Research article Received: 22 October 2013,
Revised: 17 March 2014,
Accepted: 21 March 2014
Published online in Wiley Online Library: 2 May 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2681
Flow injection analysis of thiamazole based on strong Ru(bpy)32+ co-reactant electrochemiluminescence Dexian Kong,a,b Qinglu Li,a Jiang Jiang,a Zheng Xinyu,a Zhou Xuechou,a Yuwu Chib* and Guonan Chenb ABSTRACT: Based on the strong electrochemiluminescence (ECL) reaction between thiamazole and tris(2,2′-bipyridine)ruthenium(II) (Ru(bpy)3 2+), a sensitive, simple and rapid flow injection analysis method for the determination of thiamazole was developed. When a Pt working electrode was maintained at a potential of +1.50 V (vs Ag/AgCl) in pH 12.0 H3PO4–NaOH solution containing thiamazole and Ru(bpy)3 2+ at a flow rate of 1.0 mL/min, a linear range of 2.0 × 10 7–1.0 × 10 4 mol/L with a detection limit of 5.0 × 10 8 mol/L was obtained for the detection of thiamazole. The method showed good reproducibility with a relative standard deviation (RSD) of 0.75%. The method has been successfully applied to the determination of thiamazole in spiked animal feeds. In addition, a co-reactant ECL mechanism was proposed for the thiamazole–Ru(bpy)3 2+ system. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: electrochemiluminescence; flow injection; thiamazole; antithyroid
Introduction
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As a thio-compound, thiamazole (2-mercapto-1-methylimidazole; Fig. 1) exhibits high antithyroid activities, and is thus one of the most commonly used drugs to manage hyperthyroidism associated with Graves’ disease (1), with the action of slowing iodide integration into tyrosine and hence inhibiting the production of thyroid hormones. Despite its acceptability, the use of thiamazole in the treatment of hyperthyroidism is associated with many complications, such as skin irritation, a decrease in the number of white blood cells, impaired taste, olfaction, allergies and pharyngitis with fever, and in rare occasions, nephritis and liver cirrhosis (2). Meanwhile, thiamazole is illegally added to animal feeds by some farmers in order to promote animal growth, because of its effect of enhancing water retention in the tissues of animals (3). This may lead to thiamazole accumulation in the animal and finally ingestion by humans, posing a great threat to human health. The addition of thiamazole in animal feeds is prohibited in most countries, such as the European Union (EU Directive 86/469/EEC) and Australia (Livestock Regulations 1998). Therefore, research into a simple, rapid, sensitive and dependable method for the determination of thiamazole is of great importance. To date, various methods have been established for the determination of thiamazole, including: flow-injection spectrophotometry (4), chemiluminescence (5), resonance light scattering spectroscopy (6), fluorescence (7), high-performance liquid chromatography (8–11), gas chromatography (12,13), capillary zone electrophoresis (14) and electrochemical methods (15–17). However, the available techniques have some drawbacks such as high cost, complexity, time-consuming sample preparation and low sensitivity, so the search for new methods of thiamazole determination continues. Electrochemiluminescence (ECL) was developed based on electrochemistry method. ECL involves the generation of species at electrode surfaces that then undergo electron-transfer
Luminescence 2015; 30: 12–17
reactions to form excited states that emit light. The advantages of ECL are its high sensitivity, good reproducibility and that it can be controlled easily (18). Hua and co-workers have developed a method for the determination of thiamazole based on its quenching effect on the ECL intensity of CdTe quantum dots (QDs) (19). However, this ECL method involves the use of CdTe QDs, which is a threat to human health and the environment. In the past decade, ECL based on Ru(bpy)3 2+ has attracted much interest from analysts and has been successfully combined with flow injection (FI) analysis, high-performance liquid chromatography, capillary electrophoresis, and micro-total analysis systems (20). To the best of our knowledge, there are not any research into the determination of thiamazole based on its ECL reaction with Ru(bpy)3 2+. Here, the ECL behaviors of thiamazole in the presence of Ru(bpy)3 2+ are investigated. We found that thiamazole could intensively enhance the ECL of Ru(bpy)3 2+ in alkaline media, on the basis of which we developed a sensitive, simple and rapid FI–ECL method for the determination of thiamazole. The developed method has been applied to the detection of thiamazole in spiked animal feeds and satisfactory results have been obtained.
* Correspondence to: Y. Chi, MOE Key Laboratory of Analysis and Detection Technology for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, and Department of Chemistry, Fuzhou University, Fujian 350108, China. Tel: +86-591-22866137; Fax: +86591-22866137. E-mail: [email protected] a
School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China
b
MOE Key Laboratory of Analysis and Detection Technology for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, and Department of Chemistry, Fuzhou University, Fujian, 350108, China
Copyright © 2014 John Wiley & Sons, Ltd.
Flow injection analysis of thiamazole with ECL
Figure 1. Chemical structural formula of thiamazole.
Experimental Chemicals and solutions Tris(2,2′-bipyridine)ruthenium(II) chloride hexahydrate (Ru(bpy)3 Cl2 · 6H2O) was obtained from Aldrich. Thiamazole was purchased from Sigma. Other chemicals were of analytical grade. Doubledistilled water was used to prepare sample solutions. A 0.010 mol/L stock solution of Ru(bpy)3Cl2 was prepared by dissolving 0.0749 g of Ru(bpy)3Cl2·6H2O in 10 mL of double-distilled water, and was stored under refrigeration. A 0.010 mol/L stock solution of thiamazole was prepared by adding 0.0114 g of sample to 10 mL of double-distilled water and was also stored in the refrigerator. Carrier solutions were prepared by diluting appropriate volumes of the above Ru(bpy)3Cl2 stock solutions with phosphate buffer solutions. Sample solutions were prepared by diluting the required volumes of the thiamazole stock solutions with carrier solutions. The phosphate buffer solutions (pH 2–13) were prepared by titrating 0.1 mol/L phosphoric acid solutions with sodium hydroxide to the required pH.
