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Flow-injection chemiluminescence determination of tetracyclines with in situ electrogenerated bromine as the oxidant
Analytica Chimica Acta 440 (2001) 143–149
Flow-injection chemiluminescence determination of tetracyclines with in situ electrogenerated bromine as the oxidant Xingwang Zheng a , Yang Mei b , Zhujun Zhang a,∗ a
Department of Chemistry, Shaanxi Normal University, Xi’an 710062, PR China b Liaoning Normal University, Dalian, PR China
Received 2 August 2000; received in revised form 19 March 2001; accepted 12 April 2001
Abstract By designing a novel flow-through electrolytic cell (FEC), bromine was produced near to the surface of the platinum electrode by electrochemical oxidation of acidic KBr. The fast and weak chemiluminescence signal produced by the chemical reaction of the electrogenerated bromine with H2 O2 was greatly enhanced by tetracyclines Based on these observations, a new, sensitive and simple electrogenerated chemiluminescence (ECL) method for the determination of tetracyclines was developed. Under the optimum experimental conditions, the calibration graphs are linear over the range 3.0 × 10−8 to 5.0 × 10−5 g ml−1 for tetracycline, 2.0 × 10−7 to 2.4 × 10−5 g ml−1 for oxytetracycline and 1.0 × 10−7 to 5.0 × 10−5 g ml−1 for chlortetracycline. The limits of detection (S/N = 3) are 1.0 × 10−8 g ml−1 for tetracycline, 7.0 × 10−8 g ml−1 for oxytetracycline and 1.5 × 10−7 g ml−1 for chlortetracycline. For the determination 5.0 × 10−7 g ml−1 tetracycline, the relative standard deviation was <5%. The proposed method was used to determine tetracyclines in pharmaceutical formulations. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Electrogenerated bromine; Flow-injection analysis; Tetracyclines; Chemiluminescence
1. Introduction Tetracyclines are broad-spectrum antibiotics, which possess a hydronaphthacene skeleton with a wide variety of functional groups. They are commonly used in veterinary medicine, animal nutrition and as feed additives. Tetracyclines are determined mainly by chromatographic [1–3], spectrophotometric [4–7], spectrofluorimetric [8,9] and electrochemical methods [10]. In recent years flow-injection (FI) chemiluminescence (CL) methods have received much attention and have been used for the determinations of many ∗
Corresponding author. Tel.: +86-29-530-8748; fax: +86-29-530-8748. E-mail address: [email protected] (Z. Zhang).
analytes [11–16] since this technique offer excellent sensitivity a wide linear dynamic range and only simple instrumentation is required. Usually, the CL can be achieved by direct reaction or by energy-transfer mechanisms [17,18]. Nevertheless, the energy-transfer type of CL system has received attention since it is based on the vast knowledge of spectrofluorimetry and also owned the good selectivity inherent in spectrofluorimetry. However, up to now, only a few FI-based direct CL methods have been used for the determination of tetracyclines [19–24]. For example, Alwarthan and Townshend [20] have reported a bromine-based FI–CL method for tetracycline, but it needed a complex procedure for preparing the bromine standard solution. Halvatzis et al. [22] reported another sensitive CL method for tetracyclines; this method was based on the CL reaction
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X. Zheng et al. / Analytica Chimica Acta 440 (2001) 143–149
of tetracyclines with N-bromosuccinimide, but it suffered from the instablity of N-bromosuccinimide. Although, Han et al. have reported sensitive CL methods for tetracyclines, these methods suffer from the use of the expensive CL reagent Ru(bpy)3 2+ [25]. To the best our knowledge, up to now, no energy-transfer CL method has been reported for the determination of tetracyclines. When reviewing useful CL systems it was found that most of the analytically useful CL reactions are relatively fast. For example, the CL intensity produced by hypobromite reacting with urea reaches a maximum within 20 ms after mixing and no CL is detectable after 120 ms [26]. For obtaining the maximum CL emission signal, many efforts, such as reducing the length of the mixing coil between the last sample/reagent introduction point and the photomultiplier tube (PMT) viewing window, and increasing the flow rates of the reagent and sample in the mixing coil have been proposed. But these arrangements often result in poor reproducibility and poor accuracy due to the incomplete mixing of fluids, and they also create greater potential accidental leaks in the flow system and greater reagent consumption due to the increasing flow rates. On the other hand, although various transparent cells of spiral and similar designs have been reported for carrying out mixing within the viewing volume [26,27], special materials and techniques are still needed. In this paper, it was found that the bromine produced electrochemically could react with hydrogen peroxide injected into the CL reaction cell, to produce a fast, weak CL signal. It was also found that this weak CL signal was greatly enhanced by the presence of tetracyclines, based on an energy-transfer CL mechanism and accompanied by a fast CL signal. However, due to the requirements of both the complex procedures for preparing the standard bromine solution and the lack of a useful scheme for detecting this fast CL signal, further analytical application of these observations was frustrated. For overcoming these limits, we further found that when an platinum electrode was introduced into the CL reaction cell, the bromine could be generated in situ near to the surface of the platinum electrode. In this case, the fast CL reaction procedure (including the initial mixing of electrogenerated bromine with hydrogen peroxide and the subsequent CL reaction procedure) could not only be located
near the surface of the platinum flake electrode but also occurred in front of the window of the PMT. Thus, fast CL signal could be detected effectively for analytical purposes. Based on these observations, a new, simple, sensitive and rapid energy-transfer CL method for the determination of tetracyclines is proposed and a useful scheme for effectively detecting the fast CL signal is described.
