Flow Injection Analysis System Equipped with a Newly Designed

Jan 24, 2006 - Determination of Atenolol and Metoprolol by Capillary Electrophoresis with Tris(2,2′-bipyridyl)ruthenium(II) Electrochemiluminescence...
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Anal. Chem. 2006, 78, 1568-1573

Flow Injection Analysis System Equipped with a Newly Designed Electrochemiluminescent Detector and Its Application for Detection of 2-Thiouracil Yuwu Chi, Jianping Duan, Shudan Lin, and Guonan Chen*

The MOE Key Laboratory of Analysis and Detection Technology for Food Safety, and Department of Chemistry, Fuzhou University, Fuzhou, Fujian, 350002, China

A new flow injection analysis (FIA) system equipped with an electrochemiluminescent (ECL) detector has been developed and applied for the ECL detection of 2-thiouracil. The FIA-ECL system used a specially designed flowthrough ECL thin-layer cell to reduce the dead volume, the IR drop across the cell, and the probability of accumulation of gas bubbles in the cell. It was thus envisioned to improve the detection limit of the FIA-ECL method. After being established, the new FIA-ECL system was used to investigate the ECL response of 2-thiouracil in the presence of the ECL of Ru(bpy)32+. It was found that 2-thiouracil could enhance the ECL of Ru(bpy)32+ over a wide pH range (pH 4.0-12.0). A highly sensitive method for detection of 2-thiouracil in biological samples was developed by the new FIA-ECL system after optimizing several experimental conditions, such as the applied potential of the working electrode, the pH value of the aqueous solution, the flow rate of carrier solution, and the concentration of Ru(bpy)32+. 2-Thiouracil (see Scheme 1) is a substance with potent biological and pharmacological activities. 2-Thiouracil and its derivatives exhibit high antithyroid activities and have been used for the treatment of hyperthyroidism.1 It has been found that 2-thiouracil and its derivatives readily incorporate into the nucleic acid and thus display antivirus and antitumor activities.2-4 Especially, 2-thiouracil can selectively incorporate into growing melanins; therefore, it acts as a highly specific melanoma seeker and can be used for early diagnosis and chemotherapy of disseminated melanoma.5,6 Unfortunately, it has been reported that this kind of drug might induce agranulocytosis complicated by life-threatening * Corresponding author. E-mail: [email protected]. Fax: 86-87893315. (1) Cooper, D. S. N. Engl. J. Med. 2005, 352, 905-919. (2) Lindsay, R. H.; Romine, C. J.; Wong, M. Y. Arch. Biochem. Biophys. 1968, 126, 812-820. (3) Yu, M. Y. W.; Sedlak, J.; Lindsay, R. H. Arch. Biochem. Biophys. 1973, 155, 111-119. (4) Bretner, M.; Kulokowski, T.; Dzik, J. M.; Balinska, M.; Rode, W.; Shugar, D. J. Med. Chem. 1993, 36, 3611-3617. (5) Watjen, F.; Buchardt, O.; Langvad, E. J. Med. Chem. 1982, 25, 956-960. (6) Napolitano, A.; Palumbo, A.; D’Ishchia, M.; Prota, G. J. Med. Chem. 1996, 39, 5192-5201.

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Scheme 1. Main Tautomeric Forms of 2-Thiouracil

