Journal of Electroanalytical Chemistry 708 (2013) 46–53

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Journal of Electroanalytical Chemistry

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Determination of the fungicide kresoxim-methyl in grape juices usingsquare-wave voltammetry and a boron-doped diamond electrode

1572-6657/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.jelechem.2013.09.015

⇑ Corresponding author. Fax: +55 21 3527 1637.E-mail address: aucelior@puc-rio.br (R.Q. Aucelio).

Rafael M. Dornellas a, Rômulo A.A. Franchini b, Andrea R. da Silva a, Renato C. Matos c, Ricardo Q. Aucelio a,⇑a Chemistry Department, Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, RJ 22453-900, Brazilb College of Basic Sciences, Universidade Federal Fluminense, Campus Nova Friburgo, RJ 22625-650, Brazilc Chemistry Department, Universidade Federal de Juiz de Fora, Juiz de Fora, MG 36036-900, Brazil

a r t i c l e i n f o

Article history:Received 29 June 2013Received in revised form 17 September 2013Accepted 19 September 2013Available online 29 September 2013

Keywords:Boron-doped diamond electrodeSquare-wave voltammetryKresoxim-methylGrape juice

a b s t r a c t

A square-wave voltammetric method for the determination of kresoxim-methyl in grape juices wasdeveloped using the nanocrystalline boron-doped diamond electrode where an irreversible oxidationoccurred. Measurements were made through the anodic scan from +1000 to +1750 mV (Maximum at+1420 mV) in pH 4.0 acetate buffer 0.050 mol L�1. Linear signal response (R2 > 0.999) expanded formthe 8.7 � 10�7 mol L�1 (0.27 mg L�1) up to 3.4 � 10�5 mol L�1 (11 mg L�1). The method was applied inthe analysis of analyte fortified grape juices. Solid phase extraction allowed pre-concentration of the ana-lyte and eliminated interferences imposed by sample matrix. Recoveries from 91.6% to 105.3% werefound and agreed with results achieved using high performance liquid chromatography.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction strobilurins, among them kresoxim-methyl, was performed. Three

Agrochemicals play an important role in food protection andpreservation and, in the case of pesticides, the quantification oftheir residues is crucial for proper assessment of human exposurethrough foods and during farming [1]. The strobilurins comprise animportant class of synthetic agricultural fungicides presentingoutstanding anti-fungicide action, stability and lower toxicity tohumans. Therefore, strobilurins are currently being utilized in awide range of crops throughout the world [2–4]. Nowadays, thereare several commercially available strobilurins for farming purposes[5] being kresoxim-methyl, methyl (E)-methoxyimino[a-(o-tolyl-oxy)-o-tolyl]acetate (Fig. 1), one of the most used in grapecrops. According to the Brazilian National Agency of SanitarySurveillance, which follows global standards, the acceptable dailyintake of kresoxim-methyl through grape juice or from grapeconsumption is 0.50 mg kg�1 [6].

Different analytical techniques have been applied for the deter-mination of the strobilurins and most of them focus on kresoxim-methyl because of its broad activity against pathogenic fungi andthe wide application in fruit plantations. A preliminary study onthe fate of kresoxim-methyl in grapes and wine has been per-formed by gas chromatography (GC) using mass spectrometry(MS) detection and thermionic nitrogen detection [7]. Later on, acareful validation of a GC method for the determination of three

detection approaches were tested (MS, thermionic and electroncapture) with comparable values for the achieved limits of detec-tion (down to 0.006 mg kg�1) when applied for the analysis ofwheat, apples and grapes [8]. Seven strobilurins, includingkresoxim-methyl, were determined in baby food by a GC–MSmethod based on the direct immersion solid-phase micro extrac-tion. Limit of quantification for kresoxim-methyl was 0.048 ng g�1

