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Electrochimica Acta 97 (2013) 202– 209

Contents lists available at SciVerse ScienceDirect

Electrochimica Acta

jou rn al h om epa ge: www.elsev ier .com/ locate /e lec tac ta

etermination of the fungicide picoxystrobin using anodic strippingoltammetry on a metal film modified glassy carbon electrode

afael M. Dornellasa, Rômulo A.A. Franchinib, Ricardo Q. Aucelioa,∗

Chemistry Department, Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, RJ 22453-900, BrazilCollege of Basic Sciences, Universidade Federal Fluminense, Campus Nova Friburgo, RJ, Brazil

r t i c l e i n f o

rticle history:eceived 23 November 2012eceived in revised form 17 February 2013ccepted 19 February 2013vailable online 5 March 2013

eywords:icoxystrobin

a b s t r a c t

Differential pulse anodic stripping voltammetry was used for trace determination of picoxystrobin inurine and water samples. Experimental conditions have been optimized and the measurement cyclestarted with the application of a deposition potential at −700 mV, during 60 s, for the in situ formation ofthe Bi film on a glassy carbon substrate and the accumulation of the analyte. It has been proven that, inthe conditions employed, a significant amount of Bi is still present as a film on the surface electrode whenthe anodic scan is performed from +790 to +1050 mV at a rate of 40 mV s−1 and that the presence of Bi isfundamental for the accumulation and oxidation of picoxystrobin during re-dissolution (peak maximum

−1

trobilurinsismuth filmrine analysis

at +954 mV). The supporting electrolyte was HCl 1 mol L and a cleaning procedure was developed inorder to minimize memory effects. Linear and homoscedastic analytical responses (r2 > 0.99) have beenobserved with limit of quantification of 100 �g kg−1. Solid phase extraction (on a C-18 cartridge) allowsthe pre-concentration of the analyte and eliminates interferences from urine samples. Recoveries from89.3 to 104.8% were found and interferences from azoxystrobin, dimoxistrobin, fluxastrobin, kresoxi-methyl and pyraclostrobin were evaluated.

. Introduction

Pesticides are all products and components of the physical,hemical or biological process employed to prevent, destroy, repelr mitigate any crop damaging pest [1]. They play a fundamentalole in agricultural production, however, the control of its residuesn food, biological fluids and in environmental compartments suchs water bodies are necessary to evaluate and prevent potentialarm to the population [2]. Among the several types of pesticides,here are fungicides that destroy or inhibit the action of pathogenicungi. The use of synthetic fungicides is very common in conven-ional agriculture and poses a risk to humans and the environment3].

Picoxystrobin (Fig. 1) belongs to a relatively new class of syn-hetic fungicides, known as strobilurins, which have recently beenntroduced in agriculture due to its stability and effectiveness. Theirctivity is based on the inhibition of mitochondrial activity byinding to the Qo site of the cytochrome b (Qo inhibitors) [4,5].ccording to the Brazilian National Agency of Sanitary Monitoring,

hich follows worldwide standards, the acceptable daily intake oficoxystrobin is 43 �g kg−1 [6].

∗ Corresponding author. Tel.: +55 21 35271319; fax: +55 21 35271637.E-mail address: aucelior@puc-rio.br (R.Q. Aucelio).

013-4686/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.electacta.2013.02.125

© 2013 Elsevier Ltd. All rights reserved.

Only a few methods are described in the literature to determinepicoxystrobin. One is based on capillary electrophoresis separa-tion using molecular absorption detection at 200 nm [7]. The limitof quantification (LOQ) of the order of 10−8 mol L−1 was obtainedusing a solid phase extraction and normal stacking mode on-lineanalyte concentration. Two studies employed gas chromatographywith mass spectrometric detection to analyze food. Solid phaseextraction was used to clean the sample before the analysis andthe reported LOQ was 5 �g L−1 [8]. Alternatively, the extraction ofthe analyte was assisted by ultrasound and the sample cleaningwas performed by gel permeation chromatography. The reportedlimit of detection (LOD) was 0.002 mg kg−1 [9]. Flow injection anal-ysis with chemiluminescence detection has also been used for thedetermination of picoxystrobin after ultrasound assisted extrac-tion. LOD of 0.27 ng mL−1 was achieved [10]. No electroanalyticalmethod is described in the literature for the determination ofpicoxystrobin. However, a recent work describes a method forthe determination of two strobilurins pesticides (azoxystrobin anddimoxystrobin) in food samples based on the square-wave voltam-metry using the hanging drop mercury electrode [11].