infection in a few patients.7,8 Therefore, a rapid and sensitive method to determine 2-thiouracil at a low level is of considerable interest for pharmacokinetic and clinical studies. On the other hand, the use of 2-thiouracil and its derivatives (thyreostatic drugs) for promoting animal growth is prohibited in most countries, such as the EU (Directive 86/469/EEC) and Australia (Livestock Regulations 1998) for they may be harmful to human health. To control the illegal use of this type of drug in livestock, it is also necessary to develop a sensitive method to detect them. The available methods for the determination of 2-thiouracil include spectrophotometric,9,10 electrochemical analysis,11-15 chemiluminescence,16 and mass spectrometry.17 To the best of our knowledge, no attention has been paid to determine 2-thiouracil by electrochemiluminescence (ECL).18 Since ECL has been (7) Suzuki, S.; Ichikawa, K.; Nagai, M.; Mikoshiba, M.; Mori, J.; Kaneko, A.; Sekine, R.; Asanuma, N.; Hara, N.; Nishii, Y.; Yamauchi, K.; Aizawa, T.; Hashizume, K. Arch. Intern. Med. 1997, 157, 693-696. (8) Sheng, W. H.; Hung, C. C.; Chen, Y. C.; Fang, C. T.; Hsieh, S. M.; Chang, S. C.; Hsieh, W. C. QJM 1999, 92, 455-461. (9) Moretti, G.; Betto, P.; Cammarata, P.; Fracassi, F.; Giambenedetti, M. J. Chromatogr. 1993, 616, 291-296. (10) Krivankova, L.; Krasensky, S.; Bocek, P. Electrophoresis 1996, 17, 19591963. (11) Wrona, M. Z. Bioelectrochem. Bioenerg. 1983, 10, 169-183. (12) Ahmed, Z. A.; Ahmed, M. E.; Ibrahim, M. S.; Kamal, M. M.; Temerk, Y. M. Talanta 1994, 41, 659-662. (13) Guzman, A.; Agui, L.; Pedrero, M.; Yanez-Sedeno, P.; Pingarron, J. M. Talanta 2002, 56, 577-584. (14) Shahrokhian, S.; Hamzehloei, A.; Thaghani, A.; Mousavi, S. R. Electroanalysis 2004, 16, 915-921. (15) Goyal, R. N.; Singh, U. P.; Abdullah, A. A. Bioelectrochemistry 2005, 67, 7-13. (16) Vinas, P.; Garcia, I. L.; Gil, J. A. J. M. Pharm. Biomed. 1993, 11, 15-20. (17) Zhang, L.; Liu, Y.; Xie, M. X, Qiu, Y. M. J. Chromatogr., A 2005, 1074, 1-7. (18) Bard, A. J.; Faulkner, L. R. Electrochemical methods: fundamentals and applications, 2nd ed.; Wiley: New York, 2001. 10.1021/ac051508t CCC: $33.50

