has been utilized clinically to treat rheumatoid arthritis and neuralgia [1–3]. At present, several chromatographic methods including high-performance liquid chromatography (HPLC) [4–7] and thin-layer chromatography (TLC) [8,9] have been reported for the analysis of sinomenine. However, a drawback of the methods mentioned above appears to be time-consuming due to necessary extraction, concentration and/or derivatization prior to the analysis although the high sensitivity and the good selectivity have been obtained in such procedures. Therefore, it is necessary to establish rapid and effective methods for the quantitation of sinomenine.
∗
Corresponding author. Tel.: +86 931 7669904. E-mail address: [email protected] (H. Chen).
0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2007.01.018
Capillary electrophoresis (CE) is now a widely used separation technique for analysis of alkaloids with various pharmaceutical applications because of its high efficiency, resolution potential, short analysis time and minimal sample volume [10–12]. Recently, an easy, rapid nonaqueous capillary electrophoresis method has been developed for the determination of sinomenine with a UV detector [13]. In addition, Zhai et al. [14] has also proposed another CE method for sinomenine determination by use of high frequency conductivity detector with a detection limit of 0.2 g mL−1 . In recent years, there are increasing interests in coupling CE separation with high-sensitive chemiluminescence (CL) detection for alkaloids analysis [15–18]. Especially, electrochemiluminescence (ECL) detection involving tris(2,2 bipyridyl) ruthenium(II) (Ru(bpy)3 2+ ) offers other merits with wide linear range, no derivatization and good selectivity for nitrogen-containing compounds [19,20]. Therefore, analytical procedures combining CE separation with Ru(bpy)3 2+ -based ECL detection have been paid more attention to the detection of some alkaloids [21–24]. However, as far as we know, such CE-ECL procedure has not been reported for the determination of sinomenine.
M. Zhou et al. / Analytica Chimica Acta 587 (2007) 104–109
105
at 0–4 ◦ C and filtered through a membrane of 0.45 m prior to use. 2.2. Apparatus
Fig. 1. The structure of sinomenine.
In this paper, a CE-ECL method based on Ru(bpy)3 2+ system has been developed for the determination of sinomenine in Chinese herb S. acutum Rehd. et Wils. It is worth mentioning that a europium(III)-doped prussian blue analogue (Eu-PB) film was modified chemically on the surface of a microdisk platinum working electrode to avoid the possible electrode fouling as well as to improve the detection sensitivity. 2. Experimental 2.1. Reagents and chemicals All chemicals and reagents were of analytical grade except for specific statements and used without further purification. Tris(2,2 -bipyridyl) ruthenium(II) chloride hexahydrate (98%) was obtained from Aldrich (Milwaukee, WI, USA) and prepared with doubly deionized water. Sinomenine was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) and freshly prepared with 50% methanol (Spectrum analytical grade) just before use. Dried Chinese herb S. acutum Rehd. et Wils was taken from Lanzhou Huanghe Chinese Herbs wholesale Ltd. Corp. (Lanzhou, China). Sodium phosphate (pH 5.0, G.R.) was used as the background electrolyte solution. All solutions were stored in the refrigerator
A MPI-A system (Xi’an Remax Electric Ltd. Corp., China) was employed for CE-ECL detection. The schematic diagram of the CE-ECL detection system is shown in Fig. 2. Capillary electrophoresis was performed using a 50 cm length of uncoated fused-silica capillary (25 m i.d., Yongnian Optical Fiber Factory, Hebei, China) at 15 kV with a background electrolyte of 50 mM sodium phosphate (pH 5.0). Samples were introduced from the anodic end of the capillary by electrokinetic injection for 10 s at 10 kV. The end-column ECL detection was installed with a three-electrode configuration, which was made up of a 500 m Eu-PB modifying platinum disk as a working electrode, an Ag/AgCl as a reference electrode and a platinum wire as an auxiliary electrode. The capillary-to-working electrode distance was adjusted to about 150 m. A solution of 5 mM Ru(bpy)3 2+ in 75 mM phosphate buffer (pH 8.0) was directly injected into the detection reservoir [25]. ECL emission was measured using a multichannel data collection analyzer, in which a sensitive photomultipier tube (PMT) was operated at 800 V. Prior to experiments every day, the capillary was rinsed with 0.1 M NaOH for 3 min at first, then with doubly deionized water for 3 min and finally equilibrated with the background electrolyte for 5 min. Modification of the working electrode was performed by CHI832 electrochemical analyzer (Shanghai Chenhua Apparatus Corporation, China). A solution of 10.0 mL FeCl3 , 10.0 mL K3 Fe(CN)6 , 6.5 mL HCl, 5.0 mL EuCl3 and 5.0 mL potassium hydrogen phthalate (all concentration were 0.01 M) was directly added into the electrochemical cell, the Eu-PB film was gradually electrodeposited with twenty 50 mV s−1 potential cycles between 0 and 1.4 V (versus SCE reference electrode). Then the modified electrode was immerged into the saturated KCl solution and was scanned with a rate of 50 mV s−1 for 20 potential cycles from 0 to 1.3 V. Finally, the prepared electrode was subjected in 75 mM phosphate buffer (pH 8.0) to repeat cycling
Fig. 2. Schematic diagram of the CE-ECL detection system.