Despite their excellent performance in voltammetric deter-minations and their extensive use during the last five decades,

mercury based electrodes (the mercury drop or mercury film) havebeen replaced due to their toxicity and difficulties associated withhandling and disposal of mercury [12]. Several other materials,such as modified carbon electrodes, gold, platinum, silver, iridium,

R.M. Dornellas et al. / Electrochimi

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over a working day (8 h). The quantification of the analyte was car-

ig. 1. Chemical structure of picoxystrobin methyl (E)-3-methoxy-2-{2-[6-trifluoromethyl)-2-pyridyloxymethyl] phenyl} acrylate.

arious alloys and amalgams, have been proposed to replace mer-ury in the manufacture of electrodes but none of them approachedhe excellent qualities of mercury for electrochemical analysis13–17].

Recently, the bismuth film electrode (BiFE) was proposed as promising alternative for the use of mercury [16–22]. The usef BiFE is environmentally correct since the toxicity of bismuthnd its salts is negligible. In a similar manner of mercury, bismuthay allow pre-concentration of analyte on the electrode, leading

o excellent LOQ values [22,23]. Other attractive property of BiFE ishe possibility of working without prior deoxygenation of the solu-ions [19,23]. The working range reported in the literature for theiFE is a negative one ranging from −1200 to 0 mV [18–22]. Poten-ial scanning in positive potentials is not applied since it is believedhat Bi is totally removed from the surface of the substrate due tohe total oxidation of the film. Therefore, there are no reports in theiterature describing the use of BiFE in applications in the positiveotential range (anodic voltammetry).

Several works in the literature indicate BiFE for the deter-ination of Pb, Cu, Zn, Cd, Ni, Al, Fe, Co in a direct way or

fter complexation of the metal ion with a specific organic ligand22,24–27]. However, very little work has been done aiming theetermination of organic analytes. Literature describes the use ofiFE for the determination of sulfadiazine [28], parathion [29],ulfasalazine and olsalazine [30], in all cases using cathodic elec-rochemical analysis.

In this paper, it is demonstrated that a film of Bi is still presentt the surface of the glassy carbon substrate when a positive poten-ial range is applied to determine picoxystrobin. This new approachor BiFE requires a proper potential scanning strategy to preserveart of the Bi film when working at the positive potential range. Theork also indicates the importance of bismuth in the electrocataly-

is of picoxystrobin. The adsorptive anodic stripping voltammetricnalysis conditions were adjusted to allow the determination oficoxystrobin in water samples and urine in the presence of othertrobilurins.

. Experimental

.1. Reagents and chemicals

All solutions were prepared with ultrapure water (resistivityess than 18 M� cm−1) obtained from a water purifier Milli-Q Gra-ient System A10–Millipore (USA). Stock aqueous solutions of Bi3+

ere prepared from a 1000 mg L−1 atomic absorption standardolution (Aldrich, USA). Chromatography grade acetonitrile (ACN)nd methanol (Merck, Germany) were used. Acetic, hydrochlo-

ic, sulfuric, phosphoric acids, sodium acetate and sodiumydroxide were also from Merck. Azoxystrobin (99.0%), dimoxys-robin (99.0%), fluoxastrobin (99.0%), kresoxim-methyl (99.0%),

ca Acta 97 (2013) 202– 209 203

picoxystrobin (99.0%), pyraclostrobin (99.0%), trifloxystrobin(99.0%) were from Riedel-de-Haen (Germany).

2.2. Apparatus

The method was developed in a potentiostat/galvanostat BASiCV50W from Bio Analytical System Inc., USA. The instrument isinterfaced to a personal computer. The working electrode was aglassy carbon (GCE) where a bismuth film was formed. The Ag/AgClelectrode was used as the reference of the electrochemical systemand a platinum wire was used as the auxiliary electrode. The work-ing cells were made with borosilicate, with 15 mL internal volume(10 mL volumes have been used in this work).

A 200 series HPLC system (Perkin Elmer, USA) was used tocompare the results obtained by the analysis with the proposedmethod in the determination of picoxystrobin. The system wasequipped with standard modules of the 200 series HPLC: a binarypump, a degasser, an automatic sample injector and the diode arrayUV–vis absorption molecular photometric detector. The data acqui-sition/treatment software was supplied by the manufacturer.

For sample cleaning-up, a centrifuge model BE 4000 Brushlessfrom Bio-Eng (Brazil) was used. The pH measurements were madeon a pH meter (MS Technopon MPA-210, São Paulo, Brazil) with aglass membrane electrode conjugated with an Ag/AgCl referenceelectrode.