2. Experimental 2.1. Reagents All solutions were prepared from analytical-reagent grade materials with distilled, deionized water. Stock solutions of tetracyclines (1.0 × 10−3 mol l−1 were prepared by accurately weighing the hydrochorides of tetracycline, oxytetracycline and chlortetracycline (purchased from Sigma) into 50 ml calibrated flasks and diluting to volume with water. Testing standard solutions were prepared daily by appropriate dilution of the stock solutions with water. An amount of 0.30 mol l−1 KBr solution was obtained by dissolving 35.7 g of KBr (Xi’an Chemical Reagents Factory, China) in 0.010 ml l−1 sulfuric acid and diluted to 1 l with the same concentration sulfuric acid. In addition, 1.5 × 10−3 mol l−1 hydrogen peroxide was prepared by mixing suitable volumes of 30% (v/v) hydrogen peroxide with 0.10 mol l−1 KOH and diluting to exactly 1 l with water. 2.2. Apparatus Fig. 1 shows a schematic diagram of the proposed FI–CL system for the determination of tetracyclines. An R456 PMT (Hamamatsu) was used for the detection of the CL emission signal. The constant current applied for electrolysis was achieved with a JH2C potentiostat/galvanostat (Shanghai Second Component Factory, China). The flow-through electrolytic cell (FEC) was made from a transparent glass tube (length: 2.5 cm, i.d.: 0.5 cm) and utilized a conventional two-electrode set-up (as shown in Fig. 2). An amount of 0.5 cm2 platinum flake electrode and a stainless steel tube (length 2 cm, i.d.: 3 mm) were used as the working electrode and counter electrode, respectively. The flow-cell was enclosed in a light-tight
X. Zheng et al. / Analytica Chimica Acta 440 (2001) 143–149
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Fig. 1. Flow-injection ECL manifold for tetracyclines determination. (a) Electrolyte stream; (b) carrier stream (H2 O); (c) H2 O2 solution; (d) sample solution. JH2C: potentiostat; FEC: flow-through electrolytic cell; V: injection valve; pumps 1 and 2: peristaltic pumps; PMT: R456 photomultiplier tube.
box and placed directly in front of the PMT. The output signal was recorded using an XWT-204 recorder (Shanghai Dahua Instrument and Meter Plant). All reagents were delivered by two peristaltic pumps and a six-way injection valve equipped with an 80 l injection loop to introduce the sample into the carrier stream. PTFE tubing (0.8 mm i.d.) was used to connect all components in the flow system.
H2 O2 containing tetracyclines solution were recorded as the blank CL signal or sample CL signals, respectively. The concentration of tetracyclines were quantified by the peak height of relative CL intensity, obtained by deducting the peak height of the blank CL signal from that of sample CL signals.
3. Results and discussion 2.3. Procedures Ten tablets of commercial formulations containing tetracycline and oxytetracycline were accurately weighed and pulverized. An accurately weighed amount of the powder equivalent to one tablet was dissolved in a suitable volume of water for the analysis. Thereafter, the electrode was cleaned prior to the first experiment by washing it in 3 mol l−1 HNO3 , followed by washing with distilled water. All solutions were pumped by two peristaltic pumps. Next suitable currents was applied to the FEC for a 3 min electrolysis, then 80 l of the hydrogen peroxide solution or hydrogen peroxide containing tetracyclines was injected into the carrier stream. The CL signals arising from the injection of H2 O2 solution or
Fig. 2. Construction of the flow-through electrolytic cell. W: working electrode; C: counter electrode.
In our initial experiments, it was found that the chemical reaction of bromine with H2 O2 produced a week CL signal. At the same time, it was further found that a very strong and fast CL signal was observed when a trace of tetracycline was present in the H2 O2 solution (the time profile of the enhanced CL intensity is shown in Fig. 3). However, due to the complications of preparing Br2 working solutions, the
Fig. 3. ECL intensity–time profiles. The Br2 solution was obtained by collecting the off-line electrolyte when the electrolytic current was 6 mA; H2 SO4 : 0.01 mol l−1 ; KOH: 0.05 mol l−1 . The profile was obtained in batch mode by mode by injection of 2.0 × 10−6 g ml−1 alkaline tetracycline into Br2 solution.
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possible harmful effect of Br2 on the operator as well as the shortage of the means used effectively to detect a fast CL signal, the analytical application of this fast CL reaction system for tetracyclines was frustrated. In order to overcome these limits, we found that the combination of the electrochemical method with the FI technique could achieve this purpose. So the design of the FEC and the FI manifold were vital factors for achieving this. These conditions were investigated. 3.1. The design of the flow-through electrolytic cell (FEC) In order to obtain the Br2 in an easy way and to make use of the subsequent fast CL reactions of Br2 with H2 O2 or with H2 O2 containing tetracyclines for analytical purpose, a series of initial test were made. A platinum electrode was inserted into the CL reaction cell to produce Br2 in situ by electrochemically oxidizing KBr in an acidic medium. The carrier stream channel and the electrolyte channel should be inserted parallel into the flow-through CL reaction cell to produce the subsequent fast CL reaction of Br2 with the injected species in the diffusion layer around the electrode. In this case, the reaction was not only located on the near surface of the platinum flake electrode, but also in the front of the window of the PMT. This novel FEC can thus afford greater spatial and temporal control over this fast CL reaction for analytical purposes. For achieving better analytical performance of the flow-through CL reaction cell, it was found that the distance between the working electrode and the counter electrode and the location of the counter electrode in the flow-through cell were vital parameters for the design of this FEC. These factors were investigated in detail. The results showed that when the location of the counter electrode was in the down stream of the flow channel and the distance between the two-electrode was 4 cm, the best result were obtained. So a parallel insertion mode FEC (as shown Fig. 2) was chosen for further experiments. 3.2. Effect of electrolytic current used for electrogenerating Br2 Because the electrolytic current controlled the Br2 concentration, the effect of the electrolytic current on
Fig. 4. Effect of electrolytic current on the relative ECL intensity. KBr: 0.5 mol l−1 ; H2 SO4 : 0.008 mol l−1 ; NaOH: 0.05 mol l−1 ; H2 O2 : 3.0 × 10−3 mol l−1 ; tetracycline (䊉): 5.0 × 10−7 g ml−1 ; oxytetracycline (䊏): 1.0 × 10−5 g ml−1 ; chlortetracycline (䉱): 3.0 × 10−5 g ml−1 ; blank signal (- - -).