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M. Zhou et al. / Analytica Chimica Acta 587 (2007) 104–109
under the same conditions until a reproducible voltammogram was obtained.
conditions of background electrolyte, as well as the separation parameters including separation voltage and injection parameters were optimized in this work.
2.3. Sample preparation The powder of root (3.0 mg) of S. acutum Rehd. et Wils was extracted with 5 mL methanol for 30 min in an ultrasonic bath. Extraction was repeated twice. The extracts were combined and diluted with methanol to 10 mL. Then 1 mL extracted solution was mixed with 4 mL methanol and diluted with doubly deionized water to 10 mL, following by passing through a 0.45 m membrane and being directly injected into the capillary electrophoresis system and analyzed. 3. Results and discussions 3.1. Effect of the platinum electrode modified with Eu-PB film In comparison with the response to the oxidation of Ru(bpy)3 2+ on a bare platinum electrode, the Eu-PB modifying platinum electrode exhibited higher current response, with slight negative shift ca. 20 mV for the direct oxidation peak of Ru(bpy)3 2+ (see Fig. 3A). Consequently, an enhanced ECL peak of Ru(bpy)3 2+ was obtained, as shown in Fig. 3B. Thus, the prepared electrode would benefit from the improved sensitivity and give less interfering signals from other electroactive substances in real samples. In addition, the prepared electrode was stable enough for repetitive use in the detection system within 2 weeks with no need for electrode replacement. 3.2. Optimization of system The most important variables in ECL such as the concentration of Ru(bpy)3 2+ solution, the applied potential and the
3.2.1. Effect of Ru(bpy)3 2+ concentration Ru(bpy)3 2+ was used as the ECL reagent in the system and its concentration has great effect on the ECL signal. The results showed that the ECL intensity increased markedly with increasing Ru(bpy)3 2+ concentration from 0.2 to 5.0 mM. In this work, 5 mM Ru(bpy)3 2+ in 75 mM phosphate buffer (pH 8.0) was adopted due to concerned over sensitivity and economy in use of reagent. 3.2.2. Effect of detection potential Detection potential has great effect on the ECL intensity. The result in Fig. 4 showed that the increased production of Ru(bpy)3 3+ with a rise of potential led to an increased response and the ECL intensity reached a stable maximum between 1.15 and 1.2 V. Above which, the ECL response diminished as competitive reactions involving the background electrolyte dominate. Thus, the optimal potential was 1.15 V. 3.2.3. Choice of background electrolyte Acetate, Tris–HCl, phosphate and borate buffers were tested as the optional background electrolyte and the order of the ECL intensity in different solutions was: phosphate ≥ acetate > borate > Tris–HCl. Finally, phosphate was chosen in terms of the highest ECL and the best signal to noise ratio (S/N). 3.2.4. Effect of pH of background electrolyte The pH effect of phosphate on electrophoresis separation and ECL intensity was investigated in a wide pH range of 3.5–9.5 in 0.5 pH units. As illustrated in Fig. 5, the highest ECL intensity
Fig. 3. (A) Differential pulse voltammograms of Ru(bpy)3 2+ in phosphate buffer (pH 8.0): (a) at bare Pt electrode; (b) at Eu-PB modifying Pt electrode. Amplitude, 0.05 V; pulse width, 0.05 s; pulse period, 0.2 s. Concentrations: Ru(bpy)3 2+ , 5 mM; phosphate, 75 mM. (B) ECL emission of Ru(bpy)3 2+ in phosphate buffer (pH 8.0): (a) at bare Pt electrode; (b) at Eu-PB modifying Pt electrode. Concentrations: Ru(bpy)3 2+ , 5 mM; phosphate, 75 mM.
