Voltammetry and amperometric detection of tetracyclines at multi-wall ...

Anal Bioanal Chem (2007) 389:951–958 DOI 10.1007/s00216-007-1505-7

ORIGINAL PAPER

Voltammetry and amperometric detection of tetracyclines at multi-wall carbon nanotube modified electrodes D. Vega & L. Agüí & A. González-Cortés & P. Yáñez-Sedeño & J. M. Pingarrón

Received: 26 April 2007 / Revised: 3 July 2007 / Accepted: 11 July 2007 / Published online: 2 August 2007 # Springer-Verlag 2007

Abstract The voltammetric behaviour and amperometric detection of tetracycline (TC) antibiotics at multi-wall carbon nanotube modified glassy carbon electrodes (MWCNT-GCE) are reported. Cyclic voltammograms of TCs showed enhanced oxidation responses at the MWCNTGCE with respect to the bare GCE, attributable to the increased active electrode surface area. Hydrodynamic voltammograms obtained by flow-injection with amperometric detection at the MWCNT-GCE led us to select a potential value Edet =+1.20 V. The repeatability of the amperometric responses was much better than that achieved with bare GCE (RSD ranged from 7 to 12%), with RSD values for ip of around 3%, thus demonstrating the antifouling capability of MWCNT modified electrodes. An HPLC method with amperometric electrochemical detection (ED) at the MWCNT-GCE was developed for tetracycline, oxytetracycline (OTC), chlortetracycline and doxycycline (DC). A mobile phase consisting of 18:82 acetonitrile/0.05 mol L−1 phosphate buffer of pH 2.5 was selected. The limits of detection ranged from 0.09 μmol L−1 for OTC to 0.44 μmol L−1 for DC. The possibility to carry out multiresidue analysis is demonstrated. The HPLC-ED/ MWCNT-GCE method was applied to the analysis of fish farm pool water and underground well water samples spiked with the four TCs at 2.0×10−7 mol L−1. Solid-phase extraction was accomplished for the preconcentration of the analytes and clean-up of the samples. Recoveries ranged from 87±6 to 99±3%. Under preconcentration conditions, D. Vega : L. Agüí : A. González-Cortés : P. Yáñez-Sedeño (*) : J. M. Pingarrón Department of Analytical Chemistry, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain e-mail: [email protected]

limits of detection in the water samples were between 0.50 and 3.10 ng mL−1. Keywords Tetracyclines . Water analysis . High-performance liquid chromatography . Electrochemical detection . Carbon nanotube modified electrode

Introduction Antibiotics are emergent environmental pollutants not yet included in the regulatory lists [1]. Although the expected concentrations are still very low, their continuous incorporation in the environment can cause adverse effects e.g. bacterial resistance [2]. Tetracyclines (TCs) have been commonly used in veterinary medicine and aquaculture for prevention and treatment of diseases. However, their widespread use has led to TC residues. Tetracyclines adsorb strongly onto environmental materials in which they also maintain their activity. For example, it is known that oxytetracycline remains undegraded during more that ten months when it is adsorbed on marine sediments and soils [3]. Tetracyclines are extensively used in shrimp aquaculture, as food additives, and directly added to the water. Approximately 70% of the applied drugs are not consumed and remain in the environment. This fact explains the presence of high amounts of veterinary drugs and their metabolites on sediments surrounding aquaculture areas [4]. Several high-performance liquid chromatographic (HPLC) methods have been reported for the determination of tetracyclines, using UV [5–8], fluorescence [9, 10] or chemiluminescence [11] detection. Samples analysed include foods such as fish muscle [8], chicken meat [9], honey [11] or milk [7, 10], as well as environmental samples [6] and drugs [5]. Electrochemical detection (ED)