(a)
(b)
Apparatus The ECL experiments were performed on a homemade system consisting of a flow injection unit and an ECL detection unit (schematic diagram shown in Fig. 2a). The flow injection unit was a LZ-2000 flow injection processor, the details of which have been described previously (21,22). The ECL detection unit included following sections: a GD-1 chemiluminescent detector (Ruike Electronic Instrument Ltd. Co., China), a Model 400 electrochemical detector (EG&G), a HW chromatography station (Qianpu Ltd. Co., China) and an ECL flow cell. The signal produced in the ECL flow cell was detected by the chemiluminescent detector and converted into digital signal that could be recorded by the computer. The structure of the ECL flow cell was similar to that used in our previous research (22) (Fig. 2b). It consisted of a Teflon® block, a piece of Teflon® membrane and a Perspex® block tightly fixed together by screws. The Teflon® block was mounted with a working electrode (a Pt disk with an area of 22.1 mm2). The Teflon® membrane (thickness, 50 μm) with a rectangular hole in its center was sandwiched between the Teflon® block and the Perspex® block to hold ~ 2.5 μL of thin-layer solution. The Perspex® block (1 cm thick ) not only provided an optical window with ~ 75% transmittance in the wavelength range of 400–800 nm, but also accommodated a reference electrode and a counter electrode. The reference electrode was an Ag/AgCl (3 mol/L KCl) electrode, and the counter electrode was a stainless steel pipe located at the solution outlet of the cell.
ECL measurement
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(100 μL) for 15 s using pump I. Meanwhile, the carrier solution was pumped into the ECL detector using pump II. Step 2: pump I was stopped, and the sampling valve was turned to the injection position, the carrier solution was pumped into the loop using pump II to carry the sample solution to the ECL detector for measurement. Pumps I and II were both set at a same rotary speed of 30 cycles/min (~ 1.0 mL/min). Before measurements were taken, the Pt working electrode was pretreated by polishing the surface with aqueous slurries of 0.3 μm alumina particles on a polishing micro-cloth, rinsed with water and then sonicated in ethanol, HNO3 (1 : 1, v/v) and double-distilled water, in turn. Determination of thiamazole in spiked animal feeds First, 100 g of animal feed [we chose chicken feed (sample I), duck feed (sample II) and pig feed (sample III) was spiked with thiamazole at a concentration of 1.15 μg/g, and ground with a mortar and a pestle until a homogeneous mixture was obtained. Then, 1.0 g of the homogeneous sample was mixed with 10.0 mL of methanol in a centrifuge tube, and mechanically shaken for 5 min. After centrifugation at 3600 rpm for 10 min, the supernatant solution was collected and evaporated to dryness at 40 ºC under a gentle stream of nitrogen. The dry residue was dissolved with 5 mL pentane for subsequent purification by solid phase extraction (SPE). In the SPE step, an AccuBond II Florisil SPE Cartridge (1 g, Agilent Technologies) was conditioned with 10 mL of methanol, and then the 5 mL sample was passed through the Florisil SPE Cartridge. After rinsing the column with 5 mL of pentane, elution was performed using 2 mL of methanol/dichloromethane (20 : 80 v/v),
Copyright © 2014 John Wiley & Sons, Ltd.
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ECL measurement was carried out in two steps. Step 1: the sampling valve was set at the sample position, and sample solution was pumped into the sampling valve to fill the loop
Figure 2. Schematic diagram of (a) the FI-ECL unit and (b) the ECL flow cell used in the experiment.
D. Kong et al.
ECL response of thiamazole in the presence of Ru(bpy)3
2+
From the results, we found that thiamazole in all types of buffer solution did not exhibit ECL activity. However, it could enhance the ECL emission of Ru(bpy)3 2+ at the Pt electrode when pH > 7.0 (Fig. 3). In the FI–ECL system, we used Ru(bpy)3 2+ solution as the carrier, and a mixture of Ru(bpy)3 2+ and thiamazole as the sample solution. The FI–ECL peak obtained was actually the ECL enhancement (△I) of thiamazole in the Ru(bpy)3 2+ system and its intensity depended on the thiamazole concentration. In order to investigate the ECL behavior of thiamazole in the presence of Ru(bpy)3 2+, the effects of experimental conditions such as the mode of the applied voltage, the potential of the working electrode, the pH, the flow rate of carrier solution and the concentration of Ru(bpy)3 2+ were investigated and discussed. Effect of applied potential. We experimented on different potential modes such as constant potential (DC), double step potential (rectangular wave) and linear sweep potential (triangular wave), in order to examine the ECL response of thiamazole in the presence of Ru(bpy)3 2+. The results showed that thiamazole gave the most sensitive ECL response under the constant potential mode. Thus, the constant potential mode was selected in subsequent ECL investigations. After choosing the potential mode, the effect of voltage on ECL was investigated. In a solution of PBS (pH 12.0) containing 1.0 × 10 4 mol/L Ru(bpy)3 2+, the ECL responses of thiamazole were recorded from 1000 to 1700 mV at 100 mV intervals (Fig. 4). From Fig. 4 we found that that the peak shape and peak height (ECL intensity) were both greatly affected by the applied potential. When the applied potential was too low (< 1100 mV), the ECL peak was split into two small peaks (Fig. 4a,b), which can be explained by the presence of the voltage drop as a result of
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Figure 3. Typical ECL responses obtained for thiamazole in alkaline Ru(bpy)3 5 5 solution. The concentrations of thiamazole were: (a) 1.0 × 10 mol/L, (b) 5.0 × 10 4 4 mol/L and (c) 1.0 × 10 mol/L. Carrier solution: PBS, pH 12.0, containing 1.0 × 10 mol/L 2+ Ru(bpy)3 . Sample solution: thiamazole + carrier solution. Flow rate: 1.0 mL/min. Applied potential: 1500 mV.