M. Zhou et al. / Analytica Chimica Acta 587 (2007) 104–109
Fig. 4. Effect of detection potential on ECL intensity. Separation capillary, 25 m i.d., 50 cm length; sample injection, 10 s at 10 kV; separation voltage, 15 kV; background electrolyte, 50 mM sodium phosphate (pH 5.0), phosphate in the detection cell, 75 mM at pH 8.0.
was observed at pH 5.0. As a result, pH 5.0 was selected for all the following experiments. 3.2.5. Effect of background electrolyte concentration Fixed pH value at 5.0, the concentration of phosphate was adjusted from 5 to 80 mM. It is found in Fig. 6 that working at high phosphate concentration allowed improving the sensitivity and resolution until 50 mM. Above which, both the ECL intensity and resolution decreased because of excessive heating caused by Joule effect, following an increased background signal and resulting in an unstable measurement. Hence, 50 mM phosphate (pH 5.0) was preferred as background electrolyte. 3.2.6. Effect of separation voltage and injection parameters In general, ECL intensity increased with an increase in separation voltage, injection voltage or injection time. However, both the repeatability and the resolution became worse when an excessive voltage or sample volume was introduced. So as a compromise of the high ECL intensity and the improved col-
107
Fig. 6. Effect of background electrolyte concentration. Other conditions are the same as in Fig. 4.
umn efficiency, the separation voltage of 15 kV and the injection parameters of 10 s at 10 kV were recommended. 3.3. Choice of extracting solvent The extracting solvent was chosen from ethanol and methanol, and methanol was found to give a better resolution and a higher ECL intensity. Therefore, methanol was selected for the extraction of sinomenine in herbs in order to obtain a higher extraction yield. Besides, the final concentration of methanol solution for sample injection was optimized in the range 10–100% when certain amount of herbs was extracted. The results indicated that the ECL intensity reached maximum when methanol in samples was 50–75%, and methanol solution of 50% was adopted due to concerns over greater precision and better resolution. 3.4. Calibration and detection Under the optimum conditions, the calibration graph of sinomenine concentration versus ECL intensity was linear in the range from 0.01 to 1.0 g mL−1 . The regression equation could be expressed as: I = 486.59 + 790.28 C g mL−1 with a correlation coefficient of 0.9993 (n = 5). The detection limit, defined as three times the S.D. for the reagent blank signal, was 2.0 ng mL−1 , which was equal with or lower than that obtained by other methods mentioned above [4–9,13,14]. The precision of the proposed method was determined by reduplicate injections (n = 6) of 0.1 g mL−1 sinomenine standard solution. The relative standard deviations (R.S.D.) of migration time and ECL intensity were 0.93 and 1.11%, respectively. 3.5. Applications
Fig. 5. Effect of background electrolyte pH on ECL intensity. Detection potential, 1.15 V; other conditions are the same as in Fig. 4.
To examine the application for practical analysis, the CEECL method was applied to the determination of sinomenine in Chinese herb S. acutum Rehd. et Wils. The typical electropherogram is shown in Fig. 7. The peaks were identified by
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M. Zhou et al. / Analytica Chimica Acta 587 (2007) 104–109 Table 2 Comparison of the results obtained by the present method with others Method
Linear range (g ml−1 )
LOD (g ml−1 )
SIN in Sinomenium acutum
Reference
The present method HPLC-UV
0.01–1.0
2.0 × 10−3
0.82%
–
–
[7]
CE-UV
6.25–500
7.47 mg g−1 (0.747%) 0.80 mg g−1 (0.08%) 0.70%
CE-high frequency conductivity
2.6–106
1.0–36.0
1.71 0.2
[13] [14]
system, and the relevant mechanism is regarded as a typical redox type [28]. Although both the current response and ECL intensity were enhanced by use of the Eu-PB modifying platinum electrode, no convincing evidence has been observed to indicate the existence of a new ECL mechanism till now. Thus, the production of light emission of the Ru(bpy)3 2+ /sinomenine system is considered to be similar to the pathway of the TPA/Ru(bpy)3 2+ system at a platinum electrode. Therefore, the possible mechanism can be expressed as follows: Ru(bpy)3 2+ − e− → Ru(bpy)3 3+ SINH+ − e− → SIN• Ru(bpy)3 3+ + SIN• → [Ru(bpy)3 2+ ]∗ + products Fig. 7. Electropherograms of (a) the sample solution; (b) the sample solution spiked with 0.4 g mL−1 sinomenine standard solution; other conditions are the same as in Fig. 4.