2.3. Samples and standards

Pesticide stock solutions (1 × 10−3 mol L−1) were prepared inACN and kept in the dark at 4 ◦C. Urine samples fortified withpicoxystrobin were submitted to solid phase extraction (SPE) in C-18 cartridges (3 mL and 500 mg of solid phase, Varian, USA). Beforeextraction, 2 mL of ACN was added to 5 mL of the sample followedby the adjustment of the volume to 10 mL with ultrapure water.The solution was centrifuged for 20 min at 3000 rpm to perform aclean-up. The supernatant of the urine sample were then passedthrough the C-18 cartridge previously activated with 2 mL of ACN,followed by 2 mL of water. The sample, loaded SPE cartridge, wasrinsed with 20 mL of water at 50 ◦C to remove retained impuritiesfrom urine. The retained pesticides were eluted with ACN (1 mL)and then diluted to 2 mL with ultrapure water. Samples (1 mL) ofQueen Creek and Rodrigo de Freitas Lake (both located in the Rio deJaneiro city) were fortified with picoxystrobin and diluted to 2 mLwith ultrapure water.

2.4. Procedures

All measurements were performed using differential pulseanodic stripping voltammetry. In order to perform the determi-nations using picoxystrobin, an initial potential (−700 mV) wasapplied for 60 s under continuous agitation in a cell containing HCl1 mol L−1 where 300 �L of a Bi3+ 1000 mg L−1 standard solution wasadded. During the process, the bismuth film was in situ formed andpicoxystrobin was accumulated. After 15 s equilibrium time, thepotential was scanned at 50 mV s−1 from +790 to +1050 mV withthe picoxystrobin maximum peak appearing at about +954 mV. Theprocedure adopted to obtain repetitive results for a triplicate mea-surement was to take into account only the results obtained afterthe fifth cycle of measurement. Under these conditions, repetitivemeasurements of picoxystrobin (in a cell containing HCl 1 mol L−1)indicated the stability of the analyte in this supporting electrolyte

ried out using standard addition. Calculations were made based onthe integrated peak area. Volumes of samples (urine or water) of1 mL were used for the analysis.

2 chimica Acta 97 (2013) 202– 209

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Fig. 2. Voltammograms obtained using anodic scanning from −300 to 1050 mVusing glassy carbon electrode. (A) in HCl 0.1 mol L−1 with the presence ofpicoxystrobin (9.9 × 10−6 mol L−1) (B) in HCl 0.1 mol L−1 without picoxystrobin (C)in HCl 0.1 mol L−1 with the presence of picoxystrobin (9.9 × 10−6 mol L−1) andBi3+ (4.7 × 10−5 mol L−1). Instrumental parameters: 50 mV pulse amplitude and

04 R.M. Dornellas et al. / Electro

Analyzes made with HPLC employed a C-18 column (250 mmength, 4.6 mm i.d., 5 �m particle size) from Perkin-Elmer. Chro-

atographic runs were made under isocratic regime with a mobilehase flow of 1.4 mL min−1. The mobile phase was ACN:0.1% phos-horic acid 60:40 (v/v). The injection volume was 10 �L, andetection made at 220 nm. Under such conditions, the retentionime of picoxystrobin was 8.2 min.

. Results and discussion

.1. Preliminary studies

Preliminary tests were performed using cyclic voltammetry (CV)nd differential pulse voltammetry (DPV) covering the potentialange from −1000 to +1000 mV, in both the anodic and the cathodiccanning directions. Glassy carbon was employed as the work-ng electrode with either HCl 0.1 mol L−1 or Britton–Robinson (BR)uffer at the acidic range (pH 2–4) as supporting electrolytes. Underhese circumstances, neither oxidation nor reduction characteris-ic peaks from picoxystrobin (present at the concentration levelf either 1 × 10−6 or 1 × 10−5 mol L−1) were identified. CV and DPVxperiments using the glassy carbon electrode were also performedith a higher concentration of picoxystrobin (1 × 10−3 mol L−1) in

HCl/ACN mixture and no response was observed.The test using differential pulse voltammetry was repeated

fter the addition of bismuth nitrate (final concentration of.7 × 10−5 mol L−1 in the electrochemical cell) aiming the in situormation of a Bi film on the surface of the glassy carbon electrodeGCE) before the potential scanning. The Bi deposition was made at900 mV for 60 s. Under such conditions, a peak (with maximumt +954 mV) was observed either in the BR buffer (pH 2) or in HCl.10 mol L−1 when the potential was scanned in the anodic direc-ion, in the range from +500 to +1000. As this potential range isbove the oxidation potential of bismuth (about −180 mV), furthertudies are needed to evaluate the role of Bi in this process.