[9]. High performance liquid chromatography (HPLC) with molec-ular absorption detection has also been used for the determinationof kresoxim-methyl in grapes and wine. Liquid–liquid extractionusing ethyl ether was employed. The extracted material was re-dissolved in n-hexane and the samples were submitted to aclean-up procedure using a silica cartridge. Isocratic elution withmethanol–water and a C-18 stationary phase were employed toenable a retention time of 7.5 min for the analyte and a limit ofdetection of 0.362 mg kg�1 and calculated combined uncertainty(following EURACHEM/CITAC guidelines) of 16% for the analysisof grapes and about 32% for the analysis of wines [10]. Recently,a study of the retention mechanisms of four strobilurins in differ-ent modified stationary phases was performed using experimentaldata and mathematical modeling. These calculations may predictthe conditions to separate, for instance, kresoxim-methyl fromother pesticides [11]. No electroanalytical method has beendescribed in the literature aiming the determination of kresoxim-methyl, however, recent works describe a method for the determi-nation of other two strobilurin pesticides (dimoxystrobin andazoxystrobin) in grape juice and potatoes by square-waveadsorptive stripping voltammetry using the hanging mercury drop

Fig. 1. Chemical structure of kresoxim-methyl.

R.M. Dornellas et al. / Journal of Electroanalytical Chemistry 708 (2013) 46–53 47

electrode [12] and a method for the determination of picoxystrobinin urine samples using a glassy carbon electrode covered by anin situ formed metallic film [13]. Limits of detection achieved withthese approaches were in the lg L�1 level.

Diamond presents unusual physical and chemical propertiessuch as high electrical resistivity, high thermal conductivity, highcorrosion resistance, low coefficient of friction, chemical inertness,and optical transparency [14]. The resistivity of polycrystalline dia-mond can be decreased down to 0.01 X cm after boron doping[15,16] since boron acts as an electron acceptor (due to the elec-tron deficiency in its outer shell) enabling p-type semiconductingproperties by lowering the material0s Fermi level as the numberof charge carriers is increased [14].

In recent years, boron-doped diamond (BDD) electrodes havebeen applied in various areas of research including in analyticalchemistry, for instance, aiming pharmaceutical analysis [17,18]and environmental analysis [19]. The advantageous performanceof the BDD over the glassy carbon electrode (GCE) and metallicor metallic film based electrodes relies on electrochemical featuresthat includes higher electrochemical stability, wider electrochem-ical potential window in aqueous solutions, lower capacitive back-ground current, excellent resistance to electrode fouling, goodbiocompatibility, stability of response and lower sensitivity to oxy-gen [20]. In addition, BDD electrodes allow measurements at highanodic potentials and in extreme conditions such as strongly acidicmedia. It has also been shown that the high oxidation power ofBDD promotes the effective degradation of organic substancesforming CO2 and H2O due to the production of large amounts of hy-droxyl radicals from water on BDD surface. Its larger O2 overvolt-age produces a higher concentration of adsorbed �OH and aquicker oxidation of organics [21,22].

In this work, a voltammetric method for the determination oftrace amounts of kresoxim-methyl in commercial grape juicewas developed using the BDD as the working electrode. Squarewave voltammetry (SWV) was employed to enable faster determi-nations when compared to other voltammetric pulse techniques. Itis also a cost effective method when compared to methods basedon complex analytical techniques such as GC–MS and HPLC. Themechanism of oxidation of kresoxim-methyl by BDD was studied.

2. Experimental

2.1. Instrumentation

The method was developed using a potentiostat/galvanostat (l-AUTOLAB Type III, Metrohm, Netherlands) interfaced to a personalcomputer, operating in the voltammetric analysis square-wavemode. The working electrode was a 1.0 cm2 polycrystalline bor-on-doped diamond (BDD), Adamant Technologies, Switzerland,(BDD coating: p-doped, 1–1.5 lm thick 6000–8000 mg L�1 borondoping). The Ag/AgCl(sat) electrode was used as the reference ofthe electrochemical system and a platinum wire was used as theauxiliary electrode. The working cells were made with borosilicate,with 15 mL internal volume.

A Series 200 HPLC system (Perkin Elmer, USA) was used to com-pare the analytical results. The system was equipped with a binarypump, a degassing unit, a diode array UV–visible absorptionmolecular photometric detector, an automatic sample injector

and data acquisition and data treatment software supplied by themanufacturer.

The pHmeter (Model mPA-210, MS Tecnopon LTDA, Brazil),with a glass membrane electrode conjugated with Ag/AgCl refer-ence electrode, was used.