comparison the migration times and by spiking the standards to the sample solution. In the measurement process, a series of small peaks were always detected, which was estimated to be the interference produced by flavones or glucides in the herb plant. According to the results that listed in Table 1, the content of sinomenine found in the herb was 0.82%, which corresponded with other methods for the analysis of sinomenine in S. acutum, as indicated in Table 2.
[Ru(bpy)3 2+ ]∗ → Ru(bpy)3 2+ + hν
(λ = 620 nm)
4. Conclusion A Ru(bpy)3 2+ -based CE-ECL method was studied for identification and determination of sinomenine for the first time. The developed method was found not only a good alternative for the rapid determination of sinomenine in plant extracts with good selectivity, wide linearity and reliable stability, but also an efficient supplementary technique for the preliminary investigation of other quinolizidine alkaloids in Chinese traditional herbs.
3.6. Mechanism The ECL behavior based on Ru(bpy)3 2+ system is usually related to the structure of the co-reactant. A general trend is that nitrogen-containing compounds especially tertiary amines lead to a much higher ECL intensity than others [26,27]. A typical example is the ECL in the tripropyl amine (TPA)/Ru(bpy)3 2+ Table 1 Results for the determination of sonomenine in Sinomenium acutum Present method (g ml−1 )
R.S.D (%) (n = 5)
Added (g)
Found (g ml−1 )
Recovery (%) (n = 5)
0.246
1.26
0.200 0.400
0.458 0.637
102.7 98.6
Acknowledgements We are grateful to Natural Scientific Foundation of Gansu Province, China, for supporting the research with Project 3ZS051–A25–097, and more thanks to Scientific Research Foundation of Gansu Ministry of Education, China, for the partial financial aid with Project 0501–07. References [1] J.H. Liu, W.D. Li, H.L. Teng, Acta Pharm. Sin. 40 (2005) 127. [2] Q. Liu, L.L. Zhou, R. Li, Chin. Tradit. Herb. Drugs 28 (1997) 247. [3] L. Liu, E. Buchner, D. Beitze, C.B. Schmidt-Weber, V. Kaever, F. Emmrich, R.W. Kinne, Int. J. Immunopharmacol. 18 (1996) 529.
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[16] Z. Gong, Y. Zhang, H. Zhang, J. Cheng, J. Chromatogr. A 855 (1999) 329. [17] H.L. Jiang, Y.Z. He, H.Z. Zhao, Y.Y. Hu, Anal. Chim. Acta 512 (2004) 111. [18] N.W. Barnett, B.J. Hindson, S.W. Lewis, Analyst 125 (2000) 91. [19] R.D. Gerardi, N.W. Barnett, S.W. Lewis, Anal. Chim. Acta 378 (1999) 1. [20] Y. Zhuang, D. Zhang, H. Ju, Analyst 130 (2005) (Advance article). [21] X. Yin, E. Wang, Anal. Chim. Acta 533 (2005) 113. [22] J. Yan, J. Liu, W. Cao, X. Sun, X. Yang, E. Wang, Microchem. J. 76 (2004) 11. [23] K. Tsukagoshi, N. Okuzono, R. Nakajima, J. Chromatogr. A 958 (2002) 283. [24] Y. Gao, Y. Tian, E. Wang, Anal. Chim. Acta 545 (2005) 137. [25] W. Cao, J. Liu, X. Yang, E. Kang, Electrophoresis 23 (2002) 3683. [26] A.D. Becke, J. Chem. Phys. 98 (1993) 5648. [27] X. Chen, C.Q. Yi, M.J. Li, Z. Li, X.R. Wang, Acta Chim. Sin. 60 (2002) 1662. [28] K.A. F¨ahnrich, M. Pravda, G.G. Guilbault, Talanta 54 (2001) 531.