952

Anal Bioanal Chem (2007) 389:951–958

is also frequently used owing to the well-known advantages like sensitivity and easy sample pre-treatment. Table 1 summarizes the analytical characteristics of several reported flow-injection (FI) and HPLC methods applied to the electrochemical detection of tetracyclines [12–20]. Important drawbacks, such as the high potential values required for the electrooxidation of these compounds, or fouling of the electrode surface by the products of the electrochemical reaction were observed when using conventional electrode materials as detection systems. So, relatively high detection limits were achieved when a glassy carbon electrode was employed [15]. In order to improve both the sensitivity and reproducibility of the electrochemical measurements, various approaches have been proposed. Pulsed amperometric detection (PAD) with multiple step waveforms using gold and boron-doped diamond (BDD) electrodes has been applied to avoid fouling and enhance sensitivity [12–14]. An anodized BDD electrode has also been used for the oxidation and flow-injection detection of tetracyclines [18]. Modified electrodes prepared by appropriate coating of the electrode surface with different modifier materials have also been reported. A nickel-modified glassy carbon electrode showing electrocatalytical activity towards antibiotic oxidation was applied to the flow-injection detection of TCs [19]. Moreover, a diamond thin film electrode modified with nickel (Ni-DIA) was used for the amperometric detection of various tetracyclines and their determination in shrimp samples [20]. Recently, carbon nanotubes (CNTs) have emerged as a very promising material for the preparation of amperometric sensors [21]. CNT modified GCEs provide stable and sensitive electrochemical responses for phenols [22], NADH [23], estrogenic phenolic compounds [24] and βcarboline alkaloids [25].

This work reports the voltammetric behaviour of tetracycline antibiotics at CNT modified glassy carbon electrodes and their determination by amperometric monitoring of their oxidation response at the modified electrodes. Using this approach, highly sensitive and reproducible electroanalytical responses can be achieved. Moreover, the HPLC-ED method was developed to determine TCs in water samples from a well and a fish farm pool.

Experimental Apparatus and electrodes Voltammetric measurements were carried out with a BAS 100B potentiostat provided with a BAS C2 EG-1080 cell stand. A Metrohm 6.084.010 glassy carbon electrode (3-mm ø), modified with multiwall carbon nanotubes (MWCNTs) or unmodified, was used as working electrode. The reference electrode was a BAS MF 2063 Ag/AgCl 3 M electrode, and a BAS MW-1032 Pt wire was used as the auxiliary electrode. BAS VC-2 10-mL electrochemical cells were also used. Chromatographic and flow-injection experiments were carried out using a Knauer 1000 Smartline pump fitted with a Knauer 5000 Smartline Manager stand, and a Knauer A 1357 injection valve fitted with a 50-μL coil. A Luna C18 (150×4.6-mm i.d., 5-μm particle size) (Phenomenex) chromatographic column was used. The flow-cell was a Metrohm EA-1096 wall-jet cell fitted with a Ag/AgCl reference electrode and a gold counter electrode. The potential values applied were controlled by means of a BAS Epsilon Multichannel detector, and the Chromgraph 1.0.01 software (Liquid Chromatography Control Software) from BAS was used to record data.

Table 1 Analytical characteristics of methods for the electrochemical detection of tetracyclines Compound

Technique

Electrode

Edet (V)

Linear range (μM; ng mL−1 ×10−4)

LOD (μM; ng mL−1)

Ref.

TC TC, OTC, CTC, DC DMTC, OTC, TC OTC, DC, MC, TC, CTC, MTC, DMC TC, OTC, CTC, MTC, DC OTC TC, OTC, CTC, DC TC, OTC, CTC, DC TC, OTC, CTC, DC

FI-PAD HPLC-PAD HPLC-IPAD HPLC-ED

AuE BDDE AuE GCE

1.15; 0.6; 1.6; 0.1 1.5; 2.0; 0.4 0.1–1.2; −1.5 1.2

5–2,000; 0.2–100 0.21–208; 0.01–10 (TC) 0.21–3.1; 0.01–0.15 0.21–104; 0.01–5.0 (OTC)

1.0; 480 0.10–0.21; 50–100 0.10–0.17; 50–80 0.20–2.0; 100–1,000

[12] [13] [14] [15]