DPV measurements were repeated in a cell in the absence ofi3+ using the GCE by applying 60 s deposition time at −900 mV,

ollowed by a scanning over the potential range from +790 to1050 mV. This experiment was performed to evaluate if the ana-yte could accumulate directly onto the GCE surface in order to bee-dissolved during the anodic scanning. Results obtained in the cellontaining picoxystrobin (9.9 × 10−6 mol L−1 final concentration)ere no different from the one obtained in the cell containing only

he supporting electrolyte (only HCl 0.10 mol L−1 was tested) as cane seen in voltammograms A and B of Fig. 2. However, as indicated

n the preliminary test, the addition of Bi3+ (4.7 × 10−5 mol L−1 finaloncentration in the cell) promoted the appearance of a peak with

maximum current at +954 mV whose magnitude changed in aroportional way as the experiment was repeated with differentoncentrations of picoxystrobin, which indicated that the peak isharacteristic from the analyte. The magnitude of the peak alsoecame larger as the film deposition time was increased to 90 semonstrating both that the analyte accumulated as the Bi film isormed and that the presence of Bi is important in the analyte accu-

ulation process. In addition, when repeated measurements wereerformed in the cell, the signal increased after each measurementycle (film formation at −900 mV and anodic stripping from +790o +1050 mV) indicating a memory effect on the surface of the elec-rode. As this process is not observed in the cell not containing Bi3+,t is possible that part of the Bi film containing the analyte is stillresent on the surface of the GCE after the anodic scanning.

Further tests were performed to evaluate the extension of thexidation of Bi after anodic scanning from +790 to +1050 mV aim-ng to investigate if there still bismuth left on the surface of theCE even operating at such positive potentials. First, the Bi film

25 mV s−1 scan rate.

was formed (60 s deposition time at −900 mV) in a cell containingBi3+ (at a 4.7 × 10−5 mol L−1 final concentration) and the supportingelectrolyte (HCl 0.10 mol L−1). Then, the electrode was washed withultrapure water before it was immersed in another cell containingonly the supporting electrolyte. An anodic scanning from −300 to+50 mV was performed in order to obtain the re-dissolution peakof Bi (voltammogram A of Fig. 3A). The surface of the electrode waspolished with alumina paste in order to remove all residues beforeanother ex situ film formation process was made. After the elec-trode containing the Bi film was washed with ultrapure water, itwas again immersed in a cell containing only the supporting elec-trolyte in order to perform a scanning in the positive range from+790 to +1050 mV before repeating the one from −300 to +50 mV.Surprisingly, a re-dissolution Bi peak was still observed althoughthis peak presented about 26% of the area of the one obtained inthe previous experiment (voltammogram B of Fig. 3A). After fivescans from +790 to +1050 mV, the re-dissolution peak decreasedto about 3% of the original signal. This result indicates that therestill bismuth left in the surface of the GCE even after the scanningat such high positive potentials. A similar series of experiments(scans from +790 to +1050 mV made after the formation of thebismuth film) was repeated in a cell containing the supporting elec-trolyte where picoxystrobin (1 × 10−4 mol L−1 final concentration)was added. The voltammogram made right after the oxidation ofthe Bi film indicated a Bi re-dissolution peak of about 20% of the areaof the one achieved with a fresh Bi film deposited on the GCE. Afterfive sequential scans, the Bi re-dissolution peak decreased to about1% of the original signal. Although not conclusive, this experimentmade in the presence of the analyte may indicate that picoxystrobinis affecting the oxidation process of Bi, which may imply in sometype of interaction between them. The normalized results achievedwith the experiments made in the absence and in the presence ofpicoxystrobin is shown in Fig. 3B.

The amount of Bi left on the surface of the GCE is also directlyproportional with the scan rate performed at the positive range asindicated by experiments made using anodic scans (from +790 to+1050 mV) at different scan velocities (from 5 to 50 mV s−1). Theresults (Fig. 4) indicates that the re-dissolution of the film is more

efficient as the electrode remains longer at a potentials higher thanthe one of the oxidation of bismuth.

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Fig. 3. (A) Bi redissolution peaks (measured by the scan from −300 to +50 mV)from a glass carbon coated with a Bi film (formed by applying −900 mV for 60 s)after performing (A) 0; (B) 1; and (C) 5 anodic scans from +790 to +1050 mV. (B)Normalized peak currents after performing (A) 0; (B) 1; and (C) 5 anodic scans from+790 to +1050 mV using a cell in absence (A1, B1 and C1) of picoxystrobin and a cellwith 1 × 10−4 mol L−1 picoxystrobin (A2, B2 and C2). Cell containing HCl 0.1 mol L−1

as the supporting electrolyte and Bi3+ (4.7 × 10−5 mol L−1).