Determination of sinomenine in Sinomenium acutum by capillary electrophoresis with electrochemiluminescence detection Min Zhou, Yong-Jun Ma, Xiao-Na Ren, Xiu-Ying Zhou, Li Li, Hui Chen ∗ Gansu Key Laboratory of Polymer Materials, College of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou 730070, China Received 23 October 2006; received in revised form 21 December 2006; accepted 10 January 2007 Available online 16 January 2007
Abstract A Ru(bpy)3 2+ -based electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE) has been established for the determination of sinomenine for the first time. Optimum separation was achieved with a fused-silica capillary column (50 cm × 25 m i.d.) and a background electrolyte of 50 mM sodium phosphate (pH 5.0) at a separation voltage of 15 kV. The content of sinomenine was detected by ECL at the detection voltage of 1.15 V (versus Ag/AgCl) with 5 mM Ru(bpy)3 2+ in 75 mM phosphate solution (pH 8.0) when a chemically modified platinum electrode by europium(III)-doped prussian blue analogue (Eu-PB) was used as a working electrode. Under the optimized conditions, the ECL intensity was in proportion to sinomenine concentration in the range from 0.01 to 1.0 g mL−1 with a detection limit of 2.0 ng mL−1 (3σ). The relative standard derivations of migration time and ECL intensity were 0.93 and 1.11%, respectively. The level of sinomenine in Sinomenium acutum Rehd. et Wils was easily determined with recoveries between 98.6 and 102.7%. © 2007 Elsevier B.V. All rights reserved. Keywords: Electrochemiluminescence; Capillary electrophoresis; Sinomenine
1. Introduction Sinomenine (7,8-didehydro-4-hydroxyl-3,7-dimethoxy-17methylmorphinan-6-one; SIN) is a principal alkaloid isolated from the stem and root of Chinese medical plant Sinomenium acutum Rehd. et Wils, and its chemical structure is shown in Fig. 1. Due to its analgesic and anti-inflammatory effects, sinomenine has been utilized clinically to treat rheumatoid arthritis and neuralgia [1–3]. At present, several chromatographic methods including high-performance liquid chromatography (HPLC) [4–7] and thin-layer chromatography (TLC) [8,9] have been reported for the analysis of sinomenine. However, a drawback of the methods mentioned above appears to be time-consuming due to necessary extraction, concentration and/or derivatization prior to the analysis although the high sensitivity and the good selectivity have been obtained in such procedures. Therefore, it is necessary to establish rapid and effective methods for the quantitation of sinomenine.
∗
Corresponding author. Tel.: +86 931 7669904. E-mail address: [email protected] (H. Chen).
0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2007.01.018
Capillary electrophoresis (CE) is now a widely used separation technique for analysis of alkaloids with various pharmaceutical applications because of its high efficiency, resolution potential, short analysis time and minimal sample volume [10–12]. Recently, an easy, rapid nonaqueous capillary electrophoresis method has been developed for the determination of sinomenine with a UV detector [13]. In addition, Zhai et al. [14] has also proposed another CE method for sinomenine determination by use of high frequency conductivity detector with a detection limit of 0.2 g mL−1 . In recent years, there are increasing interests in coupling CE separation with high-sensitive chemiluminescence (CL) detection for alkaloids analysis [15–18]. Especially, electrochemiluminescence (ECL) detection involving tris(2,2 bipyridyl) ruthenium(II) (Ru(bpy)3 2+ ) offers other merits with wide linear range, no derivatization and good selectivity for nitrogen-containing compounds [19,20]. Therefore, analytical procedures combining CE separation with Ru(bpy)3 2+ -based ECL detection have been paid more attention to the detection of some alkaloids [21–24]. However, as far as we know, such CE-ECL procedure has not been reported for the determination of sinomenine.
M. Zhou et al. / Analytica Chimica Acta 587 (2007) 104–109
105
at 0–4 ◦ C and filtered through a membrane of 0.45 m prior to use. 2.2. Apparatus
Fig. 1. The structure of sinomenine.
