Microchemical Journal 123 (2015) 1–8

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Microchemical Journal

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Electrochemical determination of picoxystrobin on boron-doped

diamond electrode: Square-wave voltammetry versus BIA-multiple
pulse amperometry
Rafael.M. Dornellas a,b, Rodrigo A.A. Munoz b, Ricardo Q. Aucelio a,⁎
a
Chemistry Department, Pontifical Catholic University of Rio de Janeiro (PUC-Rio), Rio de Janeiro, RJ 22451-900, Brazil
b
Chemistry Institute, Uberlandia Federal University, Uberlandia, MG 38408-100, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: This article reports and compares the performance of two new electrochemical methods for the determination
Received 7 April 2015 of picoxystrobin (a strobilurin-class fungicide). The methods were square-wave voltammetry (SWV) and
Received in revised form 10 May 2015 batch-injection analysis (BIA) with multiple-pulse amperometric detection, both employing boron-doped dia-
Accepted 11 May 2015
mond (BDD) electrode, which was cathodically treated to enhance the electrochemical activity for picoxystrobin.
Available online 16 May 2015
The SWV method presented higher sensitivity with lower detection (LOD of 0.2 μmol L−1 or 0.07 mg L−1) and
Keywords:
quantification limits (LOQ of 0.7 μmol L−1 or 0.25 mg L−1) in comparison with the BIA amperometric method
Strobilurin (LOD of 1.6 μmol L−1 or 0.6 mg L−1 and LOQ of 5.3 μmol L−1 or 2.0 mg L−1). However, the BIA-amperometric
Batch-injection analysis method provided wider linear range (from 5.3 to 100.0 μmol L−1) in comparison with the SWV method (from
Multiple-pulse amperometry 0.7 to 20.0 μmol L−1) and higher analytical frequency rate (108 measurements h−1). Interference of other
Natural water strobilurin compounds was evaluated and the presence of trifluoxystrobin and fluoxystrobin (up to 3-fold
excess) affected the SWV determination of picoxystrobin. The amperometric method undergoes interference
from the all of the tested strobilurins but a simple approach, based on multiple-pulse amperometry, can indicate
the presence of some of the potential interfering species. Hence, either SWV or BIA-multiple pulse amperometry
can be applied for picoxystrobin depending on analyte concentration, presence of interfering species, and
required analytical frequency.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction Since picoxystrobin is a recently designed pesticide only a few

Synthetic strobilurins have achieved prime importance as agricul-
tural fungicides being used for the protection of several types of crops
throughout the world [1]. Their efficiency against fungi is based on
the inhibition of the mitochondrial respiration of microorganisms.
In addition, strobulirins are attractive by their high thermal stabil-
ity and relatively lower toxicity (oral LD50 is 5.000 mg kg− 1 body
weight in rats) [2–4]. Picoxystrobin, methyl (E)-3-methoxy-2-{2-[6-
(trifluoromethyl)-2-pyridyloxymethyl] phenyl} acrylate (Fig. 1), is one
of these fungicides and its acceptable daily intake, according to the
European Food Safety Authority, is 0.042 mg kg−1 body weight day−1
[2]. Picoxystrobin was launched as a fungicide in 2001 and in 2010
started to be intensively used in soybean crops, which covers extensive
agricultural lands in countries like Unites States, Brazil, Argentina and
China. In addition, several other crops such as corn, canola, peas and
beans may benefit from the effective action of such strobulirin [3].
⁎ Corresponding author. Fax: +55 21 3527 1637.
E-mail address: aucelior@puc-rio.br (R.Q. Aucelio).
methods are described in the literature for its determination. Two stud-
ies employed gas chromatography with mass spectrometric detection
to detect this analyte in foodstuff. Solid phase extraction was used to
separate the analyte from the sample matrix before the analysis and
the reported limit of detection (LOQ) was 5 μg L−1 [4]. Alternatively,
the extraction of the analyte was assisted by ultrasound and the sample
cleaning was performed by gel permeation chromatography. The report-
ed limit of detection (LOD) was 2 μg kg−1 [5]. Capillary electrophoresis
was used to determine picoxystrobin from other two strobilurins in for-
tified urine samples before quantification by absorciometry at 200 nm
[6]. The LOQ value was in the order of 10−8 mol L−1 (32 μg L−1 for
picoxystrobin) after submitting the sample to solid phase extraction
and using normal stacking mode on-line analyte concentration. Flow in-
jection analysis with chemiluminescence detection had also been used
for the determination of picoxystrobin in fortified water samples after
ultrasound assisted extraction (LOD of 0.27 ng mL−1) [7]. Direct analysis
in real time ion-source coupled with a time-of-flight mass-spectrometer
was recently used to determine picoxystrobin in wheat [8]. Limit of
detection (LOD) of 12 mg kg−1 and recovery of 92% were obtained. A