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ECL Intensity/mV
Results and discussion
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followed by 5 mL of methanol. The collected eluent was evaporated to dryness at 40 °C under a gentle stream of nitrogen. Finally, the residue was dissolved in 1.0 mL of carrier solution (H3PO4– NaOH; PBS, pH 12.0) containing 1.0 × 10 4 mol/L Ru(bpy)3 2+.
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t /min Figure 4. ECL responses of thiamazole at various applied potentials: (a) 1000, (b) 1100, (c) 1200, (d) 1300, (e) 1400, (f) 1500, (g) 1600 and (h) 1700 mV. (Inset) Plot of the enhanced ECL intensity vs potential. Carrier solution: PBS, pH 12.0, containing 4 2+ 1.0 × 10 mol/L Ru(bpy)3 . Sample solution: thiamazole + carrier solution. Flow rate: 1.0 mL/min.
internal resistance in the thin-layer ECL cell which led to insufficient oxidation of Ru(bpy)3 2+ (23). Then, when the applied potential was > 1200 mV, the ECL peak shape was normal and ECL intensity increased with the potential (Fig. 4c–f); the ECL intensity tended to became invariable at potentials > 1500 mV. However, when the potential was > 1500 mV (Fig. 4g,h), the ECL intensity changed slightly, but more background noise was produced due to the formation of large amounts of oxygen bubbles at the Pt electrode. So an applied potential of 1500 mV was chosen in the subsequent study to obtain the highest ECL signal at the lowest background noise. Effect of pH. The properties of the buffer solution have a great influence on the ECL behavior of the Ru(bpy)3 2+ system (18). In order to choose the best buffer solution, the ECL responses of thiamazole in different media (0.1 mol/L for all test media), such as H3PO4–NaOH, NaHCO3–NaOH, H3BO3–NaOH and Britton–Robinson buffer were investigated. It was found that the ECL response of thiamazole was most sensitive in H3PO4–NaOH (PBS). Therefore, PBS was chosen as the ECL reaction medium. When the pH of the buffer solution was < 7.0, thiamazole had no enhancement effect on ECL of Ru(bpy)3 2+, whereas when the pH changed from 7.0 to 12.0, the ECL intensity increased sharply (Fig. 5). From Fig. 5, we know that thiamazole could greatly enhance the ECL intensity of Ru(bpy)3 2+ in alkaline solution (Fig. 5e,f). However, when the pH was too high, the background noise was higher and the signal-to-noise ratio decreases, which may result from a chemiluminescent reaction between Ru(bpy)3 3+ and the high-energy intermediate (HO2·) electro-generated in the alkaline solution (24). The most satisfactory ratio of signal-to-noise was found at pH 12.0, and so PBS at pH 12.0 was chosen for further ECL study. Effect of flow rate. We found that the flow rate of the carrier solution could influence the ECL behavior of thiamazole significantly. From Fig. 6, we know that the higher the flow rate, the narrower ECL peak (compare Fig. 6a–c). This phenomenon can be explained by the fact that a shorter time is needed for a given volume (100 μL) of sample to pass through the ECL cell at a higher flow rate, giving a narrower ECL peak. From Fig. 6, we also know that the ECL intensity increased obviously from
Copyright © 2014 John Wiley & Sons, Ltd.
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Flow injection analysis of thiamazole with ECL 20
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Figure 5. ECL responses of thiamazole in carrier solution at various pH values (pH ≥ 7.0) (A) 7.0, (B) 8.0, (C) 9.0, (D) 10.0, (E) 11.0 and (F) 12.0. Carrier: H3PO4–NaOH solution 4 2+ containing 1.0 × 10 mol/L Ru(bpy)3 . Sample solution: thiamazole + carrier solution. Flow rate: 1.0 mL/min. Applied potential: 1500 mV.
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Figure 6. ECL responses of thiamazole at various flow rates: (A) 0.5, (B) 1.0 and (C) 2.0 mL/min. Carrier solution: PBS, pH 12.0, containing 1.0 × 10 solution: thiamazole + carrier solution. Applied potential: 1500 mV.
0.5 to 1.0 mL/min, and then increased slightly at flow rates of 1.0–2.0 mL/min. At the same time, the ECL background noise decreased with increasing flow rate, which might result from the relatively lower flow pulse produced by the peristaltic pump at the higher flow rate. To obtain relatively high sensitivity while reducing the use of Ru(bpy)3 2+, a flow rate of 1.0 mL/min was selected.