In this paper, a CE-ECL method based on Ru(bpy)3 2+ system has been developed for the determination of sinomenine in Chinese herb S. acutum Rehd. et Wils. It is worth mentioning that a europium(III)-doped prussian blue analogue (Eu-PB) film was modified chemically on the surface of a microdisk platinum working electrode to avoid the possible electrode fouling as well as to improve the detection sensitivity. 2. Experimental 2.1. Reagents and chemicals All chemicals and reagents were of analytical grade except for specific statements and used without further purification. Tris(2,2 -bipyridyl) ruthenium(II) chloride hexahydrate (98%) was obtained from Aldrich (Milwaukee, WI, USA) and prepared with doubly deionized water. Sinomenine was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) and freshly prepared with 50% methanol (Spectrum analytical grade) just before use. Dried Chinese herb S. acutum Rehd. et Wils was taken from Lanzhou Huanghe Chinese Herbs wholesale Ltd. Corp. (Lanzhou, China). Sodium phosphate (pH 5.0, G.R.) was used as the background electrolyte solution. All solutions were stored in the refrigerator
A MPI-A system (Xi’an Remax Electric Ltd. Corp., China) was employed for CE-ECL detection. The schematic diagram of the CE-ECL detection system is shown in Fig. 2. Capillary electrophoresis was performed using a 50 cm length of uncoated fused-silica capillary (25 m i.d., Yongnian Optical Fiber Factory, Hebei, China) at 15 kV with a background electrolyte of 50 mM sodium phosphate (pH 5.0). Samples were introduced from the anodic end of the capillary by electrokinetic injection for 10 s at 10 kV. The end-column ECL detection was installed with a three-electrode configuration, which was made up of a 500 m Eu-PB modifying platinum disk as a working electrode, an Ag/AgCl as a reference electrode and a platinum wire as an auxiliary electrode. The capillary-to-working electrode distance was adjusted to about 150 m. A solution of 5 mM Ru(bpy)3 2+ in 75 mM phosphate buffer (pH 8.0) was directly injected into the detection reservoir [25]. ECL emission was measured using a multichannel data collection analyzer, in which a sensitive photomultipier tube (PMT) was operated at 800 V. Prior to experiments every day, the capillary was rinsed with 0.1 M NaOH for 3 min at first, then with doubly deionized water for 3 min and finally equilibrated with the background electrolyte for 5 min. Modification of the working electrode was performed by CHI832 electrochemical analyzer (Shanghai Chenhua Apparatus Corporation, China). A solution of 10.0 mL FeCl3 , 10.0 mL K3 Fe(CN)6 , 6.5 mL HCl, 5.0 mL EuCl3 and 5.0 mL potassium hydrogen phthalate (all concentration were 0.01 M) was directly added into the electrochemical cell, the Eu-PB film was gradually electrodeposited with twenty 50 mV s−1 potential cycles between 0 and 1.4 V (versus SCE reference electrode). Then the modified electrode was immerged into the saturated KCl solution and was scanned with a rate of 50 mV s−1 for 20 potential cycles from 0 to 1.3 V. Finally, the prepared electrode was subjected in 75 mM phosphate buffer (pH 8.0) to repeat cycling
Fig. 2. Schematic diagram of the CE-ECL detection system.
106
M. Zhou et al. / Analytica Chimica Acta 587 (2007) 104–109
under the same conditions until a reproducible voltammogram was obtained.
conditions of background electrolyte, as well as the separation parameters including separation voltage and injection parameters were optimized in this work.
2.3. Sample preparation The powder of root (3.0 mg) of S. acutum Rehd. et Wils was extracted with 5 mL methanol for 30 min in an ultrasonic bath. Extraction was repeated twice. The extracts were combined and diluted with methanol to 10 mL. Then 1 mL extracted solution was mixed with 4 mL methanol and diluted with doubly deionized water to 10 mL, following by passing through a 0.45 m membrane and being directly injected into the capillary electrophoresis system and analyzed. 3. Results and discussions 3.1. Effect of the platinum electrode modified with Eu-PB film In comparison with the response to the oxidation of Ru(bpy)3 2+ on a bare platinum electrode, the Eu-PB modifying platinum electrode exhibited higher current response, with slight negative shift ca. 20 mV for the direct oxidation peak of Ru(bpy)3 2+ (see Fig. 3A). Consequently, an enhanced ECL peak of Ru(bpy)3 2+ was obtained, as shown in Fig. 3B. Thus, the prepared electrode would benefit from the improved sensitivity and give less interfering signals from other electroactive substances in real samples. In addition, the prepared electrode was stable enough for repetitive use in the detection system within 2 weeks with no need for electrode replacement. 3.2. Optimization of system The most important variables in ECL such as the concentration of Ru(bpy)3 2+ solution, the applied potential and the