2.2. Reagents and solutions

All solutions were prepared with ultrapure water (resistivityless than 18 MX cm) obtained from a water purifier Milli-Q Gradi-ent System A10, Millipore (Billerica, MA, USA). Azoxystrobin(99.0%), fluoxastrobin (99.0%), kresoxim-methyl (99.0%), trifloxyst-robin (99.0%), picoxystrobin (99.0%) and pyrachlostrobin (99.0%)were from Riedel-de-Haen (Seelze, Germany). Acetonitrile, metha-nol (both chromatografic grade), acetic acid, boric acid, hydrochlo-ric acid, sulfuric acid, phosphoric acid, sodium acetate and sodiumhydroxide were from Merck (Darmstadt, Germany). Potassium fer-ricyanide was from Sigma–Aldrich, USA.

2.3. Sample preparation

Pesticide stock solutions (1.0 � 10�3 mol L�1) was prepared inacetonitrile and kept in the dark at 4 �C. A fraction of 5.0 mL ofgrape juice (approximately 0.9 g) was fortified with kresoxim-methyl at the concentration levels of 6.0 � 10�6 mol L�1

(1.9 mg L�1) and 1.2 � 10�5 mol L�1 (3.8 mg L�1) and the volumecompleted to 10.0 mL with ultrapure water. The sample was thenloaded into a solid phase extraction (SPE) C18 cartridge (3.0 mLand 500.0 mg of the solid phase, Varian, USA) and washed with20.0 mL of ultrapure water. The cartridge has been previously trea-ted with 2.0 mL of acetonitrile, followed by 2.0 mL of water. The re-tained analyte was eluted with acetonitrile (1.0 mL) and thendiluted to 2.0 mL with ultrapure water. An aliquot of 1.0 mL of thissolution was added to the electrochemical cell and analysis wasperformed using the analyte addition procedure. Six samples ofgrape juice were prepared and fortified with kresoxim-methyl(6.0 � 10�6 mol L�1 or 1.9 mg L�1) by the same procedure de-scribed above for analysis by HPLC and compared with the pro-posed voltammetric method. Queen Creek water samples did notrequire any treatment (except filtering through a 0.45 lm mem-brane filter) previously to the electrochemical analysis.

The masses of grape present in the selected volumes of juicesample were estimated bringing the grape juice to dryness in anoven at 110 �C for 3 h. The solid residue was weighted and usedas an estimate of the mass of the grape in grape juice samples.

2.4. Electrochemical measurements

Pretreatment of the BDD electrode was performed daily using gal-vanostatic chronopotentiometry. Initially BDD was immersed inH2SO4 0.10 mol L�1 and submitted to an anodic pretreatment byapplying a current of 0.01 A for 1000 s, then a cathodic pretreatmentwas made with �0.01 A per 1000 s. Both anodic and cathodic pre-treatments are necessary in order to achieve repetitive measure-ments with BDD. Subsequently, cyclic voltammetry was used, inthe potential range from�500 to 1500 mV, until signal stabilization.

Diagnostic studies of the redox mechanisms were made withcyclic voltammetry with the scan rate of 100 mV s�1 and step po-tential of 2 mV in a potential range from 1200 to 2000 mV, usingacetate buffer (0.01 mol L�1 pH 4.0) as supporting electrolyte.

Analytical measurements and diagnostic studies of the redoxmechanisms were performed using square-wave voltammetry(SWV). For the determination of kresoxim-methyl, acetate buffer(0.050 mol L�1 pH 4.0) was used as the supporting electrolyte.After 15 s of equilibration time, the potential was scanned from+1000 to +1900 mV with a maximum peak kresoxim-methyl

48 R.M. Dornellas et al. / Journal of Electroanalytical Chemistry 708 (2013) 46–53

appearing at about +1420 mV. Frequency of 30 Hz, step potential of15 mV and pulse amplitude of 50 mV completed the electroanalyt-ical conditions. All calculations were made based on the integratedpeak area.

Analyzes made with HPLC-UV employed a reverse phase C18column (250 mm length, 4.6 mm i.d and 5 lm average particlesize) from Perkin-Elmer, isocratic elution using a acetonitrile/phos-phoric acid (0.1%) 60/40% v/v mobile phase at a flow rate of1.4 mL min�1. Injected sample volume was 10 lL and the absorp-tion photometric detection was made at 220 nm. Under such con-ditions, kreosoxim-methyl retention time was 7.0 min.