HPLC-CD

ESA CoulArray detector CFME BDDE Ni-GCE Ni-BDDE

0.4; 0.66; 0.68; 0.7

0.10–2.08; 0.005–0.1

0.026–0.052; 12.5–25

[16]

1.6

1.0–100; 0.05–5 100–50,000; 4.8–2,404 (TC) 5.2–208; 0.25–10 (TC) 0.1–201; 0.005–10 (OTC)

0.29; 150 0.01; 4.8 0.06–3.76; 30–1,810 0.02–0.10; 10–50

[17] [18] [19] [20]

FI-ED FI-ED FI-ED HPLC-ED

0.5 1.55

CTC chlortetracycline, OTC oxytetracycline, TC tetracycline, DC doxycycline, DMC demeclocycline, MC minocycline, MTC methacycline, DMTC dimethyltetracycline, AuE gold disk electrode, BDDE boron-doped diamond electrode, GCE glassy carbon electrode, CFME carbon fibre microelectrode


Reagents and solutions Stock 1.0×10−3 mol L−1 solutions of tetracycline hydrochloride (TC, Sigma, 95%), oxytetracycline hydrochloride (OTC, Sigma, 95%), chlortetracycline hydrochloride (CTC, Sigma, 82%) and doxycycline hydrochloride (DC, Sigma, 98%) were prepared in 0.05 mol L−1 phosphate buffer solution of pH 2.0. Working solutions were prepared from these by suitable dilution with the same buffer solution, which was also used as the supporting electrolyte. A 0.1 mol L−1 Britton–Robinson buffer solution was also used as supporting solution for the study of pH effect. Other solvents and chemicals used were of analytical reagent grade and water was obtained from a Millipore Milli-Q purification system. Multi-wall carbon nanotubes (MWCNTs, 30±15 nm ø, 95% purity) were supplied from NanoLab (Brighton, MA) and were used without any pretreatment. Water samples from a well and a fish farm pool were collected in amber glass bottles and filtered through a 0.45-μm nylon membrane. The samples were then stored at 4 °C until analysis. Procedures Preparation of the MWCNT modified GCE Prior to the modification, the glassy carbon electrode was polished with 0.3-μm alumina slurries, rinsed thoroughly with doubly distilled water, sonicated for 30 s in water and 30 s in acetone, and dried in air. A 1-mg sample of MWCNTs was dispersed with the aid of ultrasonic stirring for 45 min in an aqueous 0.1% Nafion solution. A 10-μL aliquot of this dispersion was dropped on the GC electrode surface and then the solvent was evaporated under an infrared heat lamp. When the bare glassy carbon electrode was employed, it was activated by polishing with alumina as described above. HPLC with amperometric detection at the MWCNT-GCE Chromatographic separations of mixtures of tetracycline antibiotics were performed using a 18% (v/v) acetonitrile/ 0.05 mol L−1 phosphate buffer of pH 2.5 as the mobile phase, which was previously filtered through a 0.1-μm membrane. A flow rate of 1.0 mL min−1 and an injection volume of 50 μL were used. A detection potential of +1.2 V vs. Ag/AgCl was applied. Determination of tetracyclines in spiked water samples Aliquots of 50 mL from water samples spiked with OTC, TC, CTC and DC at 31 ng mL−1, 30 ng mL−1, 78 ng mL−1 and 83 ng mL−1, respectively, were passed through 30-mg

953

Waters Oasis HLB cartridges (Waters, Milford, Massachusetts) which were previously conditioned with 5 mL methanol and 5 mL of an aqueous 10−4 mol L−1 HCl solution. The cartridge was then washed with 2 mL of a 2% (v/v) methanol aqueous solution. Finally, the analytes were eluted with two 5-mL portions of methanol. The eluate was evaporated to dryness under a nitrogen atmosphere and the dried residue was reconstituted in 500 μL of mobile phase. The determination of tetracyclines was carried out by using the standard additions method, which involved the addition of appropriate aliquots of each tetracycline to the spiked water samples, which were subjected to the same preconcentration and cleanup procedure described above.