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2+
mol/L Ru(bpy)3 . Sample
mol/L. Thus, we chose 1.0 × 10 4 mol/L Ru(bpy)3 2+ in carrier solution to reduce the consumption of Ru(bpy)3 2+, but obtain the best signal-to-noise ratio. The optimum conditions for the ECL measurement of thiamazole are shown in Table 1. Linear response range and reproducibility. Under the optimum conditions as mentioned above, a new FI–ECL method for the determination of thiamazole was developed and the concentration effect of thiamazole on its ECL response was studied. The FI–ECL method exhibited a linear response for thiamazole ranging between 2.0 × 10 7 and 1.0 × 10 4 mol/L with a detection limit (S/N = 3) of 5.0 × 10 8 mol/L. The regression equation is △I = 5.2 × 106C + 5.40 with a correlation
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Effect of Ru(bpy)3 2+ concentration. The experiment showed that higher Ru(bpy)3 2+ concentrations in the carrier solution gave both a higher ECL peak and higher background noise. The signal-to-noise ratio increased as the Ru(bpy)3 2+ concentration was increased between 1.0 × 10 5 and 1.0 × 10 4 mol/L, whereas it hardly changed between 1.0 × 10 4 and 1.0 × 10 3
4
D. Kong et al. Table 1. Optimum conditions for FI-ECL response of thiamazole Experimental items
Optimum conditions
Mode of applied voltage signal Applied potential Buffer solution Flow rate Concentration of Ru(bpy)3 2+
DC +1500 mV 0.1 mol/L PBS pH 12.0 1.0 mL/min 1.0 × 10 4 mol/L
Table 2. Results for thiamazole determination in spiked animal feed sample solutions using the proposed FI-ECL method Sample
Spiked (μmol/L)
I II III
10 10 10
Found (μmol/L)a
RSD (%)
9.41 ± 0.25 9.32 ± 0.35 9.59 ± 0.11
2.76 3.99 1.25
Recovery (%)a 94.1 ± 2.5 93.2 ± 3.5 95.9 ± 1.1
a
coefficient, r = 0.9944 (n = 6). Where △I is the enhanced ECL intensity and C is the concentration of thiamazole. We also investigated the reproducibility of the method and found good reproducibility with relative standard deviations (RSD) of 0.75% for 10 successive tests of 5 × 10 7 mol/L thiamazole.
The uncertainty values were estimated using the expression ± tn 1 s/√n, where n is the number of replicate measurements, tn 1 is the statistic parameter often called Student’s t (with n = 5, at the 95% level of confidence, t = 2.132) and s is the standard deviation (SD).
Sample analysis and recovery study Possible mechanism for the enhanced ECL behavior of thiamazole From the published reviews, we know that most ECL responses of organic species in the Ru(bpy)3 2+ system result from ECL reactions that include: (1) electrochemical oxidation of the organic species and Ru(bpy)3 2+, producing strong reducing intermediates (usually neutral radical species) and strong oxidizing Ru(bpy)3 3+; (2) followed by high-energy electron transfer between the strong reducing intermediates and Ru(bpy)3 3+, resulting in generation of the excited-state luminophore, Ru(bpy)3 2+*; and (3) finally, light emission when the excited-state (Ru(bpy)3 2+*) returns to its ground state (Ru (bpy)3 2+) Ru(bpy)3 2+ (18,20,25). The ECL response of thiamazole in the presence of Ru(bpy)3 2+ probably includes a similar ECL mechanism (Fig. 7). Previous electrochemical research has demonstrated that thiamazole can be oxidized to form highly reductive radicals (Fig. 7, II) (15,26). The oxidation peak is ~ 0.45 V, which is much lower than that of Ru(bpy)3 2+ (~ 1.20 V) (27). It is apparent that under the optimum potential (1.50 V) for ECL of the thiamazole/Ru(bpy)3 2+ system, both neutral radicals (species II) and Ru(bpy)3 3+ are produced. The further ECL reaction between them produces the excited-state Ru(bpy)3 2+*, which results in the ECL emission. The final oxidation product of the neutral radical, species II, in the alkaline media might be sulfonate (species III) (15).
In order to evaluate the applicability the FI–ECL method to real samples, it was applied to the determination of thiamazole in animal feed. After purification by SPE, spiked samples were tested using the developed FI–ECL method. The average results of five replicate measurements expressed as the 95% confidence interval obtained for animal feed samples spiked with thiamazole using the proposed method are summarized in Table 2. The recoveries of the analyte in samples I, II and III were 94.1 ± 2.5, 93.2 ± 3.5 and 95.9 ± 1.1%, respectively, at 1.15 μg/g thiamazole. The limit of detection (LOD) for thiamazole determined at a signal-to-noise ratio of 3 was ~ 5.7 μg/kg for all three samples, which is lower than the LOD for many previously reported methods, such as FI–spectrophotometry (~ 340 μg/kg) (4), FI–CL (~ 1 mg/kg) (5), GC–MS (< 100 μg/kg) (12) and electrochemical methods (~ 58 μg/kg) (15), and almost matched the HPLC method (~ 3.5 μg/kg) (8). However, the LOD was higher than that obtained with resonance light-scattering spectroscopy (~ 0.2 μg/kg) (6) and CdTe QDs ECL (~ 0.1 μg/kg) (19). From these results, the FI–ECL method was deemed applicable for the determination of thiamazole in animal feed samples.
Conclusion Thiamazole, a well-known antithyroid drug has been found to have strong ECL activity in the Ru(bpy)3 2+/co-reactant ECL system. Thiamazole could produce sensitive, stable FI–ECL signals in the presence of Ru(bpy)3 2+ in alkaline media. Experimental conditions such as applied potential, buffer solution, the flow rate of the carrier solution and the concentration of Ru(bpy)3 2+ were found to influence the FI-ECL intensity. After the optimization of experimental conditions, a new sensitive, simple, rapid and accurate FI–ECL method with a wide dynamic range for the detection of thiamazole in animal feed has been established. Acknowledgements
2+
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Figure 7. Proposed ECL mechanism of thiamazole in the presence of Ru(bpy)3 .
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This study was financially supported by National Natural Science Foundation of China (21075018), Program for New Century Excellent Talents in Chinese University (NCET-10-0019), and Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1116).