http://dx.doi.org/10.1016/j.microc.2015.05.010
0026-265X/© 2015 Elsevier B.V. All rights reserved.
2 R.M. Dornellas et al. / Microchemical Journal 123 (2015) 1–8
F3C N O
O O
H3C CH3
O to establish an electrical contact with the potentiostat, was placed at the
Fig. 1. Chemical structure of picoxystrobin.
high performance liquid chromatography method with absociometric
detection employed ultrasound-assisted surfactant-enhanced emulsifi-
cation microextraction with solidification of floating organic droplet
was used to determine the pesticide in fruit juices [9]. The limit of detec-
tion was 2 μg L−1 and recovery ranged from 82.6 to 95.7%.
Only one electroanalytical approach is described in the literature
for the determination of picoxystrobin in potatoes and grape juice
[10]. The use of the bismuth film in situ formed on a glassy carbon elec-
trode enabled the use of differential-pulse voltammetry with LOQ of
100 μg kg−1. Analyte recoveries ranging from 89.3 to 104.8% in natural
waters and urine (analyte fortified samples) reflected the poor repro-
ducibility of the film formation.
Polycrystalline boron-doped diamond (BDD) is a material with out-
standing electrochemical properties such as a wide electrochemical
working potential window that enables effective measurements at
high anodic potentials. The high oxidation power of BDD promotes
the effective degradation of organic substances due to the production
of large amounts of hydroxyl radicals in aqueous solutions [11,12].
In fact, the oxidation of other strobilurins in aqueous solution was
achieved using BDD enabling a measured current response for the
voltammetric determination of kresoxy-methyl [13] and pyraclostrobin
[14] and the amperometric determination of dimoxystrobin [15].
The square wave voltammetry (SWV) is a very sensitive voltam-
metric technique because of signal sampling that ensures low back-
ground noise. In addition, the fast potential scanning achieved in SWV
may provide a high analytical frequency [16]. On the other hand, amper-
ometric measurements made at fixed potentials simplify the analytical
procedure when compared to voltammetric approaches, especially
when batch-injection analysis (BIA) is performed. BIA only requires an
electronic micropipette, for fast and high-precise sample delivery, and
a simple cell. It is a simpler alternative to flow-injection analysis (FIA)
as it does not require the use of system s with pumps and valves [17].
In this approach, a microvolume of sample is delivered close to the
surface of the electrode where the analyte is readily reduced or oxidized
generating a transient signal that fades away as the convective transport
dilutes the analyte in the bulk of the solution in the electrochemical
cell. This approach improves the analytical frequency in amperometric
analysis of simple matrices such as water [18] and pharmaceutical
samples [19].
In this work, two electroanalytical applications using the BDD elec-
trode are proposed for the determination of picoxystrobin in natural
waters. The two approaches were developed and compared, one based
on SWV and other based on the amperometric detection using the BIA
system. A critical comparison was made in terms of detection perfor-
mance, interferences and analytical frequency and cost.
2. Experimental
2.1. Instrumentation
Voltammetric measurements were made using a potentiostat/
galvanostat (μ-AUTOLAB Type III, Metrohm, Switzerland) operating
in the voltammetric analysis square wave mode. The instrument is
interfaced to a personal computer. The working electrode was a