Results and discussion Figure 1 shows cyclic voltammograms of 5.0×10−4 mol L−1 TC, OTC, CTC and DC, whose structures are also displayed, in 0.1 mol L−1 phosphate buffer of pH 2.0, recorded at a MWCNT-GCE and at a bare GCE. As shown, all the compounds tested exhibited oxidation waves at potential values higher than +1.2 V vs. Ag/AgCl. According to the literature, the electrochemical oxidation of tetracyclines occurs through the phenol group at position 10 and dimethylamino group at position 4 [15]. Similarly to that observed with an anodized BDD electrode [18], cyclic voltammograms from TCs did not show any cathodic signal, except in the case of CTC, which exhibits a broad cathodic peak in the reverse scan at +0.4 V similarly to that reported for 4-chlorophenol at carbon electrodes [26]. The currents obtained at the MWCNT-GCE are significantly larger than those obtained at the bare GCE. Considering that the overpotentials are not decreased with respect to the unmodified electrode, the enhanced voltammetric responses observed at the MWCNT-GCE were attributable to an increased active electrode surface area as a consequence of the MWCNTs coating. The effect of pH on the electrochemical oxidation of TC, OTC, CTC and DC at the MWCNT-GCE was tested by cyclic voltammetry over the 1.0 to 7.0 pH range using 0.1 mol L−1 Britton–Robinson buffer as supporting electrolyte. The voltammetric responses of tetracyclines are strongly affected by pH. Figure 2 shows, as examples, the results obtained for OTC (Fig. 2a) and CTC (Fig. 2b). As can be observed, the oxidation currents decreased with increasing pH over the whole range checked, and practically disappeared at pH values around 7. This behaviour is consistent with the proposed oxidation of tetracyclines through the phenol group at position 10. These results are in good agreement with the literature in which tetracyclines are reported to have poor stability in aqueous alkaline media [27]. No significant changes in the background voltammo-

954


Fig. 1 Cyclic voltammograms of 1.0×10−4 mol L−1 tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC) and doxytetracycline (DC): solid line at a MWCNT-GCE and dashed line at a bare GCE; dotted line background voltammogram in 0.05 mol L−1 phosphate buffer solution of pH 2.0, ν=50 mV s−1. Inset tetracycline structures

grams were observed in the whole pH range. Taking into account that the reproducibility of the measurements was lower at pH 1.0, a pH value of 2.5, which, as it is shown below, is also an appropriate pH value for the chromatographic separation of tetracyclines, was chosen for further work. Amperometric detection at the MWCNT-GCE Flow-injection with amperometric detection at the MWCNTGCE was used to obtain hydrodynamic voltammograms from 1.0×10−4 mol L−1 TC, OTC, DC and CTC. The corresponding peak current values measured at different applied potentials, when 50-μL aliquots of each solution were injected into a carrier solution consisting of a 0.05 mol L−1 phosphate buffer solution of pH 2.5, are depicted in Fig. 3. Fig. 2 Influence of pH on cyclic voltammograms of 1.0× 10−4 mol L−1 OTC and CTC at a MWCNT-GCE; 0.1 mol L−1 Britton–Robinson buffer of pH 1.5 (a) to 7.0 (g); ν=50 mV s−1. Dashed line background voltammograms at pH 2.5

As shown, the high oxidation potentials of these compounds give rise to hydrodynamic voltammograms without welldefined regions of current “plateaux”. Taking into account the most favourable signal-to-background current ratio in the potential region where the measured currents trend to level off, a potential value of +1.2 V was selected for the amperometric detection of TCs at the MWCNT-GCE. The repeatability of the amperometric flow-injection responses at this potential value was checked by successive injections of 50 μL of 1.0×10−4 mol L−1 TC, OTC, CTC and DC, and compared with those obtained at the bare GCE. Figure 4 displays the responses for TC and OTC. The relative standard deviation (RSD) for ip after 15 successive measurements for each TC was of approximately 3% for all of them at the MWCNT-GCE, whereas RSD values of between 7% (TC)