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Flow injection analysis of thiamazole with ECL
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Luminescence 2015; 30: 12–17
Copyright © 2014 John Wiley & Sons, Ltd.
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Revised: 17 March 2014,
Accepted: 21 March 2014
Published online in Wiley Online Library: 2 May 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2681
Flow injection analysis of thiamazole based on strong Ru(bpy)32+ co-reactant electrochemiluminescence Dexian Kong,a,b Qinglu Li,a Jiang Jiang,a Zheng Xinyu,a Zhou Xuechou,a Yuwu Chib* and Guonan Chenb ABSTRACT: Based on the strong electrochemiluminescence (ECL) reaction between thiamazole and tris(2,2′-bipyridine)ruthenium(II) (Ru(bpy)3 2+), a sensitive, simple and rapid flow injection analysis method for the determination of thiamazole was developed. When a Pt working electrode was maintained at a potential of +1.50 V (vs Ag/AgCl) in pH 12.0 H3PO4–NaOH solution containing thiamazole and Ru(bpy)3 2+ at a flow rate of 1.0 mL/min, a linear range of 2.0 × 10 7–1.0 × 10 4 mol/L with a detection limit of 5.0 × 10 8 mol/L was obtained for the detection of thiamazole. The method showed good reproducibility with a relative standard deviation (RSD) of 0.75%. The method has been successfully applied to the determination of thiamazole in spiked animal feeds. In addition, a co-reactant ECL mechanism was proposed for the thiamazole–Ru(bpy)3 2+ system. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: electrochemiluminescence; flow injection; thiamazole; antithyroid
Introduction
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As a thio-compound, thiamazole (2-mercapto-1-methylimidazole; Fig. 1) exhibits high antithyroid activities, and is thus one of the most commonly used drugs to manage hyperthyroidism associated with Graves’ disease (1), with the action of slowing iodide integration into tyrosine and hence inhibiting the production of thyroid hormones. Despite its acceptability, the use of thiamazole in the treatment of hyperthyroidism is associated with many complications, such as skin irritation, a decrease in the number of white blood cells, impaired taste, olfaction, allergies and pharyngitis with fever, and in rare occasions, nephritis and liver cirrhosis (2). Meanwhile, thiamazole is illegally added to animal feeds by some farmers in order to promote animal growth, because of its effect of enhancing water retention in the tissues of animals (3). This may lead to thiamazole accumulation in the animal and finally ingestion by humans, posing a great threat to human health. The addition of thiamazole in animal feeds is prohibited in most countries, such as the European Union (EU Directive 86/469/EEC) and Australia (Livestock Regulations 1998). Therefore, research into a simple, rapid, sensitive and dependable method for the determination of thiamazole is of great importance. To date, various methods have been established for the determination of thiamazole, including: flow-injection spectrophotometry (4), chemiluminescence (5), resonance light scattering spectroscopy (6), fluorescence (7), high-performance liquid chromatography (8–11), gas chromatography (12,13), capillary zone electrophoresis (14) and electrochemical methods (15–17). However, the available techniques have some drawbacks such as high cost, complexity, time-consuming sample preparation and low sensitivity, so the search for new methods of thiamazole determination continues. Electrochemiluminescence (ECL) was developed based on electrochemistry method. ECL involves the generation of species at electrode surfaces that then undergo electron-transfer
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reactions to form excited states that emit light. The advantages of ECL are its high sensitivity, good reproducibility and that it can be controlled easily (18). Hua and co-workers have developed a method for the determination of thiamazole based on its quenching effect on the ECL intensity of CdTe quantum dots (QDs) (19). However, this ECL method involves the use of CdTe QDs, which is a threat to human health and the environment. In the past decade, ECL based on Ru(bpy)3 2+ has attracted much interest from analysts and has been successfully combined with flow injection (FI) analysis, high-performance liquid chromatography, capillary electrophoresis, and micro-total analysis systems (20). To the best of our knowledge, there are not any research into the determination of thiamazole based on its ECL reaction with Ru(bpy)3 2+. Here, the ECL behaviors of thiamazole in the presence of Ru(bpy)3 2+ are investigated. We found that thiamazole could intensively enhance the ECL of Ru(bpy)3 2+ in alkaline media, on the basis of which we developed a sensitive, simple and rapid FI–ECL method for the determination of thiamazole. The developed method has been applied to the detection of thiamazole in spiked animal feeds and satisfactory results have been obtained.
* Correspondence to: Y. Chi, MOE Key Laboratory of Analysis and Detection Technology for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, and Department of Chemistry, Fuzhou University, Fujian 350108, China. Tel: +86-591-22866137; Fax: +86591-22866137. E-mail: [email protected] a
School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China
b
MOE Key Laboratory of Analysis and Detection Technology for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, and Department of Chemistry, Fuzhou University, Fujian, 350108, China
Copyright © 2014 John Wiley & Sons, Ltd.
Flow injection analysis of thiamazole with ECL
Figure 1. Chemical structural formula of thiamazole.
Experimental Chemicals and solutions Tris(2,2′-bipyridine)ruthenium(II) chloride hexahydrate (Ru(bpy)3 Cl2 · 6H2O) was obtained from Aldrich. Thiamazole was purchased from Sigma. Other chemicals were of analytical grade. Doubledistilled water was used to prepare sample solutions. A 0.010 mol/L stock solution of Ru(bpy)3Cl2 was prepared by dissolving 0.0749 g of Ru(bpy)3Cl2·6H2O in 10 mL of double-distilled water, and was stored under refrigeration. A 0.010 mol/L stock solution of thiamazole was prepared by adding 0.0114 g of sample to 10 mL of double-distilled water and was also stored in the refrigerator. Carrier solutions were prepared by diluting appropriate volumes of the above Ru(bpy)3Cl2 stock solutions with phosphate buffer solutions. Sample solutions were prepared by diluting the required volumes of the thiamazole stock solutions with carrier solutions. The phosphate buffer solutions (pH 2–13) were prepared by titrating 0.1 mol/L phosphoric acid solutions with sodium hydroxide to the required pH.