2.5. Determination of the electroactive area of the electrode

The BDD plate had an original 1 cm2 surface area. However, partof this surface is isolated with silicone glue during the constructionof the electrode. The working active area was established by per-forming a sequence of cyclic voltammetric measurements (from�250 to 650 mV) of a [Fe(CN)6]3� solution (using 0.50 mol L�1

potassium sulfate as the supporting electrolyte) at increasing scanrates ranging from 20 to 100 mV s�1. The calculation of the work-ing area is based on the graphic that relates the voltammogrampeak heights (Ip) in function of the square-root of the scan rate(v1/2). The slope of the linear response is equal to 2.687� 105 n3/2

A D1/2 C, where n is the quantity of electrons (mol) involved inthe redox reaction, A is electroactive area of the electrode (cm2),D is the diffusion coefficient of the ferricyanide ion (6.32 � 10�6

cm2 s�1) and C is the concentration of potassium ferricyanide(1.00 � 10�6 mol cm�3). The electroactive area of the workingelectrode was 0.035 cm2.

3. Results and discussion

3.1. Supporting electrolyte studies and electrochemical investigation

In order to obtain the best conditions for the determination ofkresoxim-methyl, a preliminary study was conducted. For thechoice of the supporting electrolyte, the analyte solution (at1.0 � 10�5 mol L�1) was prepared in different solutions where theBDD electrode was immersed and submitted to a potential scanover the range from �1000 to +2500 mV, using square-wave vol-tammetry (SWV) with the following initial instrumental parame-ters: 40 Hz of frequency (f), pulse amplitude (a) of 20 mV andstep potential (DEs) of 10 mV. The Britton-Robinson buffer(0.040 mol L�1), which is a mixture of the acetate, phosphate andborate buffers, was used to evaluate the response over the pHrange from 2.0 to 12.0. The peak potentials (Ep) of kresoxim-methyland their integrated areas are slightly influenced by the pH of theaqueous system. At acidic pH, the Ep value of the first most intenseoxidation peak of kresoxim-methyl remains to about +1420 mVwith much less intense oxidation peak at about +1700 mV(Fig. 2A). At the basic pH (8–12), the Ep of the first peak is shiftedto about +100 mV with a 30% signal deceasing while the secondoxidation peak disappears. Working at the acidic pH range alsopreserved the integrity of the electrode (as the as the BDD film isdeposited on a silicon substrate). The pH 4.0 value was chosensince it is in the middle of the pH acid range (from 2.0 to 6.0) with-in the analytical response is maximum and because it can bepromptly covered by the using of the acetate buffer. It was foundthat the use of acetate buffer (pH 4.0) at the concentration of0.050 mol L�1 enabled analytical signal with greater intensity (atleast two times higher than the ones observed using buffer at0.010 and 0.10 mol L�1) and therefore, it was chosen as the sup-porting electrolyte for the analytical determinations. Aqueoussolutions of HCl, H2SO4 aqueous solutions (from 0.01 to

1.00 mol L�1) were also tested as supporting electrolytes but theanalytical signals were no higher than 10% of the ones observedin Britton–Robinson buffer.

Cyclic voltammetric studies of kresoxim-methyl (Fig. 2B) wereperformed at 0.050 mol L�1 acetate buffer (pH 4.0) in the positivepotential region. Under such conditions, kresoxim-methyl pre-sented two electrochemically irreversible anodic peaks, the moreintense at 1425 mV and a smaller one at +1670 mV. The applica-tion of a sequence of cyclic voltamograms (10 cycles) clearly showsthe decreasing of both peaks (Fig. 2C) until total absence of signal.In Fig. 2D the decreasing of the most intense of the peaks is showndue to the decrease of the analyte in the interface with the elec-trode as the oxidation process is irreversible.

Focusing on the first oxidation peak, the diagnostic studies en-abled by square-wave voltammetry (SWV) [23] confirmed the irre-versibility of the process as the relationship between the peakcurrent (Ip) and the square root of the increasing applied scan rates(from 100 to 1000 mV s�1) was linear (Fig. 3A). The linear relation-ship between the analyte peak current intensity and the frequencyof the application of pulses (Fig. 3B) also confirms the irreversibil-ity of the electrochemical oxidation that occurs at +1420 mV andindicates that the process controlled by the adsorption of species.