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Fig. 4. Bi redissolution peaks (measured by the scan from −300 to +50 mV) from aglass carbon coated with a Bi film (formed by applying −900 mV for 60 s) after per-forming anodic scans (from +790 to +1050 mV) at different velocities: (A) 5; (B) 10;(C) 15; (D) 20; (E) 25; (F) 30; (G) 35; (H) 40; (I) 45; and (J) 50 mV s−1. Cell containingHCl 0.1 mol L−1 as the supporting electrolyte and Bi3+ (4.7 × 10−5 mol L−1).

ca Acta 97 (2013) 202– 209 205

The decreasing of the magnitude of the Bi re-dissolution peakis inversely proportional to the number of the anodic scans (from+790 to +1050 mV) performed before the signal measurement, atthe range from −300 to +50 mV, is made. For instance, in voltam-mogram C of Fig. 3A, the Bi re-dissolution peak observed after fivesequential anodic scans between −300 and +50 mV has only about2% of the area of the original re-dissolution peak.

These preliminary studies clearly show that a significant part ofthe Bi is still present at the surface of the electrode when the anodicscanning is made and that Bi may contribute to the electrocatalysisof picoxystrobin.

Diagnostic studies were made using square-wave voltammetryin order to obtain insights on the mechanism involved. In the firststudy, the variation of the frequency of the applied pulse (f), from 10to 50 Hz, is not linearly correlated with the value of the peak current,which is indicative of quasi-reversible systems. In addition, thereis no linear relationship between the log f value and peak potential,which ruled out the possibility of an irreversible system [31]. Fur-ther and complementary information could not be achieved by CVsince such experiment could not be performed using the bismuthfilm and since picoxystrobin does not suffer oxidation directly onthe glassy carbon.

The proton-dependence of the process has been proven as theanalyte signal significantly increased as the concentration of theHCl, used as the supporting electrolyte, was increased from 0.0050to 1.0 mol L−1. In order to maintain the ionic force of the cellfairly constant as the concentration of HCl decreased, an equivalentamount of KCl was added in order to keep constant the concentra-tion of Cl−, which was equivalent to the summation of the cations(H+ + K+). Although no mechanism can be described because of thelack of data, it is believed that picoxystrobin is reduced as it isadsorbed in the bismuth film, during the accumulation step, byreceiving protons. Then, the reduced product is oxidized.

3.2. Selection of experimental and instrumental conditions

In order to achieve the best experimental and instrumentalconditions for the differential pulse voltammetric determinationof picoxystrobin using BiFE, an exhaustive study was performedtaking into consideration the overall analysis procedure and thecritical parameters that affects the signal intensity, stability andreproducibility. The general procedure was the addition of Bi3+

standard solution and sample in a cell containing the supportingelectrolyte. The cycle measurement started with a negative depo-sition potential applied during a specific time in order to form thefilm and accumulate the analyte. Then, the anodic scanning from+790 to +1050 mV was performed to end the cycle (the cycle maybe repeated several times in order to obtain repetitive set of sig-nal measurements from the analyte in the cell). A cleaning stepwas made to let the electrode surface prepared for another set ofmeasurement cycles with another standard or sample. Therefore,crucial parameters to be optimized were: (i) type and concen-tration of the supporting electrolyte, (ii) concentration of Bi3+ inthe cell, (iii) deposition potential, (iv) deposition time, (v) scanrate, (vi) pulse amplitude and (vii) the electrode cleaning proce-dure.

The first critical choice was the supporting electrolyte to beemployed. Previous studies using BR buffer (0.04 mol L−1) in arange of pH 2–12, indicated that the current characteristic of theanalyte was observed only in pH 2. Therefore, tests were per-formed using non-buffered systems such as H2SO4 (1.0; 0.10 and0.010 mol L−1) and HCl (1.0; 0.10; 0.050 and 0.010 mol L−1). A sig-

nificant memory effect in sequential measurements was found inless concentrated acidic solutions and best signal profiles (higherintensity and sharper peaks) were achieved in HCl 1.00 mol L−1,therefore, it was chosen for analysis. Even in this condition, a

206 R.M. Dornellas et al. / Electrochimi

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issolution peak measured from +790 to +1050 mV. Instrumental parameters: HCl.00 mol L−1 supporting electrolyte, Bi3+ (6 × 10−5 mol L−1), 60 s deposition time,0 mV pulse amplitude, and 25 mV s−1 scan velocity.

eproducible analyte signal is only achieved after the about teneasurements as shown in Fig. 5. Along these four or five con-

ecutive cycle measurements, the re-dissolution signal becameonstant and stable. Such behavior was probably caused by the slowdsorption equilibrium of the analyte at the solution/Bi film inter-ace. When determining azoxystrobin and dimoxystrobin using theanging mercury drop electrode with HCl 0.10 mol L−1 as the sup-orting electrolyte, Pacheco et al. have also pointed out similarehavior [11]. Therefore, the data considered to account in sampler standard replicates were the ones after the fifth cycle measure-ent.