(a)
(b)
Apparatus The ECL experiments were performed on a homemade system consisting of a flow injection unit and an ECL detection unit (schematic diagram shown in Fig. 2a). The flow injection unit was a LZ-2000 flow injection processor, the details of which have been described previously (21,22). The ECL detection unit included following sections: a GD-1 chemiluminescent detector (Ruike Electronic Instrument Ltd. Co., China), a Model 400 electrochemical detector (EG&G), a HW chromatography station (Qianpu Ltd. Co., China) and an ECL flow cell. The signal produced in the ECL flow cell was detected by the chemiluminescent detector and converted into digital signal that could be recorded by the computer. The structure of the ECL flow cell was similar to that used in our previous research (22) (Fig. 2b). It consisted of a Teflon® block, a piece of Teflon® membrane and a Perspex® block tightly fixed together by screws. The Teflon® block was mounted with a working electrode (a Pt disk with an area of 22.1 mm2). The Teflon® membrane (thickness, 50 μm) with a rectangular hole in its center was sandwiched between the Teflon® block and the Perspex® block to hold ~ 2.5 μL of thin-layer solution. The Perspex® block (1 cm thick ) not only provided an optical window with ~ 75% transmittance in the wavelength range of 400–800 nm, but also accommodated a reference electrode and a counter electrode. The reference electrode was an Ag/AgCl (3 mol/L KCl) electrode, and the counter electrode was a stainless steel pipe located at the solution outlet of the cell.
ECL measurement
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(100 μL) for 15 s using pump I. Meanwhile, the carrier solution was pumped into the ECL detector using pump II. Step 2: pump I was stopped, and the sampling valve was turned to the injection position, the carrier solution was pumped into the loop using pump II to carry the sample solution to the ECL detector for measurement. Pumps I and II were both set at a same rotary speed of 30 cycles/min (~ 1.0 mL/min). Before measurements were taken, the Pt working electrode was pretreated by polishing the surface with aqueous slurries of 0.3 μm alumina particles on a polishing micro-cloth, rinsed with water and then sonicated in ethanol, HNO3 (1 : 1, v/v) and double-distilled water, in turn. Determination of thiamazole in spiked animal feeds First, 100 g of animal feed [we chose chicken feed (sample I), duck feed (sample II) and pig feed (sample III) was spiked with thiamazole at a concentration of 1.15 μg/g, and ground with a mortar and a pestle until a homogeneous mixture was obtained. Then, 1.0 g of the homogeneous sample was mixed with 10.0 mL of methanol in a centrifuge tube, and mechanically shaken for 5 min. After centrifugation at 3600 rpm for 10 min, the supernatant solution was collected and evaporated to dryness at 40 ºC under a gentle stream of nitrogen. The dry residue was dissolved with 5 mL pentane for subsequent purification by solid phase extraction (SPE). In the SPE step, an AccuBond II Florisil SPE Cartridge (1 g, Agilent Technologies) was conditioned with 10 mL of methanol, and then the 5 mL sample was passed through the Florisil SPE Cartridge. After rinsing the column with 5 mL of pentane, elution was performed using 2 mL of methanol/dichloromethane (20 : 80 v/v),
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ECL measurement was carried out in two steps. Step 1: the sampling valve was set at the sample position, and sample solution was pumped into the sampling valve to fill the loop
Figure 2. Schematic diagram of (a) the FI-ECL unit and (b) the ECL flow cell used in the experiment.
D. Kong et al.
ECL response of thiamazole in the presence of Ru(bpy)3
2+
From the results, we found that thiamazole in all types of buffer solution did not exhibit ECL activity. However, it could enhance the ECL emission of Ru(bpy)3 2+ at the Pt electrode when pH > 7.0 (Fig. 3). In the FI–ECL system, we used Ru(bpy)3 2+ solution as the carrier, and a mixture of Ru(bpy)3 2+ and thiamazole as the sample solution. The FI–ECL peak obtained was actually the ECL enhancement (△I) of thiamazole in the Ru(bpy)3 2+ system and its intensity depended on the thiamazole concentration. In order to investigate the ECL behavior of thiamazole in the presence of Ru(bpy)3 2+, the effects of experimental conditions such as the mode of the applied voltage, the potential of the working electrode, the pH, the flow rate of carrier solution and the concentration of Ru(bpy)3 2+ were investigated and discussed. Effect of applied potential. We experimented on different potential modes such as constant potential (DC), double step potential (rectangular wave) and linear sweep potential (triangular wave), in order to examine the ECL response of thiamazole in the presence of Ru(bpy)3 2+. The results showed that thiamazole gave the most sensitive ECL response under the constant potential mode. Thus, the constant potential mode was selected in subsequent ECL investigations. After choosing the potential mode, the effect of voltage on ECL was investigated. In a solution of PBS (pH 12.0) containing 1.0 × 10 4 mol/L Ru(bpy)3 2+, the ECL responses of thiamazole were recorded from 1000 to 1700 mV at 100 mV intervals (Fig. 4). From Fig. 4 we found that that the peak shape and peak height (ECL intensity) were both greatly affected by the applied potential. When the applied potential was too low (< 1100 mV), the ECL peak was split into two small peaks (Fig. 4a,b), which can be explained by the presence of the voltage drop as a result of
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Figure 3. Typical ECL responses obtained for thiamazole in alkaline Ru(bpy)3 5 5 solution. The concentrations of thiamazole were: (a) 1.0 × 10 mol/L, (b) 5.0 × 10 4 4 mol/L and (c) 1.0 × 10 mol/L. Carrier solution: PBS, pH 12.0, containing 1.0 × 10 mol/L 2+ Ru(bpy)3 . Sample solution: thiamazole + carrier solution. Flow rate: 1.0 mL/min. Applied potential: 1500 mV.
