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Analytica Chimica Acta 587 (2007) 104–109

Determination of sinomenine in Sinomenium acutum by capillaryelectrophoresis 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 2007Available online 16 January 2007

bstract

A Ru(bpy)32+-based electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE) has been established for the deter-

ination of sinomenine for the first time. Optimum separation was achieved with a fused-silica capillary column (50 cm × 25 �m i.d.) and aackground electrolyte of 50 mM sodium phosphate (pH 5.0) at a separation voltage of 15 kV. The content of sinomenine was detected by ECLt 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 modifiedlatinum electrode by europium(III)-doped prussian blue analogue (Eu-PB) was used as a working electrode. Under the optimized conditions, the

CL 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).he relative standard derivations of migration time and ECL intensity were 0.93 and 1.11%, respectively. The level of sinomenine in Sinomeniumcutum Rehd. et Wils was easily determined with recoveries between 98.6 and 102.7%.

2007 Elsevier B.V. All rights reserved.

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eywords: Electrochemiluminescence; Capillary electrophoresis; Sinomenine

. Introduction

Sinomenine (7,8-didehydro-4-hydroxyl-3,7-dimethoxy-17-ethylmorphinan-6-one; SIN) is a principal alkaloid isolated

rom the stem and root of Chinese medical plant Sinome-ium acutum Rehd. et Wils, and its chemical structure ishown in Fig. 1. Due to its analgesic and anti-inflammatoryffects, sinomenine has been utilized clinically to treatheumatoid arthritis and neuralgia [1–3]. At present, severalhromatographic methods including high-performance liquidhromatography (HPLC) [4–7] and thin-layer chromatographyTLC) [8,9] have been reported for the analysis of sinomenine.owever, a drawback of the methods mentioned above appears

o be time-consuming due to necessary extraction, concentra-ion and/or derivatization prior to the analysis although the high

ensitivity and the good selectivity have been obtained in suchrocedures. Therefore, it is necessary to establish rapid andffective methods for the quantitation of sinomenine.

∗ Corresponding author. Tel.: +86 931 7669904.E-mail address: Lf0522@0931mail.com (H. Chen).

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003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2007.01.018

Capillary electrophoresis (CE) is now a widely usedeparation technique for analysis of alkaloids with variousharmaceutical applications because of its high efficiency, reso-ution potential, short analysis time and minimal sample volume10–12]. Recently, an easy, rapid nonaqueous capillary elec-rophoresis method has been developed for the determination ofinomenine with a UV detector [13]. In addition, Zhai et al. [14]as also proposed another CE method for sinomenine determi-ation by use of high frequency conductivity detector with aetection limit of 0.2 �g mL−1.

In recent years, there are increasing interests in cou-ling CE separation with high-sensitive chemiluminescenceCL) detection for alkaloids analysis [15–18]. Especially,lectrochemiluminescence (ECL) detection involving tris(2,2′-ipyridyl) ruthenium(II) (Ru(bpy)3

2+) offers other merits withide linear range, no derivatization and good selectivity foritrogen-containing compounds [19,20]. Therefore, analyticalrocedures combining CE separation with Ru(bpy)3

2+-based

CL detection have been paid more attention to the detectionf some alkaloids [21–24]. However, as far as we know, suchE-ECL procedure has not been reported for the determinationf sinomenine.

M. Zhou et al. / Analytica Chimic

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Fig. 1. The structure of sinomenine.

In this paper, a CE-ECL method based on Ru(bpy)32+ sys-

em has been developed for the determination of sinomenine inhinese herb S. acutum Rehd. et Wils. It is worth mentioning

hat a europium(III)-doped prussian blue analogue (Eu-PB) filmas modified chemically on the surface of a microdisk platinumorking electrode to avoid the possible electrode fouling as well

s to improve the detection sensitivity.