In irreversible systems, the width at the half maximum of theSWV peak (DEP/2) is described by the following equation: DEP/2 =(65.5 ± 0.5)/an, where n is the number of electrons involved inthe process and a is the electronic transfer coefficient [23]. Consid-ering the system with the a value of 0.5, the equation turn intoDEP/2 = 127/n. As the calculated DEP/2 was equal to 78 mV, the va-lue for n was 1.6, suggesting the oxidation involving two electrons.This result agrees with the one that states that for totally irrevers-ible reactions with adsorbed reactants and/or product, the poten-tial dependence upon the logarithm of the applied frequency (DEversus Dlog f) is linear and has a slope equals to �2.3RT/anF [23]as seen in Fig. 4A for kresoxim-methyl. The product an (productof the electron transfer coefficient the number of electrons in-volved in the electrode reaction) was 1.04 which would lead tothe a value of 0.52 and two electrons per molecule of pesticide in-volved in the oxidation process.

The SWV Ip varies linearly in function of the a (up to 50 mV) asshown in Fig. 4B allowing the calculation of the surface concentra-tion of the adsorbed species (C) through the angular coefficient ofthe line (Eq. (2)) [24], where q is the electrode area (cm�2), n is thequantity of electrons (mol), a is the electronic transfer coefficient, Fthe Faraday constant (A s mol�1), a the pulse amplitude of square-wave (V), f the frequency (Hz) of the pulse and DEs is the step po-tential (V). Through this calculation, the surface concentration (C)of the adsorbed species was equal to 1.3 � 10�10 mol cm�2.

Ip=a ¼ ð5� 1Þ102qan2FfDEsC ð1Þ

Based on the experimental results and on information from lit-erature, a mechanism for the oxidative process of kresoxim-methyl was proposed. Since this strobilurin presents no chemicalgroups that can be easily be oxidized, a highly applied positivepotential is required to enable any chemical reaction. The mech-anism is based on the oxidation of hydrocarbon groups present inthe structure of kresoxim-methyl, in accordance with literaturedata, which describes the oxidation of aromatic and alkyl aro-matic hydrocarbons in aqueous medium [25]. In these cases, thesolvent present in the system promotes a nucleophilic attack ina carbon atom deficient in electrons, leading to the formation ofa substitution product. In Fig. 5 two possible mechanisms are pro-posed, both starting with the loss of an electron generating a rad-ical cation (in two possible positions indicated in mechanism 1and in mechanism 2). The following abstraction of a proton leadsto the radical formation. Once formed, this radical loses another

Fig. 2. (A) SWV of kresoxim-methyl in pH 4.0. (B) Cyclic voltammogram of kresoxim-methyl (solid line) and blank (dashed line) in pH 4.0 acetate buffer (0.05 mol L�1). (C) Asequence of cyclic voltammograms for kresoxim-methyl (from +1200 to +2000 mV) made without mass transport by convection. (D) A sequence of cyclic voltammograms forkresoxim-methyl (from +1200 to +1600 mV) made without mass transport by convection. Electrochemical parameters for SWV (f = 20 Hz; DEs = 5 mV; a = 30 mV; supportingelectrolyte Britton–Robinson buffer 0.40 mol L�1 pH 4.0, concentration of the standard solution 9.9 � 10�6 mol L�1) and for cyclic voltammetry (scan rate 100 mV s�1;DEs = 5 mV; supporting electrolyte acetate buffer 0.05 mol L�1 pH 4.0; concentration of the standard solution 1.5 � 10�5 mol L�1).

Fig. 3. (A) Variation of the square root of scan rate (40–280 mV s�1) in function ofthe Ip of kresoxim-methyl. (B) Integrated area peak in function of the f. Electro-chemical parameters: supporting electrolyte pH 4.0 acetate buffer (0.05 mol L�1);9.9 � 10�6 mol L�1 of final concentration of kresoxim-methyl.

Fig. 4. (A) Peak potential in function of the logarithm of the applied frequency atpulse amplitude of 30 mV. (B) Variation of peak current as a function of a atf = 30 Hz. Other electrochemical parameters: DEs = 5 mV; pH 4.0 acetate buffer(0.05 mol L�1); 9.9 � 10�6 mol L�1 of final concentration of kresoxim-methyl.