Further experiments were made to try to correlate the increas-

ng of the picoxystrobin signal, during the first runs until signaltabilization, and the variation of the Bi re-dissolution peak. ResultsFig. 6) have shown that the Bi re-dissolution peak (peaks C to H)

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ig. 6. Sequential Bi and picoxystrobin redissolution peaks from a glassy carbonoated with a Bi film (formed by applying −900 mV for 60 s). (A) Without Bi andnalyte; (B) with Bi and without analyte; (C) 1st run with both Bi and analyte; (D) 3rdun with both Bi and analyte; (E) 5th run with both Bi and analyte; (F) 6th run withoth Bi and analyte; (G) 9th run with both Bi and analyte; and (H) 12th run with bothi and analyte. Cell containing HCl 1.0 mol L−1 as the supporting electrolyte. Finaloncentration of Bi3+ (4.7 × 10−5 mol L−1) and picoxystrobin (1.0 × 10−4 mol L−1).

ca Acta 97 (2013) 202– 209

presents a tendency to increase along the runs (probably becauseof the residual Bi left on the surface of the GCE after each run) stabi-lizing when the analyte signal also stabilizes. Results indicate thatthe variation in analyte signal may be correlated to the variationsof the amount of the metal film formed onto the substrate.

A cleaning procedure, consisting on the application of a +400 mVduring 90 s, was used in order to eliminate any memory effectsfrom a previous measurement. Any cleaning potential above theoxidation potential of Bi would lead to a proper cleaning of the elec-trode no matter if the potential is below the oxidation potential ofthe analyte. The application of cleaning potentials above +1000 mV(above the oxidation potential of the analyte) causes a passivationof the electrode and a systematic decreasing of the analytical signal.Therefore, such high cleaning potential were avoided.

The choice of the film formation/analyte deposition poten-tial and time as well as the concentration of Bi3+ in the cellwere chosen after a careful study since these parameters stronglyaffected the quality of the analysis. In terms of the accumulationof picoxystrobin on the electrode surface, the analyte strippingsignal increased up to the application of 210 s of deposition timeno matter the concentration of Bi3+ in the cell (from 2.4 × 10−5

to 2.7 × 10−4 mol L−1). Such data was obtained using a fresh car-bon surface (after GCE surface polishing with alumina) for eachof the deposition times studied. However, when deposition timeslarger than 90 s were applied, the passivation of the electrode wasobserved after successive measurements using a single GCE, whicheventually made the electrode not responsive to the presence ofthe analyte. This passivation process is probably due to forma-tion of bismuth oxide which binds to the glassy carbon, therebypreventing its contact with the Bi film and with the solution. Inorder to restore the detection capability of the electrode, its sur-face was mechanically cleaned with 0.45 �m sandpaper followedby the immersion of the electrode in 10% nitric acid for 24 h. Some-times, the cleaning of the surface of the auxiliary electrode as well asthe maintenance of the reference electrode (cleaning of the porousmembrane and replacement of the internal solution) was alsorequired. In order to avoid the passivation of the electrode for theanalyses performed during a whole working-day, the depositiontime was set to a shorter value (60 s). Under such circumstances,the analysis could be performed without passivation of the surfaceelectrode for at least 60 measurement cycles before performingthe cleaning of the electrode as indicated by the repetitive voltam-mograms obtained from a sequence of measurement cycles in thesame cell.

The concentration of bismuth added into the electrochemicalcell for the in situ formation of the film was evaluated. Final con-centrations of Bi3+ in the cell, ranging from 0 to 2.7 × 10−4 mol L−1

were tested using 60 s deposition time at −900 mV. The analytesignal (picoxystrobin 7.7 × 10−6 mol L−1) increased as the concen-tration of Bi3+ reached up to 1.2 × 10−4 mol L−1 becoming stable inthe presence of higher concentrations of Bi3+ in the cell. The bis-muth concentration of 1.4 × 10−4 mol L−1 was chosen since it wasin the robust range of the analytical response (Fig. 7A). The depo-sition potentials (Edep) from 0 to −1000 mV were tested in orderto allow the measurement of intense and reproductive signals. Atthe deposition potential, the bismuth film is formed together withthe accumulation of the analyte. Picoxystrobin signal increasedup to −700 mV, decreasing when higher Edep values were applied(Fig. 7B). Results using Edep of −700 mV were also reproducible aftera series of consecutive runs.

The potential scan rate of 40 mV s−1 was chosen as it is in therobust range (from 35 to 50 mV s−1) that enabled intense analyte

re-dissolution current. Pulse amplitude (75 mV) was also chosenwithin the range that enabled maximum signal (60–120 mV). Con-ditions chosen to perform the determinations of picoxystrobin areindicated in Table 1.