. Experimental

.1. Reagents and chemicals

All chemicals and reagents were of analytical grade exceptor specific statements and used without further purification.ris(2,2′-bipyridyl) ruthenium(II) chloride hexahydrate (98%)as obtained from Aldrich (Milwaukee, WI, USA) and pre-ared with doubly deionized water. Sinomenine was purchasedrom National Institute for the Control of Pharmaceutical andiological Products (Beijing, China) and freshly prepared with0% methanol (Spectrum analytical grade) just before use. Dried

hinese herb S. acutum Rehd. et Wils was taken from Lanzhouuanghe Chinese Herbs wholesale Ltd. Corp. (Lanzhou, China).odium phosphate (pH 5.0, G.R.) was used as the backgroundlectrolyte solution. All solutions were stored in the refrigerator

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Fig. 2. Schematic diagram of the

a Acta 587 (2007) 104–109 105

t 0–4 ◦C and filtered through a membrane of 0.45 �m prior tose.

.2. Apparatus

A MPI-A system (Xi’an Remax Electric Ltd. Corp., China)as employed for CE-ECL detection. The schematic diagramf the CE-ECL detection system is shown in Fig. 2. Capillarylectrophoresis was performed using a 50 cm length of uncoatedused-silica capillary (25 �m i.d., Yongnian Optical Fiber Fac-ory, Hebei, China) at 15 kV with a background electrolyte of0 mM sodium phosphate (pH 5.0). Samples were introducedrom the anodic end of the capillary by electrokinetic injectionor 10 s at 10 kV. The end-column ECL detection was installedith a three-electrode configuration, which was made up of a00 �m Eu-PB modifying platinum disk as a working electrode,n Ag/AgCl as a reference electrode and a platinum wire as anuxiliary electrode. The capillary-to-working electrode distanceas adjusted to about 150 �m. A solution of 5 mM Ru(bpy)3

2+

n 75 mM phosphate buffer (pH 8.0) was directly injected intohe detection reservoir [25]. ECL emission was measured using

multichannel data collection analyzer, in which a sensitivehotomultipier tube (PMT) was operated at 800 V. Prior to exper-ments every day, the capillary was rinsed with 0.1 M NaOHor 3 min at first, then with doubly deionized water for 3 minnd finally equilibrated with the background electrolyte formin.

Modification of the working electrode was performed byHI832 electrochemical analyzer (Shanghai Chenhua Appara-

us Corporation, China). A solution of 10.0 mL FeCl3, 10.0 mL3Fe(CN)6, 6.5 mL HCl, 5.0 mL EuCl3 and 5.0 mL potassiumydrogen phthalate (all concentration were 0.01 M) was directlydded into the electrochemical cell, the Eu-PB film was grad-ally electrodeposited with twenty 50 mV s−1 potential cyclesetween 0 and 1.4 V (versus SCE reference electrode). Then the

odified electrode was immerged into the saturated KCl solu-

ion and was scanned with a rate of 50 mV s−1 for 20 potentialycles from 0 to 1.3 V. Finally, the prepared electrode was sub-ected in 75 mM phosphate buffer (pH 8.0) to repeat cycling

CE-ECL detection system.

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06 M. Zhou et al. / Analytica C

nder the same conditions until a reproducible voltammogramas obtained.

.3. Sample preparation

The powder of root (3.0 mg) of S. acutum Rehd. et Wilsas extracted with 5 mL methanol for 30 min in an ultrasonicath. Extraction was repeated twice. The extracts were com-ined and diluted with methanol to 10 mL. Then 1 mL extractedolution was mixed with 4 mL methanol and diluted with dou-ly deionized water to 10 mL, following by passing through a.45 �m membrane and being directly injected into the capillarylectrophoresis system and analyzed.

. Results and discussions

.1. Effect of the platinum electrode modified with Eu-PBlm

In comparison with the response to the oxidation ofu(bpy)3

2+ on a bare platinum electrode, the Eu-PB modify-ng platinum electrode exhibited higher current response, withlight negative shift ca. 20 mV for the direct oxidation peak ofu(bpy)3

2+ (see Fig. 3A). Consequently, an enhanced ECL peakf Ru(bpy)3

2+ was obtained, as shown in Fig. 3B. Thus, the pre-ared electrode would benefit from the improved sensitivity andive less interfering signals from other electroactive substancesn real samples. In addition, the prepared electrode was stablenough for repetitive use in the detection system within 2 weeksith no need for electrode replacement.