R.M. Dornellas et al. / Journal of Electroanalytical Chemistry 708 (2013) 46–53 49

Fig. 5. Proposed mechanisms for the oxidation of kresoxim-methyl.

Fig. 6. Integrated area of the peak in function of: (A) a and (B) applied f.Electrochemical parameters: pH 4.0 acetate buffer (0.05 mol L�1) and 9.9 � 10�6 -mol L�1 kresoxim-methyl final concentration.

50 R.M. Dornellas et al. / Journal of Electroanalytical Chemistry 708 (2013) 46–53

electron promoting the formation of the intermediate carbocation(structure II following mechanism 1) or (structure V followingmechanism 2). Both carbocations have good stability due to thepossibility of resonance of the positive charge along the aromaticrings [26,27]. In the presence of polar solvents, carbocations arehighly susceptible to nucleophilic attack, so it is plausible that awater molecule participates in the process generating the species(II following mechanism 1) or (VI following mechanism 2). How-ever, it is important to note that the species (II) can undergo acleavage generating substances (III) and (V) as observed in sys-tems that enable the elimination of neutral molecules [28,29].Therefore, the proposed mechanisms justify the irreversible oxi-dation, promoted by loss two electrons.

Diagnostic studies made for the smaller second oxidation peakindicated a quasi-reversible behavior. This evidence comes from:(i) the relationship between Ip and the square root of the scan ratein cyclic voltammetry (10–280 mV s�1) and (ii) the relationshipbetween Ip and f (1–50 Hz) in SWV are not linear.

3.2. Optimization of the square-wave conditions

The influence of the parameter a on the square-wave signal wastested by varying it over a range from 10 to 100 mV. The currentintensity increased as the value of a increased up to 50 mV(Fig. 6A) with no significant variations for larger amplitudes. Otherimportant parameter in SWV the f of the applied pulse since itinfluences in both the signal-to-noise ratio and the speed of theanalytical measurement. Frequencies from 10 to 50 Hz were testedand a directly proportional increase in the integrated peak areawas observed (Fig. 6B). As the peak width also increased in a directproportional way with f, a better compromise was obtained using30 Hz.

The DEs was evaluated within 5 and 35 mV. The speed of thevoltammetric analysis is directly influenced by this parameter as

R.M. Dornellas et al. / Journal of Electroanalytical Chemistry 708 (2013) 46–53 51

the scan rate is a product of the f and the DEs. The DEs may increasethe measured signal and affect the width of the voltammetric peak.The variation of the DEs did not bring any significant changes insignal intensity but directly affected the width of the peak, whichmay impact resolution of the analytical measurements. Thus theDEs value of 15 mV was chosen.

In Table 1, the optimized conditions for the determination ofkresoxim-methyl using SWV on a BDD working electrode ispresented.

Fig. 7. (a) Voltammograms of increasing concentrations of kresoxim-methyl: (A)Blank; (B) 3.9 � 10�6 mol L�1; (C) 7.9 � 10�6 mol L�1; (D) 1.2 � 10�5 mol L�1; (E)1.6 � 10�5 mol L�1; (F) 2.0 � 10�5 mol L�1; (G) 2.3 � 10�5 mol L�1; (H) 2.7 � 10�5 -mol L�1; (I) 3.1 � 10�5 mol L�1; (J) 3.4 � 10�5 mol L�1. (b) Analytical curve withY = (3.3 � 10�2 ± 2.5 � 10�4 lA L mol�1) X � (2.1 � 10�3 ± 5.3 � 10�3) andR2 = 0.9995. (c) Detail showing the difference between the signal produced by (H)concentration LOQ (8.7 � 10�7 mol L�1) and (A) Blank; Electrochemical parametersused are described in Table 1.

Table 2Evaluation of the interference from other strobilurins in the kresoxim-methyl(3.9 � 10�6 mol L�1) electroanalytical signal.

Interferent Ratio (Strobulirin/kresoxim-methyl)

i(kresoxim-methyl + Interferent)/ikresoxim-methyl

Fluoxastrobin 0:1 1.001:1 0.942:1 1.27

Trifluoxystrobin 0:1 1.001:1 1.052:1 1.053:1 1.055:1 0.9310:1 1.19

Potential scanned between +1000 to +1750 mV.Maximum peak potential measured at +1420 mV.For others conditions see Table 1.