R.M. Dornellas et al. / Electrochimica Acta 97 (2013) 202– 209 207

0.00 0.05 0.10 0.15 0.20 0.25 0.30

0

7

14

21

28

35In

tegr

ated

pea

k ar

ea (n

A)

Bi concentration in the cell (mmol L-1)

0 -200 -400 -600 -800 -1000

0

8

16

24

32

40

Inte

grat

ed p

eak

area

(nA

)

Potential (mV)

A

B

Fig. 7. (A) Integrated area of the re-dissolution peak of picoxystrobin (measuredfrom +790 to +1050 mV) in function of the concentration of Bi3+ in the cell. (B) Effectof different deposition potentials during 60 s on the picoxystobin re-dissolution peakarea (measured from +790 to +1050 mV). Instrumental parameters: HCl 1.00 mol L−1

sa

3

upistcpc

TEo

50 10 15 20

0

20

40

60

80

Inte

grat

ed a

rea

of p

eak

(nA

)

Concentration of picoxystrobin (µmol L-1)

850 900 950 1000 10500.8

1.2

1.6

2.0

2.4 F

E

D

C

B

ACur

rent

(µA

)

Potential (mV)

A

B

Fig. 8. (A) Voltammograms of increasing concentrations of picoxystrobin: (a)blank; (b) 3.9 × 10−6 mol L−1; (c) 7.7 × 10−6 mol L−1; (d) 1.2 × 10−5 mol L−1; (e)1.5 × 10−5 mol L−1; (f) 1.9 × 10−5 mol L−1. (B) Analytical curve with equation modelof Y = (4.29 × 10−3 L mol−1)X + 2.42 × 10−9 and R2 = 0.9945. Instrumental parame-ters: supporting electrolyte HCl 1.00 mol L−1, 75 mV amplitude, 35 mV s−1 scan rate,

upporting electrolyte, Bi3+ (4.7 × 10−5 mol L−1), 60 s deposition time, 50 mV pulsemplitude, and 35 mV s−1 scan velocity.

.3. Analytical performance

The analytical figures of merit of the method were obtainedsing the experimental conditions selected for determiningicoxystobin. A sequence of voltammograms of solutions contain-

ng increasing concentrations of picoxystobin is shown in Fig. 8,howing that the stripping peak area is directly and linearly propor-ional to the concentration of picoxystrobin in the electrochemical

ell. The analytical curve, Y = (4.29 × 10−3 L mol−1)X + 2.42 × 10−9,resented linear response (R2 = 0.9945) from de limit of quantifi-ation (LOQ) up to 1.9 × 10−5 mol L−1. The limit of detection (LOD)

able 1xperimental conditions chosen for the voltammetric method for the determinationf picoxystrobin.

Parameter Value

Supporting electrolyte HCl 1.00 mol L−1

Cleaning potential 400 mVCleaning time 90 sDeposition potential −700 mVDeposition time 60 sAmplitude 75 MvScan rate 40 mV s−1

deposition potential at −700 mV during 60 s.

in the analytical cell was 2.3 × 10−8 mol L−1 (8.4 �g L−1) of picoxys-trobin. The LOD was calculated using the xb + 3sb criteria, wherexb is the average value of 10 consecutive measurements of theblank and sb is the standard deviation of the blank, which was esti-mated by measuring 10 consecutive measurements of the lowestconcentration of analyte in the curve. The LOQ calculated in thecell was 3.1 × 10−8 mol L−1 (11.3 �g L−1) and it was based on thexb + 10sb criteria. LOD and LOQ values in the sample were in theorder of 10−7 mol L−1 (about 100 �g L−1) since the dilution factorof the samples in the cell, urine samples for instance, is about 10times.

The precision was evaluated through the repeatability and inter-mediary precision studies. The repeatability was evaluated byten consecutive signal measurements (after the first five or sixvoltammetric runs for signal stabilization) of a solution contain-ing 3.9 × 10−6 mol L−1 of picoxystrobin. The intermediary precisionwas evaluated by comparing the results achieved from two dif-ferent analysts measuring ten times a solution containing theanalyte (each analyst has prepared its own solution). These experi-ments were repeated in three different days using freshly prepared

analyte solutions. The results indicated repeatability of 4.1% andintermediary precision of 2.7% (value that indicated the differenceof the results achieved by the analysts).

208 R.M. Dornellas et al. / Electrochimica Acta 97 (2013) 202– 209

Table 2Evaluation of the interference from other strobilurins in the picoxystrobina analyt-ical signal.