© 2006 American Chemical Society Published on Web 01/24/2006

confirmed to be a powerful analytical method, having high sensitivity and wide response for many analytes,19-21 herein, the ECL behavior of 2-thiouracil was studied using a new flow injection analytical system equipped with an electrochemiluminescent detector (FIA-ECL), based on which a rapid and sensitive FIAECL detection method for 2-thiouracil was established. The new FIA-ECL system was established by overcoming several drawbacks of conventional FIA-ECL systems. ECL thinlayer flow cells were often used in the electrochemiluminescent detection for FIA and high-performance liquid chromatography (HPLC). Since it is rare to find ECL cells that can be commercially provided, most researchers have made their own ECL cells and then established different ECL detection systems by combining their homemade ECL cells with other commercially available parts such as potentiostats, light sensing devices (e.g., PMT), and flow injection units. The ECL thin-layer flow cells made by different researchers might be quite different in appearance;22-26 however, most of them were derived from the same basic design as shown in Figure 1. The working electrode (WE) was placed in a thinlayer solution, the counter electrode (CE) was usually a stainless steel pipe near the outlet of solution, and the reference electrode was located near the inlet (Figure 1A)22 or the outlet (Figure 1B) of the solution.23-27 This type of ECL flow cell had the following drawbacks: (1) large dead volume, especially when the reference electrode was placed near the inlet (Figure 1A), would reduce both the detection sensitivity and separation efficiency of sample in HPLC; (2) high IR drop, especially when the reference electrode was located at the downstream (Figure 1B), caused high overpotential and thus decreased the sensitivity of ECL detection; (3) high flow resistance made it difficult to remove away possible gas bubbles, which would cause significant noise.23 To overcome the above drawbacks, a new type of ECL flow cell with small dead volume, IR drop, and flow resistance was designed and used in our homemade FIA-ECL system. After being established, the new FIA-ECL system was adopted to investigate the ECL response of 2-thiouracil in the presence of the ECL of Ru(bpy)32+ and further detect 2-thiouracil in biological samples. EXPERIMENTAL SECTION Chemicals and Solutions. Tris(2,2′-bipyridine)ruthenium(II) chloride hexahydrate (Ru(bpy)3Cl2‚6H2O) was obtained from Aldrich. 2-Thiouracil was purchased from Sigma. Other chemicals were analytical grade or better. Double-distilled water was used to prepare sample solutions. A 1 × 10-3 mol L-1 stock solution of Ru(bpy)3Cl2 was prepared by dissolving 0.0749 g of Ru(bpy)3Cl2‚6H2O with 100 mL of doubledistilled water, and the resultant mixture was stored under (19) Gerardi, R. D.; Barnett, N. W.; Lewis, S. W. Anal. Chim. Acta 1999, 378, 1-41. (20) Yin, X.; Dong, S.; Wang, E.; TrAC-Trends Anal. Chem. 2004, 23, 432441. (21) Richter, M. M. Chem. Rev. 2004, 104, 3003-3036. (22) Yi, C.; Tao, Y.; Chen, X. Anal. Chim. Acta 2005, 541, 75-83. (23) Chi, Y.; Duan, J.; Zhao, Z. F.; Chen, H.; Chen, G. Electroanalysis 2003, 15, 208-218. (24) Li, F.; Cui, H.; Lin, X. Q. Anal. Chim. Acta 2002, 471, 187-194. (25) Ridlen, J. S.; Skotty, D. R.; Kissinger, P. T.; Nieman, T. A. J. Chromatogr., B 1997, 694, 393-400. (26) Ridlen, J. S.; Klopf, G. J.; Nieman, T. A. Anal. Chim. Acta 1997, 341, 195204. (27) Zhu, L.; Zhu, G. Anal. Sci. 2003, 19, 575-578.

Figure 1. Schematic diagram of the conventional ECL thin-layer flow cell.

refrigeration. A 1 × 10-2 mol L-1 stock solution of 2-thiouracil was prepared by adding 0.0128 g of sample in 10 mL of water and storing in the refrigerator before use. Carrier solution was prepared by diluting appropriate volume of the above Ru(bpy)3Cl2 stock solution with phosphate buffer solution. Sample solution was prepared by diluting the required volume of the 2-thiouracil stock solution with the carrier solution. The phosphate buffer solutions (pH 2-12) were prepared by titrating 0.1 mol L-1 phosphoric acid solutions with sodium hydroxide to the required pH. Apparatus. The electrochemiluminescent experiments were performed on a new homemade FIA-ECL system (see Figure 2), consisting of a flow injection unit and an ECL detection unit. The flow injection unit was an LZ-2000 flow injection processor, the details of which have been described previously.28 The ECL detection unit included the following sections: a GD-1 chemiluminescent detector (Ruike Electronic Instrument Ltd. Co.), a potentiostat, a HW chromatography station (Qianpu Ltd. Co.), and an ECL flow-through cell. The potentiostat was a model 400 electrochemical detector (EG&G), which could perform dc, linear, and differential pulse sweeps; The HW chromatography station was used for transferring the analog ECL signals from the output of the GD-1 chemiluminescent detector into the digital ECL signals recorded by a computer. The ECL flow cell is a new type of thinlayer flow-through cell, the detailed fabrication of which is described below. (28) Chen, G. N.; Duan, J. P.; Hu, O. F. Anal. Chim. Acta 1994, 292, 159-167.

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Figure 2. Block diagram of the components needed for the FIAECL detection.