3.3. Analytical figures of merit

Using the experimental conditions selected for the determina-tion of kresoxim-methyl, the analytical parameters of merit wereobtained. In Fig. 7, a sequence of voltammograms of increasing con-centrations of kresoxim-methyl is shown. The analytical curve rep-resent the direct linear relationship (R2 = 0.9995) between theintegrated area of the peak and the concentration of the analytein the electrochemical cell. The analytical model is described byY = (3.3� 10�2 ± 2.5� 10�4 lA L mol�1)X� (2.1� 10�3 ± 5.3� 10�3).The limit of detection (LOD) in the cell was 2.6 � 10�7 mol L�1

(0.09 mg L�1) and the limit of quantification (LOQ) was 8.7 � 10�7

mol L�1 (0.27 mg L�1). LOD and LOQ values were calculated byten consecutive measurements of the lowest concentration of theanalyte in the analyte addition curve to estimate the standarddeviation (sb) and applying it respectively in the 3 sb/m and10 sb/m equations, where ‘‘m’’ is the slope of the calibration curve.

The precision was assessed by instrumental precision and inter-mediate precision studies. The instrumental precision was evalu-ated by the relative standard deviation (RSD) of ten consecutivemeasurements of the signal from solutions containing 3.9 � 10�6

mol L�1 of the analyte. Intermediate precision was determined bycomparing the results obtained from the analysis (ten independentreplicates) of aqueous kresoxim-methyl solutions made by two dif-ferent measuring analysts (each analyst prepared its own set ofsolution). The instrumental precision was represented by a RSD va-lue of about 2% and intermediary precision results were below 5%.

Interference was evaluated through the i(kresoxim-methyl + interferent)/ikresoxim-methyl values, which is given by the ratio between the signalmeasured from the solution containing the mixture containingkresoxim-methyl and another strobilurin fungicide and the signalobtained from a solutions containing only kresoxim-methyl. Thetests were made with increasing strobilurin/kresoxim-methyl con-centration (in mol L�1) ratios. Measurements made in triplicateand calculations were based on the integrated peak area. Resultsare shown in Table 2.

As azoxystrobin, picoxystrobin and pyrachlostrobin are oxi-dized at potentials very close to the one of kresoxim-methyl, there-fore, interferences from these pesticides were observed even at thelower molar ratio (1/1) studied. A more satisfactory situation wasfound when kresoxim-methyl is measured in the presence ofeither trifluoxystrobin or fluoxystrobin as these strobulirins areoxidized at higher potential (above +1700 mV). No interferencewas observed in mixtures containing trifluoxystrobin at

Table 1Experimental conditions selected for the voltammetric determination of kresoxim-methyl using the BDD working electrode.

Parameter Value

Supporting electrolyte Acetate buffer 0.050 mol L�1 pH 4.0Amplitude (a) 50 mVStep potential (DEs) 15 mVFrequency (f) 30 HzSignal measurement At +1420 mVScanned potential range From +1000 to +1750 mV

concentrations up to five times higher than the one of the analyte.On the other hand, fluoxystrobin imposed interferences inmixtures containing two times the concentration of the analyteas the i(kresoxim-methyl + Interferent)/ikresoxim-methyl ratio increased to1.27. However, it is not a commonly practice the use mixturesof strobilurins to treat crops, therefore, samples containingkresoxim-methyl may not contain other strobilurin pesticide.

Recovery test were made by comparing the method proposed inthis paper with the one based on the use of HPLC with UV absorp-

Table 4Recoveries of kresoxim-methyl (n = 3) in analyte fortified samples of grape juice attwo levels of concentration: (I) 6.0 � 10�6 mol L�1 and (II) 1.2 � 10�5 mol L�1.