Interferent Proportion(picoxystrobin:interferent)

iPicoxystrobin/(iPicoxystrobin +iInterferent)

Fluoxastrobin 1:0 1.001:1 1.001:5 1.001:10 1.481:20 5.031:30 30.82

Pyraclostrobin 1:0 1.001:1 6.591:2 1232.20

Trifluoxystrobin 1:0 1.001:1 1.271:5 2.501:10 7.23

Kresoxim-methyl 1:0 1.001:1 1.001:5 1.001:20 3.97

okatp(u1o

mcih(acepftepsfii

3

F1melmipSw

Table 3Recoveries of picoxystrobin in analyte fortified water samples and in urine at twolevels of concentration: (I) 4.9 × 10−6 mol L−1 and (II) 1.3 × 10−5 mol L−1.

Sample Day Recovery (%), confidence limit(n = 3)

Level I Level II

Queen Creek 1 104.2 ± 0.5 98.8 ± 3.12 108.3 ± 0.6 100.4 ± 1.43 107.8 ± 1.0 100.4 ± 2.1

Rodrigo de Freitas Lake 1 94.8 ± 1.0 84.6 ± 0.82 108.1 ± 2.0 100.5 ± 2.53 106.2 ± 1.5 100.7 ± 2.1

1:30 13.09

a Peak potential measured at +954 mV (scan from +790 to +1050 mV).

The selectivity was evaluated by recovery tests. The influencef other strobilurins (azoxystrobin, dimoxistrobin, fluxastrobin,resoxi-methyl, pyraclostrobin and trifluoxystrobin) was testednd the ratios between the signal obtained from a picoxys-robin standard and the signal obtained from a mixture oficoxystrobin and increasing proportions of another strobilurinipicoxistrobin/i(picoxystrobin+interferent)) were evaluated. While ratio val-es close to unity indicated no interference, ratio values higher than

indicated a decrease of the analyte signal due the presence ofther strobilurin.

No analytical signal from pyraclostrobin, fluxastrobin, kresoxi-ethyl and trifluoxystrobin are found in the experimental

onditions defined in this paper. Fluxastrobin and kresoxi-methylmpose no interferences when present at concentrations five timesigher than the one of picoxystrobin in the electrochemical cellsee Table 2). As the proportion of these interferents increased, thenalyte signal decreased, however, even in such cases, the analyteould still be determined by the analyte addition method. The pres-nce of pyraclostrobin or trifluoxystrobin, even at the equimolarroportions, decreased the signal of picoxystrobin. Interferencesrom trifluoxystrobin can be corrected by the analyte additionechnique. However, the interference from pyraclostrobin is moreffective, eliminating the analyte signal when it is present at pro-ortions only two times higher than picoxystrobin. Under theelected experimental conditions, analytical signals were observedor dimoxistrobin and for azoxystrobin around the same character-stic potential of picoxystrobin. Therefore, these two strobilurinsmpose critical interferences for the method.

.4. Application of the method

Studies using analyte fortified urine samples were performed.ortification was made at two concentration levels (10−6 and0−5 mol L−1). It was observed a strong interference from the urineatrix that eliminated the analyte signal. Therefore, a solid phase

xtraction (on a C-18 cartridge) was employed to separate the ana-yte from the interfering matrix components. Solid phase extraction

ay also be used to pre-concentrate the analyte at least 10 times

n order to achieve the capability to analyze samples containingicoxystrobin at concentration down to 10 mg kg−1 (10 �g L−1).amples from the Queen Creek and from Rodrigo de Freitas Lakeere analyzed directly without any treatment but filtration.

Urine 1 94.8 ± 0.1 90.0 ± 1.42 92.5 ± 0.3 90.1 ± 0.83 94.1 ± 0.6 90.8 ± 1.1

Recovery tests were performed using the analyte addition tech-nique for the urine samples and by directly interpolating the signalsfrom the water samples in the analytical curve prepared on a differ-ent cell. Results are presented in Table 3, with satisfactory resultsin all cases.

The performance of the proposed method in the analysis ofwater samples fortified with picoxystrobin (6.0 × 10−6 mol L−1)was compared to the one achieved by using high performance liq-uid chromatography (HPLC) with absorption detection at 220 nm.Six authentic replicates were analyzed and the average resultsobtained by the two different methods were evaluated by Student ttest at a confidence interval of 95%. The experimental t-value (0.52)was smaller than the reference value (2.23) for 10 degrees of free-dom. Therefore, results indicated no significant difference in theresults achieved by the two different methods.

4. Conclusions

A new method for the determination of picoxystrobin wasdeveloped. The approach using BiFE is new and the importanceof Bi in the process, even at positive potentials, has been proven.The method was used to determine the analyte in urine and watersamples with satisfactory results and selective towards othersstrobilurins. Such analytical performance becomes an attractivealternative in relation to more complex and expensive techniquessuch as capillary electrophoresis, gas chromatography and liquidchromatography.

Acknowledgements

Aucélio thanks CNPq-Brazil and FAPERJ for research grants andscholarships. Dornellas thanks CNPq-Brazil for scholarship.

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