Figure 3. Schematic diagram of the new ECL thin-layer flow cell.

As shown in Figure 3, the new ECL flow cell consisted of a quartz cup, a capillary inlet, a stainless steel pipe outlet, a Ag/ AgCl reference electrode (RE), a Pt ring working electrode (WE), and a stainless steel counter electrode (CE). The quartz cup was used to contain buffer solution for the three-electrode system and served as an optical window (the bottom part). The CE was a stainless steel pipe located at the outlet of the solution, the outer wall of which was covered by a black plastic jacket to prevent undesired ECL emission on it. The working electrode was a Pt ring electrode, which was fabricated as follows: At one end of a glass capillary (0.5-mm i.d., 1.00-mm o.d.), a Pt wire (0.2 mm in diameter) was wound around the outer wall of the capillary. The wound Pt wire was then coated with an epoxy resin thin layer (0.1 mm thick) to insulate most of the Pt wire except for the surface near the outlet of the capillary. Finally, a Pt ring of 1.0mm i.d. and 1.4-mm o.d. was obtained by polishing the outlet surface.29 The thin-layer cell was installed in the following way: The compound capillary (immobilized with Pt ring) was inserted into the quartz cup containing buffer solution until its tip contacted with the bottom of the quartz cup. Then the compound capillary was shaken slightly; hence a thin-layer solution was formed between the tip of the compound capillary and the bottom of the quartz cup, the thickness of which was not more than 50 µm. The thin layer covered a ring surface area (0.5-mm i.d. and 1.6-mm (29) Chen, G.; Chi, Y.; Wu, X.; Duan, J.; Li, N. Anal. Chem. 2003, 75, 66026607.

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Figure 4. Distribution of the thin layer at the tip of Pt ringimmobilized capillary.

o.d.) from the inner wall of the capillary to epoxy resin layer (see Figure 4). ECL measurement. The FIA-ECL manifold is shown in Figure 2. The ECL measurement was carried out in two steps. Step 1: the sample solution was pumped into the sampling valve to fill the loop (100 µL) for 15 s by pump I. Simultaneously, the carrier solution was pumped into the ECL detector by pump II. Step 2: Pump I was stopped, and the sampling valve was turned to injection position; the carrier solution was pumped to the loop by pump II to take the sample solution to ECL detector for measurement. The rates for both pump I and pump II were 30 turns/min. Before experiments, the working electrode was well polished with fine aluminum powder and then cleaned by ultrasonic bath. Determination of Thiouracil in Spiked Meat. First, 250 g of previously chopped sample (pork or beef) bought from a local Walmart supermarket (Sam’s Club, Fuzhou, China) was homogenized and spiked with thiouracil at the 100 µg/kg level. Then 0.5 g of the spiked sample was accurately weighed and purified, in turn, by matrix solid-phase dispersion (MSPD)17 and solid-phase extraction (SPE).13 These purification procedures were accomplished with the help of a 10-port vacuum manifold processing station (Agilent Technologies). In MSPD step, the above 0.5 g of spiked sample and 2.0 g of silica gel of 50 µm (YMC, Kyoto, Japan) were placed into a mortar and blended with a pestle until a homogeneous mixture was obtained. After 30 min, the mixture was moved into a 10-mL syringe barrel and packed as the MSPD column. The MSPD column was washed with 10 mL of chloroform and eluted with 5 mL of methanol/chloroform (v/v ) 25:75). The collected eluate in a tube was evaporated to dryness at 40 °C under a gentle stream of nitrogen, and the dried sample was then dissolved in 5 mL of methanol. In the SPE step, an AccuBond II Florisil SPE cartridge (1 g, Agilent Technologies) was previously conditioned with 10 mL of methanol, and then the 5-mL sample was passed three times through the Florisil SPE cartridge. The efluent was collected into a tube and evaporated to dryness at 40 °C under a gentle stream of nitrogen. Finally, 0.5 mL of testing solution was prepared by dissolving the purified sample with pH 12.0 phosphate buffer solution containing 1× 10-4 mol L-1. ECL measurement for 2-thiouracil was carried out at +1.5 V after injecting 100 µL of the testing solution into the FIA-ECL system.