the relative CL emission within the 0–8 mA range was investigated with 5.0 × 10−7 g ml−1 tetracyclines. The results shown in Fig. 4 indicate that the higher the electrolytic current the higher the electrogenerated Br2 concentration obtained, and the stronger the CL signal observed. But when the electrolytic current was >6, 4 or 3 mA, the relative CL intensities for tetracycline, oxytetracycline and chlortetracycline, respectively decreased. A possible reason was the absorption of the emitted light by Br2 . 3.3. Effect of the electrolyte components Since the function of the electrolyte used in the system was not only to provide enough bromide to produce the CL oxidant Br2 but also to offer a suitable medium for the subsequent CL reaction, so the effect of both the KBr concentration and the electrolyte medium on the CL intensity for the determination of tetracyclines were investigated. It was found initially, that when the medium was alkaline, and either alkaline or acidic H2 O2 solution was injected into the FEC, no blank CL single or tetracycline enhanced CL signal was observed. The enhanced CL signals of tetracyclines were observed only when the electrolyte was acidic and the H2 O2 solution was alkaline. These results indicated that Br2 is the real CL oxidant as the Br2 needed an acidic electrolyte to generate it electrochemically. Based on these initial tests, when HCl, H3 PO4 , HClO4 , or acetic acid–acetate were used
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as the medium of electrolyte within the 0–1 mol l−1 range, none of these media proved to be better than 0.01 mol l−1 H2 SO4 for tetracycline determination. Thus, 0.01 mol l−1 H2 SO4 was selected as the electrolyte. The effect of KBr concentration in the range 0.10–1.0 mol l−1 was investigated. The results showed that 0.3 mol l−1 KBr was adequate for the subsequent CL reactions, and was selected for subsequent experiments. 3.4. Effect of the constitution of the H2 O2 solution Under the above optimum conditions several alkaline solutions (KOH, Na2 CO3 , NaHCO3 , NH4 Cl–NH3 and Na2 B4 O7 –NaOH) within a suitable concentration range were used as the mediums of 0.01 ml l−1 H2 O2 . The results showed that none of these media was better than 0.05 mol l−1 KOH which was therefore, selected as the medium for the H2 O2 solution. The H2 O2 concentration was an important factor for the determination of tetracyclines in the proposed flow CL system since it influenced the CL intensity. Under otherwise optimum conditions, the H2 O2 concentration that gave the greatest intensity for all tetracyclines studied was 1.5 × 10−3 mol l−1 , which therefore, was selected for subsequent experiments. 3.5. Effect of flow rate The flow rate of the electrolyte not only controlled the Br2 concentration near the surface of the electrode but also had a important effect on the CL intensity when the electrolytic current and flow rate of the carrier stream were fixed at 5 mA and 2.0 mol min−1 , respectively. The effect of the electrolyte flow rate on the CL signal was studied in the range 0.5–3.0 ml min−1 . The results for all the tetracyclines studied were
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similar the relative CL signal increasing with increasing electrolyte flow rate from 0.5 to 2.0 ml min−1 reaching its maximum at 2.0 ml min−1 above which, the signal decreased. The 2.0 ml min−1 was selected as the optimum flow rate of electrolyte. 3.6. Performance of the CL system for tetracyclines measurement All three tetracyclines give rise to enhanced CL under the optimized conditions. The analytical parameters for each tetracycline examined are summarized in Table 1. At a flow rate of 2.0 ml min−1 , it took <1 min to complete one measurement, so the injection frequency could be 60 h−1 . All relative standard deviations were <5% for repetitive (n = 5) measurements of 5.0 × 10−7 g ml−1 of each antibiotic. 3.7. Interference study The influence of foreign species was studied by analyzing a solution of 2.0×10−3 mol l−1 H2 O2 to which increasing amounts of potential interfering species were added. The tolerable limit of a foreign species was taken as relative error not greater than 5%. The tolerable ratio of foreign ions to 2.0 × 10−6 g ml−1 tetracyclines was 1000 for K+ , Na+ , F− , Cl− , NO3 − , SO4 2− , HCO3 − , Ac− , Al3+ , Ca2+ , Ba2+ , Mg2+ , Zn2+ , glucose, maltose, cellulose, sucrose and starch; 200 for S2− , NO2 − , lactose and galactose. Equal concentration of ascorbic acid and NH4 + interfered with the determination of tetracyclines. 3.8. Analysis of samples The proposed CL method was applied to the analysis of some commercial formulations containing tetracycline and oxytetracycline. The results are
Table 1 Analytical characteristics for the determination of tetracyclines by the proposed methoda Species
Linear concentration range (g ml−1 )
Regression equation (I)
Correlation coefficient (n = 5)
DL (g ml−1 )
Tetracycline Oxytetracyline Chlortetracycline
0.03–50 0.2–24 0.1–50
0.442 + 96C 0.368 + 26C 0.698 + 8.5C
0.9992 0.9997 0.9998
0.01 0.07 0.15
a
DL: 3σ detection limit; I: relative ECL intensity; C: concentration of tetracycline in g ml−1 .
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Table 2 Determination of tetracycline and oxytetracycline in some commercial formulations Formulationa component
Tetracycline HCl (g ml−1 ) Initially present
Added
Recovered
Recovery tetracycline (%) n = 3
Amount of tetracycline HCl (g)
Tetracycline HCl
20.4 40.8
20.0 40.0
40.2 81.5
98 102
0.253 0.255
Oxytetracycline HCl
19.8 39.5
20.0 40.0
40.1 79.4
101 99
0.249 0.247
a
All formulation are tablets.