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ECL Intensity/mV
Results and discussion
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followed by 5 mL of methanol. The collected eluent was evaporated to dryness at 40 °C under a gentle stream of nitrogen. Finally, the residue was dissolved in 1.0 mL of carrier solution (H3PO4– NaOH; PBS, pH 12.0) containing 1.0 × 10 4 mol/L Ru(bpy)3 2+.
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t /min Figure 4. ECL responses of thiamazole at various applied potentials: (a) 1000, (b) 1100, (c) 1200, (d) 1300, (e) 1400, (f) 1500, (g) 1600 and (h) 1700 mV. (Inset) Plot of the enhanced ECL intensity vs potential. Carrier solution: PBS, pH 12.0, containing 4 2+ 1.0 × 10 mol/L Ru(bpy)3 . Sample solution: thiamazole + carrier solution. Flow rate: 1.0 mL/min.
internal resistance in the thin-layer ECL cell which led to insufficient oxidation of Ru(bpy)3 2+ (23). Then, when the applied potential was > 1200 mV, the ECL peak shape was normal and ECL intensity increased with the potential (Fig. 4c–f); the ECL intensity tended to became invariable at potentials > 1500 mV. However, when the potential was > 1500 mV (Fig. 4g,h), the ECL intensity changed slightly, but more background noise was produced due to the formation of large amounts of oxygen bubbles at the Pt electrode. So an applied potential of 1500 mV was chosen in the subsequent study to obtain the highest ECL signal at the lowest background noise. Effect of pH. The properties of the buffer solution have a great influence on the ECL behavior of the Ru(bpy)3 2+ system (18). In order to choose the best buffer solution, the ECL responses of thiamazole in different media (0.1 mol/L for all test media), such as H3PO4–NaOH, NaHCO3–NaOH, H3BO3–NaOH and Britton–Robinson buffer were investigated. It was found that the ECL response of thiamazole was most sensitive in H3PO4–NaOH (PBS). Therefore, PBS was chosen as the ECL reaction medium. When the pH of the buffer solution was < 7.0, thiamazole had no enhancement effect on ECL of Ru(bpy)3 2+, whereas when the pH changed from 7.0 to 12.0, the ECL intensity increased sharply (Fig. 5). From Fig. 5, we know that thiamazole could greatly enhance the ECL intensity of Ru(bpy)3 2+ in alkaline solution (Fig. 5e,f). However, when the pH was too high, the background noise was higher and the signal-to-noise ratio decreases, which may result from a chemiluminescent reaction between Ru(bpy)3 3+ and the high-energy intermediate (HO2·) electro-generated in the alkaline solution (24). The most satisfactory ratio of signal-to-noise was found at pH 12.0, and so PBS at pH 12.0 was chosen for further ECL study. Effect of flow rate. We found that the flow rate of the carrier solution could influence the ECL behavior of thiamazole significantly. From Fig. 6, we know that the higher the flow rate, the narrower ECL peak (compare Fig. 6a–c). This phenomenon can be explained by the fact that a shorter time is needed for a given volume (100 μL) of sample to pass through the ECL cell at a higher flow rate, giving a narrower ECL peak. From Fig. 6, we also know that the ECL intensity increased obviously from
Copyright © 2014 John Wiley & Sons, Ltd.
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Flow injection analysis of thiamazole with ECL 20
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Figure 5. ECL responses of thiamazole in carrier solution at various pH values (pH ≥ 7.0) (A) 7.0, (B) 8.0, (C) 9.0, (D) 10.0, (E) 11.0 and (F) 12.0. Carrier: H3PO4–NaOH solution 4 2+ containing 1.0 × 10 mol/L Ru(bpy)3 . Sample solution: thiamazole + carrier solution. Flow rate: 1.0 mL/min. Applied potential: 1500 mV.
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Figure 6. ECL responses of thiamazole at various flow rates: (A) 0.5, (B) 1.0 and (C) 2.0 mL/min. Carrier solution: PBS, pH 12.0, containing 1.0 × 10 solution: thiamazole + carrier solution. Applied potential: 1500 mV.
0.5 to 1.0 mL/min, and then increased slightly at flow rates of 1.0–2.0 mL/min. At the same time, the ECL background noise decreased with increasing flow rate, which might result from the relatively lower flow pulse produced by the peristaltic pump at the higher flow rate. To obtain relatively high sensitivity while reducing the use of Ru(bpy)3 2+, a flow rate of 1.0 mL/min was selected.