RESULTS AND DISCUSSION Characteristics of the Proposed FIA-ECL System. By comparison with those conventional ECL flow cells (see introduction), our designed ECL flow cell (described in Experimental Section) for FIA has several apparent advantages. The new ECL flow cell has overcome the drawbacks of the conventional ECL flow cells as follows: First, the new ECL flow cell has a very small dead volume. The volume of the thin layer is estimated to be less than 100 nL, which is ∼1-2 orders less than those of the conventional ECL flow cells (normally varying from 1 to 30 µL).22-27,30 Therefore, the newly designed ECL flow cell will decrease the dilution of samples and thus improve the detection sensitivity for FIA; moreover, it can be used as an ECL detector for HPLC and even high-performance capillary electrophoresis (HPCE).29 Second, the new ECL flow cell has a very low IR drop. In the new design (see Figure 3), the reference electrode and counter electrode are set in the bulk solution, and only the working electrode is located in the narrow thin layer (the distance between the edge of working electrode and bulk solution is ∼0.1 mm), resulting in a much lower IR drop in comparison with the conventional ECL flow cells (see Figure 1). Apparently, the overpotential will be greatly decreased and the efficiency of ECL reaction will be thus improved. Third, the new ECL flow cell has a low flow resistance. The low flow resistance resulting from the use of the narrow thin layer in the ECL flow cell (see Figure 4) makes it difficult for the electrochemically generated gas (usually, oxygen) to accumulate in the thin layer. Therefore, it is favorable to remove the significant noise problem when using strongly alkaline media in ECL measurements23 and enlarges application of ECL in critical pH conditions. To demonstrate the advantages of the proposed FIA-ECL system, especially the improvement of sensitivity over existing methods, a drug of study interest, thiouracil, was detected by the FIA-ECL system. Observation of ECL Behavior of 2-Thiouracil by the FIAECL System. The primary experimental results showed that 2-thiouracil itself did not exhibit ECL activity over the examined pH range (pH 2-13). However, it could enhance the ECL emission of Ru(bpy)32+ at the Pt electrode at pH >4.0. The enhanced ECL intensity (∆I) was obtained by subtracting the ECL background of Ru(bpy)32+ (in the absence of 2-thiouracil) from the total ECL (in the presence of both Ru(bpy)32+ and 2-thioracil). In the FIAECL measurements, Ru(bpy)32+ solution was used as the carrier solution and 2-thiouracil diluted with the Ru(bpy)32+ carrier solution was used as the sample solution. Therefore, the FIA-ECL intensity obtained was actually the relative (enhanced) intensity, ∆I. In the present study, the FIA-ECL measurement system shown in Figure 2 was used to investigate the ECL response of 2-thiouracil in the presence of Ru(bpy)32+ under various experimental conditions. (1) Effect of Applied Potential. Several potential signals such as a constant potential (dc), a triangular potential scanning, and a double-step pulse voltage scanning were used to examine the ECL response of 2-thiouracil in the presence of Ru(bpy)32+. The experimental results show that 2-thiouracil has a more sensitive ECL response under the constant-potential mode than the others. Thus, the constant-potential mode was adopted for further ECL measurements.