shown in Table 2. The results obtained compare favorably with that reported by the manufacturer (0.250 g pill) and also had a good recovery. This suggested that the proposed CL method is accurate. 3.9. Discussion of the possible ECL reaction mechanism In order to explain the possible CL reaction scheme, a series of experiments were carried out. First, when no electrolytic current was applied to the FEC and the other experimental conditions was the same with those of the optimum conditions, it was found that no CL signal was observed. However, when the electrolyte stream contained acidic Br2 the CL signal or the enhanced CL signal from the reaction of H2 O2 or H2 O2 containing tetracyclines, respectively, with acidic Br2 , can be observed. These results suggest that electrogenerated Br2 is the real oxidant or the precursor of the real CL oxidant in the proposed CL system. Secondly, neither the chemical reaction between BrO− and H2 O2 containing tetracyclines in alkaline medium or the chemical reaction between Br2 and H2 O2 containing tetracyclines in acidic medium was not accompanied by any CL signal. In addition, when the electrolyte medium was 0.05 mol l−1 KOH and the medium of the H2 O2 solution was 0.01 mol l−1 H2 SO4 in the proposed CL system, no CL signal was detected. These results suggest that BrO− was not the actual CL oxidant in the proposed system and the optimum medium of this CL reaction (between the in situ electrogenerated Br2 and H2 O2 ) was not H2 SO4 . Based on the results mentioned above, we think that the actual CL oxidant in the proposed system was Br2 and the optimum medium for this CL reaction was
alkaline KOH. This alkaline medium should be produced by the injection of alkaline H2 O2 solution. Thirdly, for obtaining information about the possible emitter, the spectrum of the CL emission and the fluorescence spectrum of the tetracycline was obtained by a rf-540 fluorescence spectrophotometer. The results revealed that the CL emission spectra of tetracyclines overlapped with the fluorescence spectra of the tetracyclines in an alkaline medium. So the emitter could be an excited state tetracycline, its excitation energy being obtained from excited oxygen [28,29] (produced by the CL reaction of Br2 with H2 O2 ) based on an energy-transfer CL mechanism. All these results suggest that this electrogenerated chemiluminescence (ECL) reaction scheme is to be attributed to the following reactions: 1. 2Br − − 2e → Br 2 (electrochemical oxidation in acidic medium); 2. Br 2 + H2 O2 → O2 ∗ (in alkaline medium, O2 ∗ excited oxygen); 3. O2 ∗ + Tc → Tc∗ + O2 (Tc: tetracyclines, Tc∗ : excited state tetracyclines); 4. Tc∗ → Tc + light.
4. Conclusions In this paper, based on designing of a FEC, Br2 , the useful CL oxidant, was easily and simply prepared by an electrochemical method, and was used for the determination of tetracyclines by the proposed CL method. Some problems, such as the reagent (Br2 ) solution addition [30], the complex procedure for preparing the Br2 standard solution and other problems such as the instability of a Br2 standard solution [31] were
X. Zheng et al. / Analytica Chimica Acta 440 (2001) 143–149
eliminated. At the same time, this FEC described in this paper offers a useful means of detecting effectively the fast CL signal. In addition, this CL reaction cell also offer a chance both to develop new CL oxidants, which have been used in coulometric titration analysis, and explore their use in CL analytical procedures.
Acknowledgements This study was supported by the National Natural Science Foundation of China (39730160). References [1] L.M. Hirschy, E.V. Dove, J.D. Winefordner, Anal. Chim. Acta 147 (1983) 311. [2] T. Hasan, B.S. Cooperman, J. Chromatogr. 321 (1985) 462. [3] E. Ragazzi, G. Veronese, J. Chromatogr. 134 (1977) 223. [4] F. Salinas, J.J. Berzas Nevado, A. Espinosa, Analyst 114 (1989) 1141. [5] M.S. Mahrous, M.M. Abbdel-Khalek, Talanta 31 (1984) 289. [6] S.M. Sultan, F.E.O. Suliman, S.O. Duffnaa, I.I. Abuabdoun, Analyst 117 (1992) 1179. [7] A.A. Alwarthan, S.A. Al-Tamrah, S.M. Sultan, Analyst 116 (1991) 183. [8] F. Salinas, A. Munoz de la Pena, I. Duran Meras, Anal. Lett. 23 (1990) 863. [9] W.B. Chang, Y.B. Zhao, Y.X. Li, L.Y. Hu, Analyst 117 (1992) 1377.
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[10] M.A. Ghandour, A.M.M. Alt, Anal. Lett. 24 (1991) 2171. [11] K. Robards, P.J. Worsfold, Anal. Chim. Acta 266 (1992) 147–173. [12] A. Townshend, Analyst 115 (1990) 495–500. [13] I.I. Koukli, A.C. Calokerinos, Anal. Chim. Acta 236 (1990) 463. [14] I.I. Koukli, A.C. Calokerinos, T.P. Hadjiioannou, Analyst 114 (1989) 711. [15] Z.D. Zhang, W.R.G. Baeyens, X.R. Zhang, G. van der Weken, J. Pharmacol. Biomed. Anal. 14 (1996) 939–945. [16] X.R. Zhang, W.R.G. Baeyens, G. van der Weken, A.C. Calokerinos, Anal. Chim. Acta 303 (1995) 121–125. [17] I.I. Kouki, A.C. Calokerinos, Analyst 115 (1990) 1553. [18] I.M. Psarellis, N.T. Deftereos, E.G. Sarantonis, A.C. Calokerinos, Anal. Chim. Acta 272 (1993) 265. [19] T. Owa Tmasujima, H. Yoshida, K. Imai, Bunseki Kagaku 33 (1984) 568. [20] A.A. Alwarthan, A. Townshend, Anal. Chim. Acta 205 (1988) 261. [21] X.R. Zhang, W.R.G. Baeyens, A. van den Borre, G. van der Weken, Analyst 120 (1995) 463. [22] S.A. Halvatzis, M.M. Timotheou-Potamia, A.C. Calokerinos, Analyst 118 (1993) 633–637. [23] Z. Li, M.L. Feng, J.R. Lu, Anal. Lett. 30 (1997) 797–807. [24] A. Pena, L.P. Palilis, C.M. Lino, M.I. Silveira, A.C. Calokerinos, Anal. Chim. Acta 405 (2000) 51–56. [25] H.Y. Han, Z.K. He, Y.E. Zeng, Anal. Sci. 15 (1999) 476. [26] X. Hu, N. Takenaka, S. Takasuna, Anal. Chem. 65 (1992) 3489. [27] J. Li, P.K. Dasgupta, Anal. Chim. Acta 398 (1999) 33–39. [28] J. Lin, T. Hobo, Anal. Chem. Acta 69 (1996) 323. [29] F. Zhang, Q. Lin, Talanta 40 (1993) 1557. [30] J.S. Littig, T.A. Nieman, Anal. Chem. 64 (1992) 1140–1144. [31] Z.F. Zhao, Analytical Chemistry Experiments, High Education Press, Beijing, 1987, p. 54.