.2. Optimization of system

The most important variables in ECL such as the concen-ration of Ru(bpy)3

2+ solution, the applied potential and the

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ig. 3. (A) Differential pulse voltammograms of Ru(bpy)32+ in phosphate buffer (pH

.05 V; pulse width, 0.05 s; pulse period, 0.2 s. Concentrations: Ru(bpy)32+, 5 mM;

.0): (a) at bare Pt electrode; (b) at Eu-PB modifying Pt electrode. Concentrations: R

a Acta 587 (2007) 104–109

onditions of background electrolyte, as well as the separationarameters including separation voltage and injection parame-ers were optimized in this work.

.2.1. Effect of Ru(bpy)32+ concentration

Ru(bpy)32+ was used as the ECL reagent in the system and

ts concentration has great effect on the ECL signal. The resultshowed that the ECL intensity increased markedly with increas-ng Ru(bpy)3

2+ concentration from 0.2 to 5.0 mM. In this work,mM Ru(bpy)3

2+ in 75 mM phosphate buffer (pH 8.0) wasdopted due to concerned over sensitivity and economy in usef reagent.

.2.2. Effect of detection potentialDetection potential has great effect on the ECL intensity.

he result in Fig. 4 showed that the increased production ofu(bpy)3

3+ with a rise of potential led to an increased responsend the ECL intensity reached a stable maximum between.15 and 1.2 V. Above which, the ECL response diminisheds competitive reactions involving the background electrolyteominate. Thus, the optimal potential was 1.15 V.

.2.3. Choice of background electrolyteAcetate, Tris–HCl, phosphate and borate buffers were

ested as the optional background electrolyte and the orderf the ECL intensity in different solutions was: phos-hate ≥ acetate > borate > Tris–HCl. Finally, phosphate washosen in terms of the highest ECL and the best signal to noiseatio (S/N).

.2.4. Effect of pH of background electrolyteThe pH effect of phosphate on electrophoresis separation and

CL intensity was investigated in a wide pH range of 3.5–9.5 in.5 pH units. As illustrated in Fig. 5, the highest ECL intensity

8.0): (a) at bare Pt electrode; (b) at Eu-PB modifying Pt electrode. Amplitude,phosphate, 75 mM. (B) ECL emission of Ru(bpy)3

2+ in phosphate buffer (pHu(bpy)3

2+, 5 mM; phosphate, 75 mM.

M. Zhou et al. / Analytica Chimica Acta 587 (2007) 104–109 107

Fig. 4. Effect of detection potential on ECL intensity. Separation capillary,21i

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5 �m i.d., 50 cm length; sample injection, 10 s at 10 kV; separation voltage,5 kV; background electrolyte, 50 mM sodium phosphate (pH 5.0), phosphaten the detection cell, 75 mM at pH 8.0.

as observed at pH 5.0. As a result, pH 5.0 was selected for allhe following experiments.

.2.5. Effect of background electrolyte concentrationFixed pH value at 5.0, the concentration of phosphate was

djusted from 5 to 80 mM. It is found in Fig. 6 that workingt high phosphate concentration allowed improving the sensi-ivity and resolution until 50 mM. Above which, both the ECLntensity and resolution decreased because of excessive heatingaused by Joule effect, following an increased background sig-al and resulting in an unstable measurement. Hence, 50 mMhosphate (pH 5.0) was preferred as background electrolyte.

.2.6. Effect of separation voltage and injection parametersIn general, ECL intensity increased with an increase in sep-

ration voltage, injection voltage or injection time. However,oth the repeatability and the resolution became worse whenn excessive voltage or sample volume was introduced. So ascompromise of the high ECL intensity and the improved col-

ig. 5. Effect of background electrolyte pH on ECL intensity. Detection poten-ial, 1.15 V; other conditions are the same as in Fig. 4.

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ig. 6. Effect of background electrolyte concentration. Other conditions are theame as in Fig. 4.

mn efficiency, the separation voltage of 15 kV and the injectionarameters of 10 s at 10 kV were recommended.

.3. Choice of extracting solvent

The extracting solvent was chosen from ethanol andethanol, and methanol was found to give a better resolution

nd a higher ECL intensity. Therefore, methanol was selectedor the extraction of sinomenine in herbs in order to obtain

higher extraction yield. Besides, the final concentration ofethanol solution for sample injection was optimized in the

ange 10–100% when certain amount of herbs was extracted.he results indicated that the ECL intensity reached maximumhen methanol in samples was 50–75%, and methanol solutionf 50% was adopted due to concerns over greater precision andetter resolution.