Additionally, the ECL response of 2-thiouracil is strongly dependent on the voltage of the applied potential at the Pt ring working electrode. First, there is no FIA-ECL peak of 2-thiouracil until the potential is higher than + 900 mV (Figure 5A a). Then, a further increase in the voltage of the applied potential leads to a sharp increase in ECL intensity in the voltage range of +1000 to +1400 mV (Figure 5A b-d). Finally, the increase of ECL peak height is slowed after reaching +1500 mV (Figure 5A e, f). To avoid the formation of a large amount of oxygen gas at the Pt ring electrode at higher potential, +1500 mV was selected for further ECL investigation. (2) Effect of pH. The effect of pH on the ECL response of 2-thiouracil was studied using various pH solutions. The FIA-ECL signals of 2-thiouracil in nine different phosphate buffer solutions (pH 4.0-12.0) are shown in Figure 6 A-I, respectively. Figure 6 demonstrates that 2-thiouracil has an enhanced ECL response over a very wide pH range. The FIA-ECL intensity varies with pH. In weakly acidic, neutral, and weakly alkaline media (pH 4.0-10.0), 2-thiouracil has a relatively weaker FIA-ECL response. In contrast, in strongly alkaline media (pH >10.0), 2-thiouracil gives a strong FIA-ECL response. The ECL intensity was found to be increased sharply from pH 10.0 to pH 12.0. This phenomenon implies that the strongly alkaline media favor the formation of deprotonated radical essential to the ECL.19,31 It can be known from Figure 6 that the FIA-ECL response is quite strong at pH 12.0. This suggests that a sensitive FIA-ECL method for detecting 2-thiouracil can be developed under this pH condition. Although the ECL intensity can be further increased using higher pH, to avoid the corrosion of FIA-ECL system by strongly alkaline solution, pH 12.0 PBS was selected for further ECL measurements. (3) Effect of Flow Rate. The effect of flow rate on the ECL response of 2-thiouracil was investigated using different flow rates. The FIA-ECL response at the flow rates of 0.5, 1.0, and 2.0 mL min-1 are shown in Fig. 7. It can be known from Figure 7 that a higher flow rate produces narrower FIA-ECL peaks (e.g., compare Figure 7A with Figure 7C). 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, thus giving rise to the narrower FIA-ECL peaks. The ECL peak height is also dependent on the flow rate. The ECL intensity is increased obviously from 0.5 to 1.0 mL min-1 and then increased relatively slowly in the flow rate range of 1.0-2.0 mL min-1. Additionally, the ECL background (noise) was found to be decreased with increasing flow rate, which might result from that of a relatively lower flow pulse was produced by the peristaltic pump at the higher flow rate. To obtain a relatively high sensitivity while saving the use of Ru(bpy)32+, the flow rate of 1.0 mL min-1 was selected. (4) Effect of Ru(bpy)32+ Concentration. An increase in the concentration of Ru(bpy)32+ results in the enhancement of ECL intensity of 2-thiouracil. However, it also leads to an increase of ECL background (from Ru(bpy)32+ itself). The ratio of signal-tonoise was found to be significantly increased with increasing Ru(bpy)32+ concentration in a relatively lower concentration range (1.0 × 10-6-1.0 × 10-4 mol L-1) but was slightly improved in the higher Ru(bpy)32+ concentration domain (1.0 × 10-4-1.0 × 10-3

(30) Martin, A. F.; Nieman, T. A. Biosens. Bioelectron. 1997, 12, 479-489.

(31) Wu, X.; Huang, F.; Duan, J.; Chen, G. Talanta 2005, 65, 1279-1285.

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Figure 5. Effect of the applied potential on the ECL response of 2-thiouracil. (A) ECL responses at (a) 900, (b) 1000, (c) 1100, (d) 1200, (e) 1300, (f) 1400, (g) 1500, and (h) 1600 mV. (B) Plot of the enhanced ECL intensity vs potential. Sample: 1 × 10-4 mol L-1 2-thiouracil + 1 × 10-4 mol L-1 Ru(bpy)32+ (pH 12). Carrier solution: 1 × 10-4 mol L-1 Ru(bpy)32+ (pH 12); flow rate 1 mL min-1.