Flow-injection chemiluminescence determination of tetracyclines with in situ electrogenerated bromine as the oxidant Xingwang Zheng a , Yang Mei b , Zhujun Zhang a,∗ a
Department of Chemistry, Shaanxi Normal University, Xi’an 710062, PR China b Liaoning Normal University, Dalian, PR China
Received 2 August 2000; received in revised form 19 March 2001; accepted 12 April 2001
Abstract By designing a novel flow-through electrolytic cell (FEC), bromine was produced near to the surface of the platinum electrode by electrochemical oxidation of acidic KBr. The fast and weak chemiluminescence signal produced by the chemical reaction of the electrogenerated bromine with H2 O2 was greatly enhanced by tetracyclines Based on these observations, a new, sensitive and simple electrogenerated chemiluminescence (ECL) method for the determination of tetracyclines was developed. Under the optimum experimental conditions, the calibration graphs are linear over the range 3.0 × 10−8 to 5.0 × 10−5 g ml−1 for tetracycline, 2.0 × 10−7 to 2.4 × 10−5 g ml−1 for oxytetracycline and 1.0 × 10−7 to 5.0 × 10−5 g ml−1 for chlortetracycline. The limits of detection (S/N = 3) are 1.0 × 10−8 g ml−1 for tetracycline, 7.0 × 10−8 g ml−1 for oxytetracycline and 1.5 × 10−7 g ml−1 for chlortetracycline. For the determination 5.0 × 10−7 g ml−1 tetracycline, the relative standard deviation was <5%. The proposed method was used to determine tetracyclines in pharmaceutical formulations. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Electrogenerated bromine; Flow-injection analysis; Tetracyclines; Chemiluminescence
1. Introduction Tetracyclines are broad-spectrum antibiotics, which possess a hydronaphthacene skeleton with a wide variety of functional groups. They are commonly used in veterinary medicine, animal nutrition and as feed additives. Tetracyclines are determined mainly by chromatographic [1–3], spectrophotometric [4–7], spectrofluorimetric [8,9] and electrochemical methods [10]. In recent years flow-injection (FI) chemiluminescence (CL) methods have received much attention and have been used for the determinations of many ∗
Corresponding author. Tel.: +86-29-530-8748; fax: +86-29-530-8748. E-mail address: [email protected] (Z. Zhang).
analytes [11–16] since this technique offer excellent sensitivity a wide linear dynamic range and only simple instrumentation is required. Usually, the CL can be achieved by direct reaction or by energy-transfer mechanisms [17,18]. Nevertheless, the energy-transfer type of CL system has received attention since it is based on the vast knowledge of spectrofluorimetry and also owned the good selectivity inherent in spectrofluorimetry. However, up to now, only a few FI-based direct CL methods have been used for the determination of tetracyclines [19–24]. For example, Alwarthan and Townshend [20] have reported a bromine-based FI–CL method for tetracycline, but it needed a complex procedure for preparing the bromine standard solution. Halvatzis et al. [22] reported another sensitive CL method for tetracyclines; this method was based on the CL reaction
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of tetracyclines with N-bromosuccinimide, but it suffered from the instablity of N-bromosuccinimide. Although, Han et al. have reported sensitive CL methods for tetracyclines, these methods suffer from the use of the expensive CL reagent Ru(bpy)3 2+ [25]. To the best our knowledge, up to now, no energy-transfer CL method has been reported for the determination of tetracyclines. When reviewing useful CL systems it was found that most of the analytically useful CL reactions are relatively fast. For example, the CL intensity produced by hypobromite reacting with urea reaches a maximum within 20 ms after mixing and no CL is detectable after 120 ms [26]. For obtaining the maximum CL emission signal, many efforts, such as reducing the length of the mixing coil between the last sample/reagent introduction point and the photomultiplier tube (PMT) viewing window, and increasing the flow rates of the reagent and sample in the mixing coil have been proposed. But these arrangements often result in poor reproducibility and poor accuracy due to the incomplete mixing of fluids, and they also create greater potential accidental leaks in the flow system and greater reagent consumption due to the increasing flow rates. On the other hand, although various transparent cells of spiral and similar designs have been reported for carrying out mixing within the viewing volume [26,27], special materials and techniques are still needed. In this paper, it was found that the bromine produced electrochemically could react with hydrogen peroxide injected into the CL reaction cell, to produce a fast, weak CL signal. It was also found that this weak CL signal was greatly enhanced by the presence of tetracyclines, based on an energy-transfer CL mechanism and accompanied by a fast CL signal. However, due to the requirements of both the complex procedures for preparing the standard bromine solution and the lack of a useful scheme for detecting this fast CL signal, further analytical application of these observations was frustrated. For overcoming these limits, we further found that when an platinum electrode was introduced into the CL reaction cell, the bromine could be generated in situ near to the surface of the platinum electrode. In this case, the fast CL reaction procedure (including the initial mixing of electrogenerated bromine with hydrogen peroxide and the subsequent CL reaction procedure) could not only be located
near the surface of the platinum flake electrode but also occurred in front of the window of the PMT. Thus, fast CL signal could be detected effectively for analytical purposes. Based on these observations, a new, simple, sensitive and rapid energy-transfer CL method for the determination of tetracyclines is proposed and a useful scheme for effectively detecting the fast CL signal is described.