3.2.1. Effect of Ru(bpy)3 2+ concentration Ru(bpy)3 2+ was used as the ECL reagent in the system and its concentration has great effect on the ECL signal. The results showed that the ECL intensity increased markedly with increasing Ru(bpy)3 2+ concentration from 0.2 to 5.0 mM. In this work, 5 mM Ru(bpy)3 2+ in 75 mM phosphate buffer (pH 8.0) was adopted due to concerned over sensitivity and economy in use of reagent. 3.2.2. Effect of detection potential Detection potential has great effect on the ECL intensity. The result in Fig. 4 showed that the increased production of Ru(bpy)3 3+ with a rise of potential led to an increased response and the ECL intensity reached a stable maximum between 1.15 and 1.2 V. Above which, the ECL response diminished as competitive reactions involving the background electrolyte dominate. Thus, the optimal potential was 1.15 V. 3.2.3. Choice of background electrolyte Acetate, Tris–HCl, phosphate and borate buffers were tested as the optional background electrolyte and the order of the ECL intensity in different solutions was: phosphate ≥ acetate > borate > Tris–HCl. Finally, phosphate was chosen in terms of the highest ECL and the best signal to noise ratio (S/N). 3.2.4. Effect of pH of background electrolyte The pH effect of phosphate on electrophoresis separation and ECL intensity was investigated in a wide pH range of 3.5–9.5 in 0.5 pH units. As illustrated in Fig. 5, the highest ECL intensity
Fig. 3. (A) Differential pulse voltammograms of Ru(bpy)3 2+ in phosphate buffer (pH 8.0): (a) at bare Pt electrode; (b) at Eu-PB modifying Pt electrode. Amplitude, 0.05 V; pulse width, 0.05 s; pulse period, 0.2 s. Concentrations: Ru(bpy)3 2+ , 5 mM; phosphate, 75 mM. (B) ECL emission of Ru(bpy)3 2+ in phosphate buffer (pH 8.0): (a) at bare Pt electrode; (b) at Eu-PB modifying Pt electrode. Concentrations: Ru(bpy)3 2+ , 5 mM; phosphate, 75 mM.
M. Zhou et al. / Analytica Chimica Acta 587 (2007) 104–109
Fig. 4. Effect of detection potential on ECL intensity. Separation capillary, 25 m i.d., 50 cm length; sample injection, 10 s at 10 kV; separation voltage, 15 kV; background electrolyte, 50 mM sodium phosphate (pH 5.0), phosphate in the detection cell, 75 mM at pH 8.0.
was observed at pH 5.0. As a result, pH 5.0 was selected for all the following experiments. 3.2.5. Effect of background electrolyte concentration Fixed pH value at 5.0, the concentration of phosphate was adjusted from 5 to 80 mM. It is found in Fig. 6 that working at high phosphate concentration allowed improving the sensitivity and resolution until 50 mM. Above which, both the ECL intensity and resolution decreased because of excessive heating caused by Joule effect, following an increased background signal and resulting in an unstable measurement. Hence, 50 mM phosphate (pH 5.0) was preferred as background electrolyte. 3.2.6. Effect of separation voltage and injection parameters In general, ECL intensity increased with an increase in separation voltage, injection voltage or injection time. However, both the repeatability and the resolution became worse when an excessive voltage or sample volume was introduced. So as a compromise of the high ECL intensity and the improved col-
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Fig. 6. Effect of background electrolyte concentration. Other conditions are the same as in Fig. 4.
umn efficiency, the separation voltage of 15 kV and the injection parameters of 10 s at 10 kV were recommended. 3.3. Choice of extracting solvent The extracting solvent was chosen from ethanol and methanol, and methanol was found to give a better resolution and a higher ECL intensity. Therefore, methanol was selected for the extraction of sinomenine in herbs in order to obtain a higher extraction yield. Besides, the final concentration of methanol solution for sample injection was optimized in the range 10–100% when certain amount of herbs was extracted. The results indicated that the ECL intensity reached maximum when methanol in samples was 50–75%, and methanol solution of 50% was adopted due to concerns over greater precision and better resolution. 3.4. Calibration and detection Under the optimum conditions, the calibration graph of sinomenine concentration versus ECL intensity was linear in the range from 0.01 to 1.0 g mL−1 . The regression equation could be expressed as: I = 486.59 + 790.28 C g mL−1 with a correlation coefficient of 0.9993 (n = 5). The detection limit, defined as three times the S.D. for the reagent blank signal, was 2.0 ng mL−1 , which was equal with or lower than that obtained by other methods mentioned above [4–9,13,14]. The precision of the proposed method was determined by reduplicate injections (n = 6) of 0.1 g mL−1 sinomenine standard solution. The relative standard deviations (R.S.D.) of migration time and ECL intensity were 0.93 and 1.11%, respectively. 3.5. Applications
Fig. 5. Effect of background electrolyte pH on ECL intensity. Detection potential, 1.15 V; other conditions are the same as in Fig. 4.