955

HPLC with amperometric detection at the MWCNT-GCE

Fig. 3 Hydrodynamic voltammograms obtained by flow-injection with amperometric detection at a MWCNT-GCE. 50-μL aliquots of 1.0×10−4 mol L−1 TC (¸), OTC ()), CTC (▲) and DC (●) solutions were injected into a carrier consisting of a 0.05 mol L−1 phosphate buffer solution of pH 2.5

and 12% (DC) were obtained at the bare GCE. Furthermore, in this case, a continuous decrease in the peak current was observed due to the surface fouling associated with the electrooxidation process of tetracyclines through the phenol moiety. Conversely, the unique properties of MWCNTs, with the exposed edge-plane ends or defects as electrochemically active sites [21], minimize electrode surface fouling, similarly to that shown for the oxidation of molecules such as NADH [22]. Fig. 4 Successive flow-injection amperometric responses obtained at a CNT-GCE (left) and at a bare GCE (right) after injection of 50 μL of 1.0×10−4 mol L−1 TC (a) or OTC (b)

An isocratic elution using a C18 reversed-phase column was employed for the HPLC separation of tetracycline antibiotics. The use of this stationary phase was recommended in the literature for this type of compound [11–13, 17]. The mobile phase used to investigate the separation of TCs consisted of an acetonitrile/0.05 mol L−1 phosphate buffer solution of pH 2.5. Firstly, the influence of the acetonitrile percentage on the separation of OTC, TC, CTC and DC was evaluated. Both the retention time and peak area decreased as the acetonitrile percentage increased from 10 to 20% (v/v). Therefore, a mobile phase with 18% (v/v) acetonitrile was selected to obtain adequate retention times for the less polar compounds (DC and CTC), as well as a good sensitivity. The effect of the mobile phase flow rate was tested in the 0.4–1.0 mL min−1 range. As expected, retention times and peak widths decreased as the flow rate increased for all the compounds. The highest flow rate checked, 1.0 mL min−1, was chosen for further work because higher values produced a leaching of the electrode coating. Under these conditions, a good resolution was observed for all the compounds. Retention times were 5.0, 6.7, 17.8 and 24.8 min for OTC, TC, CTC and DC, respectively. Figure 5 shows a chromatogram obtained for a mixture of these compounds at 1.0×10−4 mol L−1 each. Calibration graphs constructed for OTC, TC, CTC and DC by measuring the peak areas as the analytical signals, exhibited the analytical characteristics summarized in Table 2. The detection limits were calculated according to the 3 sb/m criterion, where m is the slope of the corresponding calibration graph for each analyte, and sb was

956


Fig. 5 Chromatograms obtained from a standard solution containing 10−4 mol L−1 each of 1 OTC, 2 TC, 3 CTC and 4 DC with amperometric detection at a MWCNT-GCE. Mobile phase, 18:82 acetonitrile/0.05 mol L−1 phosphate buffer of pH 2.5; flow rate, 1.0 mL min−1; Eapp =+1.20 V

Fig. 6 Chromatograms obtained from a water sample containing 2.0× 10−5 mol L−1 each of 1 sulfadiazine, 2 sulfamerazine, 3 OTC, 4 TC and 5 sufamethoxazol, with amperometric detection at a MWCNTGCE. Other conditions as in Fig. 4

Multiresidue analysis estimated as the standard deviation (n=10) of the peak area values obtained for the lowest concentration of the respective linear range. Detection limits ranged from 0.09 μmol L−1 (OTC) to 0.44 μmol L−1 (DC). When these values are compared with those reported previously using electrochemical detection (see Table 1), it can be observed that, in general, they are better than those achieved using other electrode materials, as amperometric detectors. For example, the limit of detection achieved for TC is approximately ten times lower than that obtained at a AuE using pulsed amperometric detection [12]. Also, the LOD for OTC is almost three times lower than that obtained by flow-injection with electrochemical detection at a carbon fibre microelectrode in an organic working medium and with an applied potential 400 mV higher [17]. An important additional advantage is the abovementioned minimization of the electrode surface fouling, which improves the repeatability of the measurements with no need to apply pulsed-based techniques for TCs detection.