2. Experimental 2.1. Reagents All solutions were prepared from analytical-reagent grade materials with distilled, deionized water. Stock solutions of tetracyclines (1.0 × 10−3 mol l−1 were prepared by accurately weighing the hydrochorides of tetracycline, oxytetracycline and chlortetracycline (purchased from Sigma) into 50 ml calibrated flasks and diluting to volume with water. Testing standard solutions were prepared daily by appropriate dilution of the stock solutions with water. An amount of 0.30 mol l−1 KBr solution was obtained by dissolving 35.7 g of KBr (Xi’an Chemical Reagents Factory, China) in 0.010 ml l−1 sulfuric acid and diluted to 1 l with the same concentration sulfuric acid. In addition, 1.5 × 10−3 mol l−1 hydrogen peroxide was prepared by mixing suitable volumes of 30% (v/v) hydrogen peroxide with 0.10 mol l−1 KOH and diluting to exactly 1 l with water. 2.2. Apparatus Fig. 1 shows a schematic diagram of the proposed FI–CL system for the determination of tetracyclines. An R456 PMT (Hamamatsu) was used for the detection of the CL emission signal. The constant current applied for electrolysis was achieved with a JH2C potentiostat/galvanostat (Shanghai Second Component Factory, China). The flow-through electrolytic cell (FEC) was made from a transparent glass tube (length: 2.5 cm, i.d.: 0.5 cm) and utilized a conventional two-electrode set-up (as shown in Fig. 2). An amount of 0.5 cm2 platinum flake electrode and a stainless steel tube (length 2 cm, i.d.: 3 mm) were used as the working electrode and counter electrode, respectively. The flow-cell was enclosed in a light-tight
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Fig. 1. Flow-injection ECL manifold for tetracyclines determination. (a) Electrolyte stream; (b) carrier stream (H2 O); (c) H2 O2 solution; (d) sample solution. JH2C: potentiostat; FEC: flow-through electrolytic cell; V: injection valve; pumps 1 and 2: peristaltic pumps; PMT: R456 photomultiplier tube.
box and placed directly in front of the PMT. The output signal was recorded using an XWT-204 recorder (Shanghai Dahua Instrument and Meter Plant). All reagents were delivered by two peristaltic pumps and a six-way injection valve equipped with an 80 l injection loop to introduce the sample into the carrier stream. PTFE tubing (0.8 mm i.d.) was used to connect all components in the flow system.
H2 O2 containing tetracyclines solution were recorded as the blank CL signal or sample CL signals, respectively. The concentration of tetracyclines were quantified by the peak height of relative CL intensity, obtained by deducting the peak height of the blank CL signal from that of sample CL signals.
3. Results and discussion 2.3. Procedures Ten tablets of commercial formulations containing tetracycline and oxytetracycline were accurately weighed and pulverized. An accurately weighed amount of the powder equivalent to one tablet was dissolved in a suitable volume of water for the analysis. Thereafter, the electrode was cleaned prior to the first experiment by washing it in 3 mol l−1 HNO3 , followed by washing with distilled water. All solutions were pumped by two peristaltic pumps. Next suitable currents was applied to the FEC for a 3 min electrolysis, then 80 l of the hydrogen peroxide solution or hydrogen peroxide containing tetracyclines was injected into the carrier stream. The CL signals arising from the injection of H2 O2 solution or
Fig. 2. Construction of the flow-through electrolytic cell. W: working electrode; C: counter electrode.
In our initial experiments, it was found that the chemical reaction of bromine with H2 O2 produced a week CL signal. At the same time, it was further found that a very strong and fast CL signal was observed when a trace of tetracycline was present in the H2 O2 solution (the time profile of the enhanced CL intensity is shown in Fig. 3). However, due to the complications of preparing Br2 working solutions, the
Fig. 3. ECL intensity–time profiles. The Br2 solution was obtained by collecting the off-line electrolyte when the electrolytic current was 6 mA; H2 SO4 : 0.01 mol l−1 ; KOH: 0.05 mol l−1 . The profile was obtained in batch mode by mode by injection of 2.0 × 10−6 g ml−1 alkaline tetracycline into Br2 solution.
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possible harmful effect of Br2 on the operator as well as the shortage of the means used effectively to detect a fast CL signal, the analytical application of this fast CL reaction system for tetracyclines was frustrated. In order to overcome these limits, we found that the combination of the electrochemical method with the FI technique could achieve this purpose. So the design of the FEC and the FI manifold were vital factors for achieving this. These conditions were investigated. 3.1. The design of the flow-through electrolytic cell (FEC) In order to obtain the Br2 in an easy way and to make use of the subsequent fast CL reactions of Br2 with H2 O2 or with H2 O2 containing tetracyclines for analytical purpose, a series of initial test were made. A platinum electrode was inserted into the CL reaction cell to produce Br2 in situ by electrochemically oxidizing KBr in an acidic medium. The carrier stream channel and the electrolyte channel should be inserted parallel into the flow-through CL reaction cell to produce the subsequent fast CL reaction of Br2 with the injected species in the diffusion layer around the electrode. In this case, the reaction was not only located on the near surface of the platinum flake electrode, but also in the front of the window of the PMT. This novel FEC can thus afford greater spatial and temporal control over this fast CL reaction for analytical purposes. For achieving better analytical performance of the flow-through CL reaction cell, it was found that the distance between the working electrode and the counter electrode and the location of the counter electrode in the flow-through cell were vital parameters for the design of this FEC. These factors were investigated in detail. The results showed that when the location of the counter electrode was in the down stream of the flow channel and the distance between the two-electrode was 4 cm, the best result were obtained. So a parallel insertion mode FEC (as shown Fig. 2) was chosen for further experiments. 3.2. Effect of electrolytic current used for electrogenerating Br2 Because the electrolytic current controlled the Br2 concentration, the effect of the electrolytic current on
Fig. 4. Effect of electrolytic current on the relative ECL intensity. KBr: 0.5 mol l−1 ; H2 SO4 : 0.008 mol l−1 ; NaOH: 0.05 mol l−1 ; H2 O2 : 3.0 × 10−3 mol l−1 ; tetracycline (䊉): 5.0 × 10−7 g ml−1 ; oxytetracycline (䊏): 1.0 × 10−5 g ml−1 ; chlortetracycline (䉱): 3.0 × 10−5 g ml−1 ; blank signal (- - -).