Figure 6. ECL response of 2-thiouracil at various pH: (A) 4.0, (B) 5.0, (C) 6.0, (D) 7.0, (E) 8.0, (F) 9.0, (G) 10.0, (H) 11.0, and (I) 12.0. Sample: 1 × 10-4 mol L-1 2-thiouracil + 1 × 10-4 mol L-1 Ru(bpy)32+. Carrier solution: 1 × 10-4 mol L-1 Ru(bpy)32+. Flow rate: 1 mL min-1. Applied potential: +1500 mV.

mol L-1). Therefore, 1.0 × 10-4 mol L-1 Ru(bpy)32+ was used for the later ECL measurements. Finally, the optimum conditions for the ECL measurement of 2-thiouracil are listed in Table 1. (5) Effect of 2-Thiouracil Concentration and Linear Response Range. Under the optimum experimental conditions shown in Table 1, the dependence of the ECL response of 2-thiouracil on its concentration was investigated. It was found 1572

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Figure 7. ECL response of 2-thiouracil at various flow rates: (A) 0.5, (B) 1.0, and (C) 2.0 mL min-1. Sample: 1 × 10-4 mol L-1 2-thiouracil + 1 × 10-4 mol L-1 Ru(bpy)32+ (pH 12). Carrier solution: 1 × 10-4 mol L-1 Ru(bpy)32+ (pH 12). Applied potential: +1500 mV.

that there was a good linear relationship between the enhanced ECL intensity and the concentration of 2-thiouracil in the range of 1.0 × 10-6-1.0 × 10-4 mol L-1. The regression equation is shown in eq 1 with a correlation coefficient, r ) 0.997 (n ) 10).

∆I/mV ) (32.7 ( 1.0) + (8.62 ( 0.43)(C/10-6 mol L-1) (1) where ∆I is the enhanced ECL intensity and C is the concentration

Table 1. Optimum Conditions for FI-ECL Response of 2-Thiouracil experimental items

optimum conditions

mode of applied voltage signal applied potential buffer flow rate concentration of Ru(bpy)32+