1.0 cm2 polycrystalline boron-doped diamond (BDD), Adamant Tech-
nologies, Switzerland, (BDD coating: p-doped, 1–1.5 micrometer thick
6000–8000 mg L−1 boron doping). The Ag/AgCl(sat) electrode was
used as the reference of the electrochemical system and a platinum
wire was used as the auxiliary electrode. The solutions were placed in
a laboratory-made cylindrical cell made of Teflon (15.00 mL volume).
The auxiliary and the reference electrodes are immersed in the solution
from the top of the cell while the BDD electrode, set onto a copper plate
bottom of the cell.
Amperometric recordings using the batch injection analysis (BIA)
cell were made using the same equipment operating at a fixed potential
for amperometric measurements. Injections of standard solutions or
samples were conducted using an BRND705000 electronic micropipette
(Handy Step®) that allows injection of volumes from 10 to 1000 μL
(using a 1.00 mL Combitip®) at a programmable dispensing rate from
5.7 to 13.3 mL min−1. The distance of the Combitip® from the working
electrode was kept constant (about 2 mm). BIA measurements were
carried out using a homemade PVC cell with the BDD electrode fixed
in the bottom. The cell has been previously described in detail [20].
The reference and auxiliary electrodes were a miniaturized Ag/AgCl
(saturated KCl) electrode and a platinum wire, respectively.
A Series 200 HPLC system (Perkin Elmer, USA) was used to compare
analytical results. The system was equipped with a binary pump, a
degassing unit, a UV–visible absorption molecular photometric detec-
tor, an automatic sample injector and data acquisition and/treatment
software supplied by the manufacturer. A pH meter (Model mPA-210,
MS Tecnopon special equipment LTDA, Brazil), with a glass membrane
electrode, was used.
2.2. Reagents and solutions
All solutions were prepared with ultrapure water (18 MΩ cm−1) ob-
tained from a water purifier Milli-Q Gradient System A10—MilliPore
(USA). The solvents acetonitrile and methanol (both LC grade) were
from Meck (Germany). Britton–Robinson buffer was prepared using
acetic, boric, sodium acetate and sodium borate all from Merck. Phos-
phoric and sulfuric acids and sodium hydroxide were also from Merck.
Azoxystrobin (99.0%), picoxystrobin (99.0%), fluoxastrobin (99.0%),
dimoxystrobin (99.0%), pyraclostrobin (99.0%), kresoxim-methyl
(99.0%), and trifloxystrobin (99.0%), were from Riedel-de-Haen (Seelze,
Germany).
2.3. Solutions and sample preparation
Pesticide stock solutions (about 10− 3 mol L−1) were prepared in
acetonitrile and kept in the dark at 4 °C. The samples of mineral water
and the water from the Rodrigo de Freitas Lagoon did not require any
pre-treatment for analysis besides fortification with the analyte stock
solution prior to either voltammetric or amperometric analysis. For
HPLC analysis (comparison studies) water samples were fortified with
picoxystrobin and then filtered (0.45 μm filter) previously to the
injection.
Volumes of 1.00 mL of water samples were directly introduced into
the electrochemical cell containing 10.0 mL of supporting electrolyte
consisting of a Britton–Robinson (BR) buffer (pH 4.0; 0.04 mol L− 1)
for determination by SWV. For analysis by BIA, 5.00 mL of each water
sample was mixed with 3.00 mL of BR buffer (pH 2.0; 0.04 mol L−1)
and 2.00 mL of acetonitrile.
2.4. Analytical procedures
Prior to the first use of the working day, the BDD was subjected to an
anodic pre-treatment using 0.01 A for 1000 s followed by a cathodic
 R.M. Dornellas et al. / Microchemical Journal 123 (2015) 1–8 3