the relative CL emission within the 0–8 mA range was investigated with 5.0 × 10−7 g ml−1 tetracyclines. The results shown in Fig. 4 indicate that the higher the electrolytic current the higher the electrogenerated Br2 concentration obtained, and the stronger the CL signal observed. But when the electrolytic current was >6, 4 or 3 mA, the relative CL intensities for tetracycline, oxytetracycline and chlortetracycline, respectively decreased. A possible reason was the absorption of the emitted light by Br2 . 3.3. Effect of the electrolyte components Since the function of the electrolyte used in the system was not only to provide enough bromide to produce the CL oxidant Br2 but also to offer a suitable medium for the subsequent CL reaction, so the effect of both the KBr concentration and the electrolyte medium on the CL intensity for the determination of tetracyclines were investigated. It was found initially, that when the medium was alkaline, and either alkaline or acidic H2 O2 solution was injected into the FEC, no blank CL single or tetracycline enhanced CL signal was observed. The enhanced CL signals of tetracyclines were observed only when the electrolyte was acidic and the H2 O2 solution was alkaline. These results indicated that Br2 is the real CL oxidant as the Br2 needed an acidic electrolyte to generate it electrochemically. Based on these initial tests, when HCl, H3 PO4 , HClO4 , or acetic acid–acetate were used
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as the medium of electrolyte within the 0–1 mol l−1 range, none of these media proved to be better than 0.01 mol l−1 H2 SO4 for tetracycline determination. Thus, 0.01 mol l−1 H2 SO4 was selected as the electrolyte. The effect of KBr concentration in the range 0.10–1.0 mol l−1 was investigated. The results showed that 0.3 mol l−1 KBr was adequate for the subsequent CL reactions, and was selected for subsequent experiments. 3.4. Effect of the constitution of the H2 O2 solution Under the above optimum conditions several alkaline solutions (KOH, Na2 CO3 , NaHCO3 , NH4 Cl–NH3 and Na2 B4 O7 –NaOH) within a suitable concentration range were used as the mediums of 0.01 ml l−1 H2 O2 . The results showed that none of these media was better than 0.05 mol l−1 KOH which was therefore, selected as the medium for the H2 O2 solution. The H2 O2 concentration was an important factor for the determination of tetracyclines in the proposed flow CL system since it influenced the CL intensity. Under otherwise optimum conditions, the H2 O2 concentration that gave the greatest intensity for all tetracyclines studied was 1.5 × 10−3 mol l−1 , which therefore, was selected for subsequent experiments. 3.5. Effect of flow rate The flow rate of the electrolyte not only controlled the Br2 concentration near the surface of the electrode but also had a important effect on the CL intensity when the electrolytic current and flow rate of the carrier stream were fixed at 5 mA and 2.0 mol min−1 , respectively. The effect of the electrolyte flow rate on the CL signal was studied in the range 0.5–3.0 ml min−1 . The results for all the tetracyclines studied were
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similar the relative CL signal increasing with increasing electrolyte flow rate from 0.5 to 2.0 ml min−1 reaching its maximum at 2.0 ml min−1 above which, the signal decreased. The 2.0 ml min−1 was selected as the optimum flow rate of electrolyte. 3.6. Performance of the CL system for tetracyclines measurement All three tetracyclines give rise to enhanced CL under the optimized conditions. The analytical parameters for each tetracycline examined are summarized in Table 1. At a flow rate of 2.0 ml min−1 , it took <1 min to complete one measurement, so the injection frequency could be 60 h−1 . All relative standard deviations were <5% for repetitive (n = 5) measurements of 5.0 × 10−7 g ml−1 of each antibiotic. 3.7. Interference study The influence of foreign species was studied by analyzing a solution of 2.0×10−3 mol l−1 H2 O2 to which increasing amounts of potential interfering species were added. The tolerable limit of a foreign species was taken as relative error not greater than 5%. The tolerable ratio of foreign ions to 2.0 × 10−6 g ml−1 tetracyclines was 1000 for K+ , Na+ , F− , Cl− , NO3 − , SO4 2− , HCO3 − , Ac− , Al3+ , Ca2+ , Ba2+ , Mg2+ , Zn2+ , glucose, maltose, cellulose, sucrose and starch; 200 for S2− , NO2 − , lactose and galactose. Equal concentration of ascorbic acid and NH4 + interfered with the determination of tetracyclines. 3.8. Analysis of samples The proposed CL method was applied to the analysis of some commercial formulations containing tetracycline and oxytetracycline. The results are
Table 1 Analytical characteristics for the determination of tetracyclines by the proposed methoda Species
Linear concentration range (g ml−1 )
Regression equation (I)
Correlation coefficient (n = 5)
DL (g ml−1 )
Tetracycline Oxytetracyline Chlortetracycline
0.03–50 0.2–24 0.1–50
0.442 + 96C 0.368 + 26C 0.698 + 8.5C
0.9992 0.9997 0.9998
0.01 0.07 0.15
a
DL: 3σ detection limit; I: relative ECL intensity; C: concentration of tetracycline in g ml−1 .
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Table 2 Determination of tetracycline and oxytetracycline in some commercial formulations Formulationa component
Tetracycline HCl (g ml−1 ) Initially present
Added
Recovered
Recovery tetracycline (%) n = 3
Amount of tetracycline HCl (g)
Tetracycline HCl
20.4 40.8
20.0 40.0
40.2 81.5
98 102
0.253 0.255
Oxytetracycline HCl
19.8 39.5
20.0 40.0
40.1 79.4
101 99
0.249 0.247
a
All formulation are tablets.