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2+
mol/L Ru(bpy)3 . Sample
mol/L. Thus, we chose 1.0 × 10 4 mol/L Ru(bpy)3 2+ in carrier solution to reduce the consumption of Ru(bpy)3 2+, but obtain the best signal-to-noise ratio. The optimum conditions for the ECL measurement of thiamazole are shown in Table 1. Linear response range and reproducibility. Under the optimum conditions as mentioned above, a new FI–ECL method for the determination of thiamazole was developed and the concentration effect of thiamazole on its ECL response was studied. The FI–ECL method exhibited a linear response for thiamazole ranging between 2.0 × 10 7 and 1.0 × 10 4 mol/L with a detection limit (S/N = 3) of 5.0 × 10 8 mol/L. The regression equation is △I = 5.2 × 106C + 5.40 with a correlation
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Effect of Ru(bpy)3 2+ concentration. The experiment showed that higher Ru(bpy)3 2+ concentrations in the carrier solution gave both a higher ECL peak and higher background noise. The signal-to-noise ratio increased as the Ru(bpy)3 2+ concentration was increased between 1.0 × 10 5 and 1.0 × 10 4 mol/L, whereas it hardly changed between 1.0 × 10 4 and 1.0 × 10 3
4
D. Kong et al. Table 1. Optimum conditions for FI-ECL response of thiamazole Experimental items
Optimum conditions
Mode of applied voltage signal Applied potential Buffer solution Flow rate Concentration of Ru(bpy)3 2+
DC +1500 mV 0.1 mol/L PBS pH 12.0 1.0 mL/min 1.0 × 10 4 mol/L
Table 2. Results for thiamazole determination in spiked animal feed sample solutions using the proposed FI-ECL method Sample
Spiked (μmol/L)
I II III
10 10 10
Found (μmol/L)a
RSD (%)
9.41 ± 0.25 9.32 ± 0.35 9.59 ± 0.11
2.76 3.99 1.25
Recovery (%)a 94.1 ± 2.5 93.2 ± 3.5 95.9 ± 1.1
a
coefficient, r = 0.9944 (n = 6). Where △I is the enhanced ECL intensity and C is the concentration of thiamazole. We also investigated the reproducibility of the method and found good reproducibility with relative standard deviations (RSD) of 0.75% for 10 successive tests of 5 × 10 7 mol/L thiamazole.
The uncertainty values were estimated using the expression ± tn 1 s/√n, where n is the number of replicate measurements, tn 1 is the statistic parameter often called Student’s t (with n = 5, at the 95% level of confidence, t = 2.132) and s is the standard deviation (SD).
Sample analysis and recovery study Possible mechanism for the enhanced ECL behavior of thiamazole From the published reviews, we know that most ECL responses of organic species in the Ru(bpy)3 2+ system result from ECL reactions that include: (1) electrochemical oxidation of the organic species and Ru(bpy)3 2+, producing strong reducing intermediates (usually neutral radical species) and strong oxidizing Ru(bpy)3 3+; (2) followed by high-energy electron transfer between the strong reducing intermediates and Ru(bpy)3 3+, resulting in generation of the excited-state luminophore, Ru(bpy)3 2+*; and (3) finally, light emission when the excited-state (Ru(bpy)3 2+*) returns to its ground state (Ru (bpy)3 2+) Ru(bpy)3 2+ (18,20,25). The ECL response of thiamazole in the presence of Ru(bpy)3 2+ probably includes a similar ECL mechanism (Fig. 7). Previous electrochemical research has demonstrated that thiamazole can be oxidized to form highly reductive radicals (Fig. 7, II) (15,26). The oxidation peak is ~ 0.45 V, which is much lower than that of Ru(bpy)3 2+ (~ 1.20 V) (27). It is apparent that under the optimum potential (1.50 V) for ECL of the thiamazole/Ru(bpy)3 2+ system, both neutral radicals (species II) and Ru(bpy)3 3+ are produced. The further ECL reaction between them produces the excited-state Ru(bpy)3 2+*, which results in the ECL emission. The final oxidation product of the neutral radical, species II, in the alkaline media might be sulfonate (species III) (15).
In order to evaluate the applicability the FI–ECL method to real samples, it was applied to the determination of thiamazole in animal feed. After purification by SPE, spiked samples were tested using the developed FI–ECL method. The average results of five replicate measurements expressed as the 95% confidence interval obtained for animal feed samples spiked with thiamazole using the proposed method are summarized in Table 2. The recoveries of the analyte in samples I, II and III were 94.1 ± 2.5, 93.2 ± 3.5 and 95.9 ± 1.1%, respectively, at 1.15 μg/g thiamazole. The limit of detection (LOD) for thiamazole determined at a signal-to-noise ratio of 3 was ~ 5.7 μg/kg for all three samples, which is lower than the LOD for many previously reported methods, such as FI–spectrophotometry (~ 340 μg/kg) (4), FI–CL (~ 1 mg/kg) (5), GC–MS (< 100 μg/kg) (12) and electrochemical methods (~ 58 μg/kg) (15), and almost matched the HPLC method (~ 3.5 μg/kg) (8). However, the LOD was higher than that obtained with resonance light-scattering spectroscopy (~ 0.2 μg/kg) (6) and CdTe QDs ECL (~ 0.1 μg/kg) (19). From these results, the FI–ECL method was deemed applicable for the determination of thiamazole in animal feed samples.
Conclusion Thiamazole, a well-known antithyroid drug has been found to have strong ECL activity in the Ru(bpy)3 2+/co-reactant ECL system. Thiamazole could produce sensitive, stable FI–ECL signals in the presence of Ru(bpy)3 2+ in alkaline media. Experimental conditions such as applied potential, buffer solution, the flow rate of the carrier solution and the concentration of Ru(bpy)3 2+ were found to influence the FI-ECL intensity. After the optimization of experimental conditions, a new sensitive, simple, rapid and accurate FI–ECL method with a wide dynamic range for the detection of thiamazole in animal feed has been established. Acknowledgements
2+
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Figure 7. Proposed ECL mechanism of thiamazole in the presence of Ru(bpy)3 .
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This study was financially supported by National Natural Science Foundation of China (21075018), Program for New Century Excellent Talents in Chinese University (NCET-10-0019), and Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1116).
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Flow injection analysis of thiamazole with ECL
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