pre-treatment by applying −0.01 A for 1000 s in a 0.1 mol L−1 H2SO4

solution.
Amperometric measurements (coupled with the BIA system) were
made at a constant potential. Picoxystrobin standard solutions used
to construct analytical curves were prepared in BR buffer (pH 2.0;
0.04 mol L−1). Prior to quantification, multiple-pulse amperometry
was used to obtain the hydrodynamic voltammogram of picoxystrobin.
Ten sequential potential pulses (from 1100 to 2000 mV, 70 ms each)
were applied for triplicate injections of picoxystrobin standard solution
through the BIA system. This technique was also applied to detect the
presence of possible electroactive interfering molecules in water sam-
ples. For this purpose, two potential pulses were selected based on the
electrochemical oxidation of picoxystrobin (1600 and 1900 mV). The
determination was made by using an analytical curve.
For the SWV determination of picoxystrobin, acetate buffer (pH 4.0;
0.01 mol L−1) was used as the supporting electrolyte. After 15 s of equil-
ibration time, the potential was scanned from + 1100 to + 2100 mV
with a maximum peak of picoxystrobin appearing at about +1450 mV.
Frequency of 30 Hz, step potential of 5 mV and pulse amplitude of
50 mV completed the electroanalytical conditions. All calculations were
made based on the integrated peak area. Quantification was performed
using the analyte addition procedure.
Diagnostic studies for redox mechanisms made by cyclic voltamme-
try were made with the scan rate of 100 mV s−1 and step potential of
2 mV in a potential range from 1200 to 2000 mV, using acetate buffer
(pH 4,0; 0.01 mol L− 1) as supporting electrolyte. Other diagnostic
studies for redox mechanisms were performed using square-wave volt- Fig. 2. (A) SWV of picoxystrobin in pH 4.0; (B) cyclic voltammogram of picoxystrobin
(solid line) and blank (dashed line) in pH 4.0 BR buffer (0.040 mol L−1). Electrochemical
ammetry (SWV).
parameters for SWV (f = 20 Hz; step potential = 15 mV; amplitude of pulse = 30 mV;
HPLC analyses employed C18 column (250 mm length, 4.6 mm i.d) supporting electrolyte BR buffer 0.04 mol L−1 pH 4.0, concentration of the standard
from Perkin-Elmer. Isocratic elution using an acetonitrile: 0.1% phos- solution 9.9 μmol L−1) and for cyclic voltammetry (scan rate 100 mV s−1; ΔEs = 5 mV;
phoric acid 60:40 (v/v) mobile phase was used at a flow rate of supporting electrolyte BR buffer 0.04 mol L−1 pH 4.0; concentration of the standard
solution 15.0 μmol L−1).
1.4 mL min− 1. Injected sample volume of 10 μL and detection at
220 nm were used. Under such conditions, picoxystrobin retention
time was 8.2 min. the electrode. Using SWV, the linear relationship (R2 = 0.993) between
Ip of the analyte and the applied the frequency of the pulses (f) (Fig. 3B)
3. Results and discussion confirmed the irreversibility of the oxidation process that occurs at
about 1450 mV.
3.1. Determination of picoxystrobin by square-wave voltammetry (SWV) In irreversible systems, the maximum width at half maximum (ΔEP/2)
can be estimated by the following equation: ΔEP/2 = (65.5 ± 0.5) / αn,
3.1.1. Preliminary studies where n is the number of electrons involved in the process. Consid-
Solutions of BR buffer (0.040 mol L−1) were used to evaluate the re- ering the electron transfer coefficient (α) as 0.5, the equation be-
sponse over the pH range from 2.0 to 12.0. By applying a square-wave comes ΔEP/2 = 127/n. As the experimental ΔEP/2 was 80 mV, the value
scanning over the potential range from −1000 to +2500 mV, a peak of n was 1.6, suggesting an oxidation process involving two electrons.
of the highest intensity was found in about 1450 mV with a second Diagnostic studies for the second oxidation peak (the less intense
peak, of lower intensity, at about 1950 mV (Fig. 2A). The peak potential peak) also indicated an electrochemical irreversible process controlled
(Ep) of picoxystrobin and their integrated areas were slightly influenced by the analyte mass transport to the surface of the electrode, as linear
by pH in the aqueous system, with the acidic pH range enabling the best relationships were obtained (R2 N 0.99) to the square root of the scan
results. At pH 4.0 it was obtained the largest value of peak current (Ip) rate of the voltammetric cyclic (10–240 mV s−1) and to the intensity
for picoxystrobin, being 24% more intense than at pH 2.0. At pH 6.0, Ip of the analyte and a function f (10 to 50 Hz). Using ΔEP/2 = 127/n
the signal decreased significantly. It has been found that the use of ace- equation with the measured ΔEP/2 equal to 74 mV, the value for n was
tate buffer (pH 4.0) as the supporting electrolyte (at concentrations 1.7, suggesting two electrons also involved in the oxidation.
varying from 0.01 to 1.00 mol L− 1) and aqueous solutions of HCl or A mechanism for the irreversible oxidation process proposed
H2SO4 (from 0.01 to 1.00 mol L− 1) enabled less intense signals than kresoxim-methyl [13] and can be extended to picoxystrobin. The mech-