Pharmaceutical products present as undesirable pollutants in wastewaters may belong to different families of veterinary drugs and therefore multiresidue analytical methods are necessary to prevent their presence and to optimize water treatments [1]. In order to evaluate the applicability of the developed method to the analysis of water samples containing various types of drugs, HPLC-ED/MWCNT-GCE was applied to mixtures of tetracycline antibiotics and sulfonamide drugs. As an example, Fig. 6 shows the chromatogram obtained from a sample containing 2.0× 10−5 mol L−1 each of sulfadiazine, sulfamerazine, OTC, TC and sulfamethoxazol. As can be observed, using the same experimental conditions employed for tetracycline separations, a good resolution was achieved for all the analytes tested, with retention times of 6.0, 8.3, 10.0, 13.0 and 29.9 min, respectively. Therefore, it is possible, in principle, to perform quantification of these sulfonamides together with that of TCs under the experimental conditions used.

Table 2 Analytical characteristics of the calibration graphs for different tetracyclines obtained by HPLC with amperometric detection at MWCNT-GCE Compound

Linear range (μM; ng mL−1 ×10−3)

Slope (μC M−1 ×104; μC ng−1 mL×107)

r

Intercept (μC)

LOD (μM; ng mL−1)

OTC TC CTC DC

2.5–100; 1–50 2.5–100; 1–50 1–100; 0.5–50 1–100; 0.5–50

8.2±0.2; 8.1±0.3; 6.7±0.4; 4.7±0.3;

0.993 0.995 0.990 0.990

−0.07±0.15 −0.01±0.12 −0.15±0.17 −0.12±0.12

0.09; 0.12; 0.31; 0.44;

3.9±0.1 4.0±0.1 3.4±0.2 2.3±0.1

41.4 53.3 149 206


957 Table 3 Recovery studies for the determination of TCs in spiked water samples using HPLC with amperometric detection at CNTsGCE Compound

Fish farm pool water

Well water

Milli-Q water

OTC TC CTC DC

95±2 99±3 87±6 93±4

98±4 97±5 95±2 89±2

97±2 88±5 93±5 95±3

were satisfactory, independently of the type of water analyzed, including the spiked Milli-Q water, thus demonstrating the suitability of the method for the determination of tetracyclines in water samples.

Conclusions Fig. 7 Chromatograms obtained from: a well water sample; b fish farm water spiked with OTC, TC, CTC and DC at a 2.0×10−7 mol L−1 each using amperometric detection at a MWCNT-GCE; 50-mL portions of samples were subjected to SPE before chromatographic analysis (see text)

Determination of tetracyclines in spiked water samples The developed HPLC-ED/MWCNT-GCE method was applied to the analysis of fish farm pool water and underground well water, which were both spiked with mixtures of OTC, TC, CTC and DC at 2.0×10−7 mol L−1. These spiking levels corresponded to those which may be found in the samples [5]. The sample treatment described in the Experimental was adapted from Ref. [1], a recent method developed for the determination of veterinary pharmaceuticals in wastewater. Considering that the detection limits achieved (see Table 2) are near the concentration values to be determined, solid-phase extraction using Oasis HLB cartridges was carried out for the preconcentration of the analytes and cleanup of the samples. This extraction was accomplished in a maximum period of 48 h. Moreover, a blank sample prepared with spiked Milli-Q water was also analysed to test the efficiency of the method. Following the procedure described above in Determination of tetracyclines in spiked water samples, a 100-fold preconcentration factor was achieved. Under these conditions, the detection limits in the water samples were estimated from five independent complete analyses of both fish farm pool and well water samples, and using the 3 sb/m criterion. The results obtained were between 0.75 and 3.10 ng mL−1 for fish farm pool water and between 0.50 and 0.70 ng mL−1 for well water. Figure 7 shows one of the chromatograms obtained for both kinds of samples. The results obtained for the analysis of five replicates from each sample are summarized in Table 3. As can be deduced, recoveries