dc mode +1500 mV pH 12.0 PBS 1.0 mL min-1 1.0 × 10-4 mol L-1

of 2-thiouracil. The detection limit of 2-thiouracil was found to be 3.7 × 10-8 mol L-1 when the ratio of signal-to-noise was 3. Possible Mechanism for the Enhanced ECL Response of 2-Thiouracil. It has been well demonstrated that most ECL responses of organic species in the Ru(bpy)33+ system result from that the electrochemical oxidation of these species producing strong reducing intermediates, usually neutral radical species, which react with Ru(bpy)33+ to generate excited-state Ru(bpy)32+* and lead to light emission.19,31-42 The ECL response of 2-thiouracil in the presence of Ru(bpy)32+ probably includes a similar neutral radical mechanism. It has been reported that the electrochemical oxidation of 2-thiouracil and its derivatives readily produces highly reductive radicals (see III in Scheme 2).15,43-45 The oxidation peak potential of 2-thiouracil is ∼+0.30 V at pH 12,15 which is much lower than that of Ru(bpy)32+, ∼+1.20 V.31 It is apparent that under the optimum potential, +1.50 V, for the ECL of the 2-thiouracil/ Ru(bpy)32+ system, both neutral radical (species III) and Ru(bpy)33+ are produced. The further ECL reaction between them produces the excited-state Ru(bpy)32+*, which results in the ECL emission. The final oxidation product of neutral radical, species III, in the alkaline media might be sulfonate (species IV).15 Determination of 2-Thiouracil in Spiked Meat by the FIAECL System. To demonstrate the applicability of the proposed FIA-ECL system for detecting 2-thiouracil in biological system, 2-thiouracil-spiked samples (pork and beef) were prepared and analyzed. After purified in turn by MSPD and SPE, the spiked samples were tested by our proposed FIA-ECL system. The standard addition method was used to calibrate the concentration of 2-thiouracil in samples for decreasing the effect of matrix. The mean recoveries of 2-thiouracil in pork and beef were found to be 87 and 91%, respectively, at the 100 µg/kg 2-thiouracil level. The relative standard deviations were 4.2 and 3.7% (n ) 5) for detecting that amount of 2-thiouracil in pork and beef samples, (32) Kirch, M.; Lehn, J.-M.; Sauvage, J.-P. Helv. Chim. Acta 1979, 62, 13451384. (33) DeLaive, P. J.; Sullivan, B. P.; Meyer, T. J.; Whitten, D. G. J. Am. Chem. Soc. 1979, 101, 4007-4008. (34) Chandrasekaran, K.; Whitten, D. G. J. Am. Chem. Soc. 1980, 102, 51195120. (35) Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc. 1981, 103, 512-516. (36) Noffsinger, J. B.; Danielson, N. D. Anal. Chem. 1987, 59, 865-868. (37) Noffsinger, J. B.; Danielson, N. D. J. Chromatogr. 1987, 387, 520-524. (38) Brune, S. N.; Bobbitt, D. R. Talanta 1991, 38, 419-424. (39) Laloo, D.; Mahanti, M. K. J. Chem. Soc., Dalton Trans. 1990, 311-313. (40) Brune, S. N.; Bobbitt, D. R. Anal. Chem. 1992, 64, 166-170. (41) Chen, X.; Sato, M. Anal. Sci. 1995, 11, 749. (42) Chen, X.; Jia, L.; Wang, X.; Hu, G. Anal. Sci. 1997, 13, 71. (43) Po, H. N.; Lo, C. F.; Jones, N.; Lee, R. W. Inorg. Chim. Acta 1980, 46, 185-189. (44) Holzer, K. P.; Wrona, M. Z. Bioelectrochem. Bioeng. 1983, 10, 199-212. (45) Holzer, K. P.; Wrona, M. Z. Bioelectrochem. Biogeng. 1983, 11, 3-13.

Scheme 2. Proposed Mechanism for the ECL Reaction of 2-Thiouracil with Ru(bpy)32+

respectively. The limit of detection (LOD) for 2-thiouracil determined at a signal-to-noise ratio of 3 was found to be 5 µg/kg for both pork and beef samples. The LOD for 2-thiouracil by this proposed method is better than other reported methods, such as CE-UV (100 µg/kg),10 FIA-EC (37 µg/kg),13 and GC.MS (10 µg/ kg).17 Apparently, the method is able to detect 2-thiouracil in animal meat at less than 20 µg/kg, the Action Level of antithyroid drugs defined by the U.K. and other countries. CONCLUSIONS A new FIA-ECL system has been developed to overcome some drawbacks of conventional FIA-ECL systems. The new FIA-ECL system used a specially designed flow-through ECL thin-layer cell to reduce the dead volume, the IR drop, and the probability of accumulation of gas bubbles in the cell. Hence, the new FIA-ECL system was envisioned to improve the detection limit of FIA-ECL and probably be used as the ECL detector for both HPLC and HPCE. After being established, the new FIA-ECL system was used to investigate the ECL behavior of 2-thiouracil. The experimental results showed that 2-thiouracil itself had an ECL response in the presence of Ru(bpy)32+ over a wide pH range (pH 4.0-12.0). The ECL response of 2-thiouracil was influenced by the applied potential of the working electrode, pH value of the media, flow rate of the carrier solution, and concentration of Ru(bpy)32+ and 2-thiouracil. After optimizing these experimental conditions, a sensitive FIA-ECL method has been developed for the determination of 2-thiouracil in some biological samples. ACKNOWLEDGMENT This project was financially supported by the National Nature Sciences Funding of China (20377007, 20575011), the Science Foundation of State Education Department (20040386002). Received for review August 23, 2005. Accepted December 26, 2005. AC051508T Analytical Chemistry, Vol. 78, No. 5, March 1, 2006

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