the observed in BR buffer (pH 4.0), which was chosen as the supporting anism is based on the oxidation of aromatic groups present in the struc-
electrolyte for voltammetric determinations. ture. In both cases, the solvent (water) present in the system promotes
Cyclic voltammetry studies of picoxystrobin (Fig. 2B) were per- nucleophilic attack of a carbon atom electron deficient, leading to the
formed in BR buffer (pH 4.0; 0.040 mol L−1) in the region of positive formation of a replacement product, with subsequent cleavage. These
potential. As expected, picoxystrobin presented two oxidation peaks processes lead to loss of two electrons.
at 1450 mV (most intense) and at 1950 mV. Focusing on the most in- The electroanalytical performance of BDD electrodes depends on
tense peak, diagnostic studies using cyclic voltammetry [21] confirmed their surface termination: hydrogen or oxygen terminated following
the irreversibility of the redox process as no reduction peaks were ob- cathodic or anodic pretreatments, respectively [22]. In Fig. 4, the elec-
served in the reverse scans of the cyclic voltammetry experiments. trochemical response for picoxystrobin is presented using SWV at the
The relationship between the Ip and the square root of the increasing BDD electrode treated either anodically or cathodically. No differences
applied scanning rates (from 10 to 240 mV s− 1) was linear (R2 = on the maximum peak potentials were observed, comparing both BDD
0.99) as indicated in Fig. 3A, which indicates that the electrochemical surfaces. However, a higher current intensity was obtained at the
reaction is controlled by the mass transport of analyte to the surface of cathodically pretreated BDD (50% higher than that obtained at the
4 R.M. Dornellas et al. / Microchemical Journal 123 (2015) 1–8
Fig. 3. (A) Integrated area peak in function of f. (B) Variation of the square root of scan rate in function of Ip of picoxystrobin. Electrochemical parameters: BR buffer (pH 4.0; 0.040 mol L−1);
analyte concentration of 9.9 × 10−6 mol L−1. 1—Oxidation peak with maximum at +1450 mV and 2—Oxidation peak with maximum at +1950 mV.
anodically pretreated BDD). Therefore, the cathodic pretreatment of the
BDD electrode obviously resulted into an improved electrochemical
activity towards picoxystrobin oxidation, which was also observed for
the electrochemical oxidation of other analytes [22–27].
3.1.2. Optimization of the square-wave conditions
The influence of the pulse amplitude (a) of the square wave signal
(integrated peak area) was tested in the range from 10 to 100 mV
with the current intensity increasing as a increased to 50 mV without
significant changes at higher pulse amplitudes. The voltammetric signal
increased with the applied f (from 10 to 50 Hz) in a directly proportional
way along with the increasing of peak width, thus, a compromise
between the speed of the voltammetric analysis and peak width was
1.00
0.75
B 10 − 4 μA L mol− 1) X + (1.1 × 10− 2 ± 8.4 × 10 − 3). The limit of
I (µA)
0.50
A
0.25
0.00
1700 1800 1900 2000
E (mV)
Fig. 4. SWV of 9.9 μmol L−1 picoxytrobin at (A) anodically and (B) cathodically pretreated
BDD electrode. Electrochemical parameters: f = 20 Hz; step potential = 15 mV; amplitude
of pulse = 30 mV; supporting electrolyte BR buffer 0.04 mol L−1 pH 4.0.
obtained using 30 Hz. As the speed of the voltammetric analysis is influ-
enced by the product of f and the step potential (ΔEs), the influence
of the magnitude of the later was evaluated between 5 and 25 mV.
The variation of ΔEs did not bring any significant changes in signal in-
tensity but the peak width was increased at the higher tested values.
In order to keep the peak profile as straight as possible, the ΔEs value
of 5 mV was chosen. The optimized conditions for the determination
of picoxystrobin using SWV on the cathodically pretreated BDD working
electrode are presented in Table 1.
3.1.3. Analytical parameters, interferences and application
Using the experimental conditions selected for the determination
of picoxystrobin, analytical figures of merit were obtained. A sequence
of voltammograms of increasing concentration of picoxystrobin can be
seen in Fig. 5. The calibration curve, shown in the detail of Fig. 5, pre-
sented linear relationship (R2 = 0.9941) between the integrated peak
area and concentration of the analyte in the electrochemical cell as
described by the mathematical model Y = (2.0 × 10 − 2 ± 7.1 ×
detection (LOD) in the cell was 2.0 × 10− 7 mol L− 1 (0.07 mg L− 1)
and the limit of quantification (LOQ) was 6.7 × 10− 7 mol L− 1
(0.25 mg L− 1). LOD and LOQ values were calculated based on ten
Table 1
Experimental conditions selected for the voltammetric determination of picoxystrobin
using the BDD working electrode.
Parameter Value
Supporting electrolyte 0.040 mol L−1 BR buffer pH 4.0
Amplitude (a) 50 mV
Step potential (ΔEs) 5 mV
Frequency (f) 30 Hz
Signal measurement At 1450 mV
Scanned potential range From 1100 to 1800 mV
 R.M. Dornellas et al. / Microchemical Journal 123 (2015) 1–8 5