The use of multi-wall carbon nanotube modified electrodes produced an enhancement of the electrochemical oxidation responses obtained for tetracycline antibiotics. This allowed amperometric detection of these compounds at the modified electrode to be performed, exhibiting some advantages with respect to the use of other electrochemical detection methodologies. This improved electroanalytical performance was exploited to develop an HPLC-EC method useful for the determination of tetracycline antibiotics in water samples. Furthermore, the possibility of multiresidue analysis is also demonstrated. Acknowledgements Financial support from the Ministerio de Educación y Ciencia (Projects CTQ2006-02905 and CTQ200602743), and PR27/05-13860-BSCH is gratefully acknowledged.

References 1. Babić S, Ašperger D, Mutavdžić D, Horvat AJM, Kaštelan-Macan M (2006) Talanta 70:732–738 2. Díaz-Cruz MS, López de Alda MJ, Barceló D (2003) Trends Anal Chem 22:340–351 3. Rabolle M, Spliid NH (2000) Chemosphere 40:715–722 4. Kim S-C, Carlson K (2006) Water Res 40:2549–2560 5. Monser L, Darghouth F (2000) J Pharm Biomed Anal 23:353–362 6. Delépée R, Pouliquen H (2003) Anal Chim Acta 475:117–123 7. Furusawa N (2003) Talanta 59:155–159 8. Wen Y, Wang Y, Feng YQ (2006) Talanta 70:153–159 9. Pena ALS, Lino CM, Silveira IN (1999) J AOAC Int 82:55–62 10. Ferraz Spisso B, de Oliveira e Jesus AL, Gonçalves de Araújo Júnior MA, Alves Monteiro M (2007) Anal Chim Acta 581:108–117 11. Wan G, Cui H, Zeng H, Zhou J, Liu L, Yu X (2005) J Chromatogr B 824:57–64 12. Palaharn S, Charoenraks T, Wangfuengkanagul N, Grudpan K, Chailapakul O (2003) Anal Chim Acta 499:191–197 13. Charoenraks T, Chuanuwatanakul S, Honda K, Yamaguchi Y, Chailapakul O (2005) Anal Sci 21:241–246

958 14. Cai Y, Cai Y, Shi Y, Mou S, Lu Y (2006) J Chromatogr A 1118:35–40 15. Kazemifard AG, Moore DE (1997) J Pharm Biomed Anal 16:689–693 16. Zhao F, Zhang X, Gan Y (2004) J Chromatogr A (2004) 1055:109–114 17. Guzmán A, Agüí L, Pedrero M, Yáñez-Sedeño P, Pingarrón JM (2003) Electroanalysis 15:601–607 18. Wangfuengkanagul W, Siangproh W, Chailapakul O (2004) Talanta 64:1183–1188 19. Oungpipat W, Southwell-Keely P, Alexander PW (1995) Analyst 120:1559–1565 20. Treetepvijit S, Preechaworapun A, Praphairaksit N, Chuanuwatanakul S, Einaga Y, Chailapakul O (2006) Talanta 68:1329–1335

Anal Bioanal Chem (2007) 389:951–958 21. Wang J, Li M, Shi Z, Li N, Gu Z (2002) Electroanalysis 4:225– 230 22. Wang J, Deo RP, Musameh M (2003) Electroanalysis 15:1830– 1834 23. Musameh M, Wang J, Merkoci A, Lin Y (2002) Electrochem Commun 4:743–746 24. Vega D, Agüí L, González-Cortés A, Yáñez-Sedeño P, Pingarrón JM (2007) Talanta 71:1031–1038 25. Agüí L, Peña-Farfal C, Yáñez-Sedeño P, Pingarrón JM (2007) Anal Chim Acta 585:323–330 26. Ureta-Zañartu MS, Bustos P, Berríos C, Diez MC, Mora ML, Gutiérrez C (2002) Electrochim Acta 47:2399–2406 27. Bryan PD, Stewent JT (1993) J Pharm Biomed Anal 11:971– 976