.4. Calibration and detection

Under the optimum conditions, the calibration graph ofinomenine concentration versus ECL intensity was linear inhe range from 0.01 to 1.0 �g mL−1. The regression equationould be expressed as: �I = 486.59 + 790.28 C �g mL−1 withcorrelation coefficient of 0.9993 (n = 5). The detection limit,

efined as three times the S.D. for the reagent blank signal, was.0 ng mL−1, which was equal with or lower than that obtainedy other methods mentioned above [4–9,13,14].

The precision of the proposed method was determined byeduplicate injections (n = 6) of 0.1 �g mL−1 sinomenine stan-ard solution. The relative standard deviations (R.S.D.) ofigration time and ECL intensity were 0.93 and 1.11%, respec-

ively.

.5. Applications

To examine the application for practical analysis, the CE-CL method was applied to the determination of sinomenine

n Chinese herb S. acutum Rehd. et Wils. The typical electro-herogram is shown in Fig. 7. The peaks were identified by

108 M. Zhou et al. / Analytica Chimica Acta 587 (2007) 104–109

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Table 2Comparison of the results obtained by the present method with others

Method Linear range(�g ml−1)

LOD(�g ml−1)

SIN inSinomeniumacutum

Reference

The presentmethod

0.01–1.0 2.0 × 10−3 0.82% –

HPLC-UV 2.6–106 – 7.47 mg g−1

(0.747%)[7]

CE-UV 6.25–500 1.71 0.80 mg g−1

(0.08%)[13]

CE-high 1.0–36.0 0.2 0.70% [14]

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ig. 7. Electropherograms of (a) the sample solution; (b) the sample solutionpiked with 0.4 �g mL−1 sinomenine standard solution; other conditions are theame as in Fig. 4.

omparison the migration times and by spiking the standardso the sample solution. In the measurement process, a series ofmall peaks were always detected, which was estimated to be thenterference produced by flavones or glucides in the herb plant.ccording to the results that listed in Table 1, the content of

inomenine found in the herb was 0.82%, which correspondedith other methods for the analysis of sinomenine in S. acutum,

s indicated in Table 2.

.6. Mechanism

The ECL behavior based on Ru(bpy)32+ system is usually

elated to the structure of the co-reactant. A general trend is thatitrogen-containing compounds especially tertiary amines leado a much higher ECL intensity than others [26,27]. A typicalxample is the ECL in the tripropyl amine (TPA)/Ru(bpy)3

2+

able 1esults for the determination of sonomenine in Sinomenium acutum

resent method�g ml−1)

R.S.D (%)(n = 5)

Added(�g)

Found(�g ml−1)

Recovery(%) (n = 5)

.246 1.26 0.200 0.458 102.70.400 0.637 98.6

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frequencyconductivity

ystem, and the relevant mechanism is regarded as a typicaledox type [28]. Although both the current response and ECLntensity were enhanced by use of the Eu-PB modifying plat-num electrode, no convincing evidence has been observed tondicate the existence of a new ECL mechanism till now. Thus,he production of light emission of the Ru(bpy)3

2+/sinomenineystem is considered to be similar to the pathway of thePA/Ru(bpy)3

2+ system at a platinum electrode. Therefore, theossible mechanism can be expressed as follows:

u(bpy)32+ − e− → Ru(bpy)3

3+

INH+ − e− → SIN•

u(bpy)33+ + SIN• → [Ru(bpy)3

2+]∗ + products

Ru(bpy)32+]∗ → Ru(bpy)3

2+ + hν (λ = 620 nm)

. Conclusion

A Ru(bpy)32+-based CE-ECL method was studied for identi-

cation and determination of sinomenine for the first time. Theeveloped method was found not only a good alternative for theapid determination of sinomenine in plant extracts with goodelectivity, wide linearity and reliable stability, but also an effi-ient supplementary technique for the preliminary investigationf other quinolizidine alkaloids in Chinese traditional herbs.

cknowledgements

We are grateful to Natural Scientific Foundation of Gansurovince, China, for supporting the research with ProjectZS051–A25–097, and more thanks to Scientific Researchoundation of Gansu Ministry of Education, China, for theartial financial aid with Project 0501–07.

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