A 5 fluoxystrobin at concentrations up to three times higher than that of

the analyte. However, it is not a common practice to use mixtures of
F
E strobilurins to treat crops therefore samples containing picoxystrobin
4 D may not contain another strobilurin pesticide.
C Recovery test were made by comparing the proposed voltammetric
B
method with the one based on the use of HPLC with UV absorption pho-
I (µA)

3 tometric detection. Six analyte fortified mineral water samples were

A used. Fortification was made at a concentration of 6.0 × 10−6 mol L−1
and the average results obtained by using the two techniques were
2 compared using the two-tailed Student t-test at a confidence level of
95%. The experimental t-value (1.67) was smaller than the reference
value (2.23) for 10 degrees of freedom. Therefore, results indicated no
1 significant difference in the results achieved by the two different
methods.
1000 1200 1400 1600 1800 The proposed method was applied in the analysis natural water
samples. The original samples did not indicate the presence of
E (mV)
picoxystrobin above the LOD of the method. Thus, the samples were for-
B tified with the analyte at two different concentration levels (5.4 × 10−6
0.4 and 9.0 × 10−6 mol L−1). Measurements were performed in triplicate
Integrated peak area

on three separate days. The recoveries are shown in Table 2 and satisfac-
tory results were obtained in all cases.
0.3