To examine the application for practical analysis, the CEECL method was applied to the determination of sinomenine in Chinese herb S. acutum Rehd. et Wils. The typical electropherogram is shown in Fig. 7. The peaks were identified by
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M. Zhou et al. / Analytica Chimica Acta 587 (2007) 104–109 Table 2 Comparison of the results obtained by the present method with others Method
Linear range (g ml−1 )
LOD (g ml−1 )
SIN in Sinomenium acutum
Reference
The present method HPLC-UV
0.01–1.0
2.0 × 10−3
0.82%
–
–
[7]
CE-UV
6.25–500
7.47 mg g−1 (0.747%) 0.80 mg g−1 (0.08%) 0.70%
CE-high frequency conductivity
2.6–106
1.0–36.0
1.71 0.2
[13] [14]
system, and the relevant mechanism is regarded as a typical redox type [28]. Although both the current response and ECL intensity were enhanced by use of the Eu-PB modifying platinum electrode, no convincing evidence has been observed to indicate the existence of a new ECL mechanism till now. Thus, the production of light emission of the Ru(bpy)3 2+ /sinomenine system is considered to be similar to the pathway of the TPA/Ru(bpy)3 2+ system at a platinum electrode. Therefore, the possible mechanism can be expressed as follows: Ru(bpy)3 2+ − e− → Ru(bpy)3 3+ SINH+ − e− → SIN• Ru(bpy)3 3+ + SIN• → [Ru(bpy)3 2+ ]∗ + products Fig. 7. Electropherograms of (a) the sample solution; (b) the sample solution spiked with 0.4 g mL−1 sinomenine standard solution; other conditions are the same as in Fig. 4.
comparison the migration times and by spiking the standards to the sample solution. In the measurement process, a series of small peaks were always detected, which was estimated to be the interference produced by flavones or glucides in the herb plant. According to the results that listed in Table 1, the content of sinomenine found in the herb was 0.82%, which corresponded with other methods for the analysis of sinomenine in S. acutum, as indicated in Table 2.
[Ru(bpy)3 2+ ]∗ → Ru(bpy)3 2+ + hν
(λ = 620 nm)
4. Conclusion A Ru(bpy)3 2+ -based CE-ECL method was studied for identification and determination of sinomenine for the first time. The developed method was found not only a good alternative for the rapid determination of sinomenine in plant extracts with good selectivity, wide linearity and reliable stability, but also an efficient supplementary technique for the preliminary investigation of other quinolizidine alkaloids in Chinese traditional herbs.
3.6. Mechanism The ECL behavior based on Ru(bpy)3 2+ system is usually related to the structure of the co-reactant. A general trend is that nitrogen-containing compounds especially tertiary amines lead to a much higher ECL intensity than others [26,27]. A typical example is the ECL in the tripropyl amine (TPA)/Ru(bpy)3 2+ Table 1 Results for the determination of sonomenine in Sinomenium acutum Present method (g ml−1 )
R.S.D (%) (n = 5)
Added (g)
Found (g ml−1 )
Recovery (%) (n = 5)
0.246
1.26
0.200 0.400
0.458 0.637
102.7 98.6
Acknowledgements We are grateful to Natural Scientific Foundation of Gansu Province, China, for supporting the research with Project 3ZS051–A25–097, and more thanks to Scientific Research Foundation of Gansu Ministry of Education, China, for the partial financial aid with Project 0501–07. References [1] J.H. Liu, W.D. Li, H.L. Teng, Acta Pharm. Sin. 40 (2005) 127. [2] Q. Liu, L.L. Zhou, R. Li, Chin. Tradit. Herb. Drugs 28 (1997) 247. [3] L. Liu, E. Buchner, D. Beitze, C.B. Schmidt-Weber, V. Kaever, F. Emmrich, R.W. Kinne, Int. J. Immunopharmacol. 18 (1996) 529.
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