shown in Table 2. The results obtained compare favorably with that reported by the manufacturer (0.250 g pill) and also had a good recovery. This suggested that the proposed CL method is accurate. 3.9. Discussion of the possible ECL reaction mechanism In order to explain the possible CL reaction scheme, a series of experiments were carried out. First, when no electrolytic current was applied to the FEC and the other experimental conditions was the same with those of the optimum conditions, it was found that no CL signal was observed. However, when the electrolyte stream contained acidic Br2 the CL signal or the enhanced CL signal from the reaction of H2 O2 or H2 O2 containing tetracyclines, respectively, with acidic Br2 , can be observed. These results suggest that electrogenerated Br2 is the real oxidant or the precursor of the real CL oxidant in the proposed CL system. Secondly, neither the chemical reaction between BrO− and H2 O2 containing tetracyclines in alkaline medium or the chemical reaction between Br2 and H2 O2 containing tetracyclines in acidic medium was not accompanied by any CL signal. In addition, when the electrolyte medium was 0.05 mol l−1 KOH and the medium of the H2 O2 solution was 0.01 mol l−1 H2 SO4 in the proposed CL system, no CL signal was detected. These results suggest that BrO− was not the actual CL oxidant in the proposed system and the optimum medium of this CL reaction (between the in situ electrogenerated Br2 and H2 O2 ) was not H2 SO4 . Based on the results mentioned above, we think that the actual CL oxidant in the proposed system was Br2 and the optimum medium for this CL reaction was
alkaline KOH. This alkaline medium should be produced by the injection of alkaline H2 O2 solution. Thirdly, for obtaining information about the possible emitter, the spectrum of the CL emission and the fluorescence spectrum of the tetracycline was obtained by a rf-540 fluorescence spectrophotometer. The results revealed that the CL emission spectra of tetracyclines overlapped with the fluorescence spectra of the tetracyclines in an alkaline medium. So the emitter could be an excited state tetracycline, its excitation energy being obtained from excited oxygen [28,29] (produced by the CL reaction of Br2 with H2 O2 ) based on an energy-transfer CL mechanism. All these results suggest that this electrogenerated chemiluminescence (ECL) reaction scheme is to be attributed to the following reactions: 1. 2Br − − 2e → Br 2 (electrochemical oxidation in acidic medium); 2. Br 2 + H2 O2 → O2 ∗ (in alkaline medium, O2 ∗ excited oxygen); 3. O2 ∗ + Tc → Tc∗ + O2 (Tc: tetracyclines, Tc∗ : excited state tetracyclines); 4. Tc∗ → Tc + light.
4. Conclusions In this paper, based on designing of a FEC, Br2 , the useful CL oxidant, was easily and simply prepared by an electrochemical method, and was used for the determination of tetracyclines by the proposed CL method. Some problems, such as the reagent (Br2 ) solution addition [30], the complex procedure for preparing the Br2 standard solution and other problems such as the instability of a Br2 standard solution [31] were
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eliminated. At the same time, this FEC described in this paper offers a useful means of detecting effectively the fast CL signal. In addition, this CL reaction cell also offer a chance both to develop new CL oxidants, which have been used in coulometric titration analysis, and explore their use in CL analytical procedures.
Acknowledgements This study was supported by the National Natural Science Foundation of China (39730160). References [1] L.M. Hirschy, E.V. Dove, J.D. Winefordner, Anal. Chim. Acta 147 (1983) 311. [2] T. Hasan, B.S. Cooperman, J. Chromatogr. 321 (1985) 462. [3] E. Ragazzi, G. Veronese, J. Chromatogr. 134 (1977) 223. [4] F. Salinas, J.J. Berzas Nevado, A. Espinosa, Analyst 114 (1989) 1141. [5] M.S. Mahrous, M.M. Abbdel-Khalek, Talanta 31 (1984) 289. [6] S.M. Sultan, F.E.O. Suliman, S.O. Duffnaa, I.I. Abuabdoun, Analyst 117 (1992) 1179. [7] A.A. Alwarthan, S.A. Al-Tamrah, S.M. Sultan, Analyst 116 (1991) 183. [8] F. Salinas, A. Munoz de la Pena, I. Duran Meras, Anal. Lett. 23 (1990) 863. [9] W.B. Chang, Y.B. Zhao, Y.X. Li, L.Y. Hu, Analyst 117 (1992) 1377.
149
[10] M.A. Ghandour, A.M.M. Alt, Anal. Lett. 24 (1991) 2171. [11] K. Robards, P.J. Worsfold, Anal. Chim. Acta 266 (1992) 147–173. [12] A. Townshend, Analyst 115 (1990) 495–500. [13] I.I. Koukli, A.C. Calokerinos, Anal. Chim. Acta 236 (1990) 463. [14] I.I. Koukli, A.C. Calokerinos, T.P. Hadjiioannou, Analyst 114 (1989) 711. [15] Z.D. Zhang, W.R.G. Baeyens, X.R. Zhang, G. van der Weken, J. Pharmacol. Biomed. Anal. 14 (1996) 939–945. [16] X.R. Zhang, W.R.G. Baeyens, G. van der Weken, A.C. Calokerinos, Anal. Chim. Acta 303 (1995) 121–125. [17] I.I. Kouki, A.C. Calokerinos, Analyst 115 (1990) 1553. [18] I.M. Psarellis, N.T. Deftereos, E.G. Sarantonis, A.C. Calokerinos, Anal. Chim. Acta 272 (1993) 265. [19] T. Owa Tmasujima, H. Yoshida, K. Imai, Bunseki Kagaku 33 (1984) 568. [20] A.A. Alwarthan, A. Townshend, Anal. Chim. Acta 205 (1988) 261. [21] X.R. Zhang, W.R.G. Baeyens, A. van den Borre, G. van der Weken, Analyst 120 (1995) 463. [22] S.A. Halvatzis, M.M. Timotheou-Potamia, A.C. Calokerinos, Analyst 118 (1993) 633–637. [23] Z. Li, M.L. Feng, J.R. Lu, Anal. Lett. 30 (1997) 797–807. [24] A. Pena, L.P. Palilis, C.M. Lino, M.I. Silveira, A.C. Calokerinos, Anal. Chim. Acta 405 (2000) 51–56. [25] H.Y. Han, Z.K. He, Y.E. Zeng, Anal. Sci. 15 (1999) 476. [26] X. Hu, N. Takenaka, S. Takasuna, Anal. Chem. 65 (1992) 3489. [27] J. Li, P.K. Dasgupta, Anal. Chim. Acta 398 (1999) 33–39. [28] J. Lin, T. Hobo, Anal. Chem. Acta 69 (1996) 323. [29] F. Zhang, Q. Lin, Talanta 40 (1993) 1557. [30] J.S. Littig, T.A. Nieman, Anal. Chem. 64 (1992) 1140–1144. [31] Z.F. Zhao, Analytical Chemistry Experiments, High Education Press, Beijing, 1987, p. 54.