3.2. Determination of picoxystrobin by batch injection amperometric

0.2 analysis (BIA-amperometry)

0.1 3.2.1. Optimization of parameters for measurements in the BIA cell

The pH of the supporting electrolyte (0.040 mol L−1 BR buffer) in the
range from 2.0 to 12.0 was studied through hydrodynamic voltammetry
0.0 over a range of potential between 1000 and 2000 mV. In Fig. 6, current
measurements of picoxystrobin (60 μmol L−1) at different pH values are
0 5 10 15 20 shown. The signals were the average of three analyte injections in the
Concentration (µmol L-1) BIA cell. The hydrodynamic voltammograms show different profiles
for the oxidation of picoxystrobin at different pH conditions. The
Fig. 5. (A) Voltammograms of increasing concentrations of picoxystrobin: (A) Blank; picoxystrobin oxidation started at around +1100 mV in all conditions
(B) 3.9 × 10−6 mol L−1; (C) 7.9 × 10−6 mol L−1; (D) 1.2 × 10−5 mol L−1; tested. Smaller currents were obtained in alkaline buffered solutions
(E) 1.6 × 10−5 mol L−1; (F) 2.0 × 10−5 mol L−1. (B) Analytical curve with Y = (pH range between 8.0 and 12.0). A significant increase in current was
(2.0 × 10−2 ± 7.1 × 10−4 μA L mol−1) × + (1.1 × 10−2 ± 8.4 × 10−3) and R2 = observed in acid buffered solution of pH 2.0 at the potential of
0.9941. Electrochemical parameters used are described in Table 1.
+ 1900 mV. At the potential region of + 1500 mV (near the Ep of the
first oxidation peak for picoxystrobin observed in SWV measurements),
slightly higher current was verified in acid buffered solution of pH 4.0,
consecutive measurements of the lowest concentration of the analyte
which is in agreement with the highest analytical response observed
in the analytical curve to estimate the standard deviation (sb) and
applying it respectively in the 3 sb/m and 10 sb/m equations, where
“m” is the slope of the calibration curve.
Table 2
The precision was assessed by repeatability and intermediate preci- Recoveries of picoxystrobin (n = 3) in analyte fortified samples of mineral water
sion studies. The repeatability was evaluated by the relative standard and Rodrigo de Freitas Lagoon at two levels of concentration: (I) 5.4 μ mol L−1 and
deviation (RSD) of ten consecutive measurements of the signal from (II) 9.0 μ mol L−1.
solutions containing 3.9 × 10−6 mol L−1 of the analyte. Intermediate Sample Picoxystrobin Recovery (%)
precision was determined by comparing the results obtained from the (μmol L−1)
analysis (ten independent replicates) of aqueous picoxystrobin solu- Added Found Day
tions measured by two different analysts. The repeatability was about
Mineral water 0.0 bLOD 1 –
5% and intermediary precision results were about 6%.
2 –
Interference was evaluated through the i(picoxystrobin + interferent) / 3 –
ipicoxystrobin values, which is given by the ratio between the signal 5.4 5.7 ± 0.3 1 104.3 + 6.3
measured from the solution containing containing picoxystrobin and 5.1 ± 0.1 2 94.7 ± 2.0
another strobulirin fungicide (i(picoxystrobin + interferent)) and the signal 5.0 ± 0.1 3 93.1 + 0.9
9.0 8.6 ± 0.0 1 96.0 + 0.5
obtained from solutions containing only picoxystrobin (i picoxystrobin). 9.0 ± 0.6 2 100.3 ± 6.3
Tests were made with increasing strobilurin/picoxystrobin concentra- 9.1 ± 0.5 3 100.5 ± 5.6
tion ratio (in mol L−1). Rodrigo de Freitas 0.0 bLOD 1 –
As azoxystrobin, kresoxim-methyl and pyraclostrobin are oxidized Lagoon 2 –
3 –