Sample Day Recovery (%)

Level I Level II

Fermented grape juice 1 96.3 + 9.0 97.1 + 4.12 96.0 + 7.6 101.5 + 4.53 93.1 + 6.5 96.1 + 2.3

Not fermented grape juice 1 100.0 + 5.7 105.3 + 8.22 91.6 + 8.6 94.3 + 7.03 105.3 + 5.4 102.9 + 14.5

Grape juice with soy milk 1 100.0 + 5.7 102.6 + 2.72 91.6 + 5.3 93.6 + 10.13 100.9 + 6.2 101.7 + 5.9

52 R.M. Dornellas et al. / Journal of Electroanalytical Chemistry 708 (2013) 46–53

tion photometric detection (with instrumental LOQ of 8 � 10�6 -mol L�1). Six analyte fortified stream water samples (collectedfrom the Rainha stream that cross the University campus) wasused. The study was also made with analyte fortified non-fer-mented grape juice samples. Fortification, in both types of samples,was made at a concentration of 6.0 � 10�5 mol L�1 (19 mg L�1). Forthe voltammetric method a 10-fold dilutions was made to enabledetection. The average results obtained by using the two tech-niques were compared using the two-tailed Student t-test at a con-fidence level of 95%. The experimental t-value (1.11 and 2.14 forwater and grape juice respectively) was smaller than the referencevalue (2.23) for 10 degrees of freedom. Therefore, results indicatedno significant difference using both methods. The voltammogramsof kresoxim-methyl obtained from a fortified grape juice sampleand from a standard of the analyte are shown in Fig. 8, indicatingno matrix interferences and insignificant losses of analyte afterthe samples is treated using the SPE procedure. In Table 3, analyterecoveries achieved in fortified non-fermented grape juice samplesusing HPLC-UV and the proposed method are shown, indicating thefeasibility of the voltammetric method.

3.4. Application of the method

Commercial samples of fermented grape juice, unfermentedgrape juice and a soy milk based grape juice were analyzed bythe proposed method. Original samples did not indicate the pres-ence of kresoxim-methyl above the detectable level. Thus the sam-ples were fortified with the analyte at two different concentrationlevels. Measurements were made in triplicate in three differentdays using the standard addition method. It is important to pointout that a strong decrease of analyte signal occurs when juice sam-ples are directly introduced in the cell without previous SPE treat-ment. The recoveries are shown in Table 4 and satisfactory results

Fig. 8. Voltammograms of kresoxim-methyl (6.0 � 10�6 mol L�1 in the cell) afterthe addition of (A) in a aqueous of kresoxim-methyl standard, (B) in the ofkresoxim-methyl fortified grape juice sample.

Table 3Analysis results in kresoxim-methyl fortified non-fermented grape juice samples.

Kresoxim-methyl Kresoxim-methyla Kresoxim-methylb

Expectedconcentration

HPLC-UV (Recovery) Voltammetry (Recovery)

6.0 � 10�5 mol L�1 (6.5 ± 0.1) � 10�5 mol L�1 (6.3 ± 0.2) � 10�5 mol L�1

(108%) (105%)

Standard deviations calculated with t = 2.57 (95% confidence level, n = 6).a Sample analyzed after solid phase extraction.b Sample analyzed after solid phase extraction and 10-fold dilution with ultra-

pure water.

were achieved in all cases. LOD and LOQ values in the sample wereof the order of 10�6 mol L�1 because of the dilution factor of sam-ples (about 10 times). However, as indicated in the sample proce-dure, the use of the SPE cartridge for cleaning of the sample mayalso be used to pre-concentrate the analyte may compensate thisdilution factor.

4. Conclusions

An electrochemical method has been developed for the quanti-fication of kresoxim-methyl in grape juice using the boron-dopeddiamond (BDD) working electrode. The oxidation of kresosim-methyl was studied and a mechanism proposed. Experimental con-ditions were adjusted in order to enable a limit of quantification inthe mg L�1, which is appropriate to determine concentrations atthe allowed pesticide maximum daily intake value. The use ofBDD is very simple and it is a very resistant and resilient materialfor such type of analytical determinations.

Acknowledgements

Dr. Aucélio thanks CNPq-Brazil and FAPERJ for research grantsand scholarships. Dornellas thanks CNPq-Brazil for scholarship.Dr. Da Silva Thanks CAPES/PNPD for scholarship. Dr. Matos thanksFAPEMIG for financial support. Authors thank Dr. MariaAuxiliadora C. Matos e Ms Mellina D.R. Santos for attempts onGC-MS studies.

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