in potentials very close to the ones of picoxystrobin, interference
5.4 5.2 ± 0.1 1 95.8 ± 2.2
of these pesticides was observed even in the 1:1 molar ratio. A more 5.3 ± 0.1 2 97.9 ± 2.0
satisfactory situation was found when picoxystrobin is measured at 5.3 ± 0.2 3 98.0 ± 4.1
1450 mV in the presence of trifluoxystrobin or fluoxystrobin as these 9.0 9.3 ± 0.3 1 103.5 + 3.8
strobilurin are oxidized at a higher potential (above 1700 mV). No inter- 8.8 ± 0.4 2 97.5 ± 4.0
8.9 ± 0.3 3 98.3 + 3.0
ference was observed in mixtures containing either trifluoxystrobin or
6 R.M. Dornellas et al. / Microchemical Journal 123 (2015) 1–8
20
pH 2.0
pH 4.0
pH 6.0
15 pH 8.0
pH 10.0
pH 12.0
Ip (µA)
10
5 no memory effect was observed before the addition of different concen-
0 solutions during analysis, increasing the analytical frequency. Also
1000 1200 1400 1600 1800 2000
E (mV)
Fig. 6. Hydrodynamic voltammograms of picoxystrobin (60 μmol L−1) in 0.04 mol L−1
Britton–Robinson buffer solution with pH adjusted to different values (from 2.0 to 12.0)
obtained by plotting peak current values as function of the applied potential pulses.
Dispensing rate: 9.6 mL min−1; injection volume: 100 μL.
by SWV under the same buffered solution. The potential selected for fur-
ther amperometric measurements was + 1900 mV. The potential of
+2000 mV was not chosen because of the background signal probably
resulting from the oxidation of acetonitrile (solvent used in the prepara-
tion of picoxystrobin solutions and injected into the cell in order of μL).
The electrochemical reduction of picoxystrobin was investigated and no
reduction peaks were observed as expected.
The BIA system with amperometric detection (constant application
at +1900 mV) was investigated to determine picoxystrobin. Ampero-
metric measurements for sequential injections of picoxystrobin pro-
duced highly repetitive signals (relative standard deviation (RSD) of
4%), which indicates that neither picoxystrobin nor its oxidation prod-
ucts adsorbs on the electrode surface. This behavior is also consistent
with the results obtained by cyclic voltammetry experiments. Stirring
of the solution during amperometric measurements was tested to verify
the influence on the measured signal, but no significant change was ob-
served. Thus, picoxystrobin analytical measurements were performed
without stirring of the solution at the BIA cell.
Consecutive measurements using the BIA-amperometric system
allowed RSD values of 4.9 and 4.4% for ten successive injections of a
standard solution picoxystrobin respectively at concentration levels of
10 and 20 μmol L−1. Operational parameters were optimized for the
BIA system, such as dispensing rate, the solution and the injection vol-
ume. The Ip value increased as the dispensing rate was increased, thus
the maximum value of dispensing rate allowed by the automatic pipette
(13.3 mL min−1) was selected. The injected volume promoted the
increase of Ip up to 40 μL. Then, the Ip value became stable as the injec-
tion volume was increased to 100 μL. Thus, injection volume of 50 μL
was selected for determination by BIA of picoxystrobin. The experimen-
tal parameters chosen for the BIA-amperometry system for the determi-
nation of picoxystrobin are described in Table 3.
Table 3
Experimental conditions chosen for the BIA-amperometric determination method for
picoxystrobin.
Parameter Value
Supporting electrolyte 0.040 mol L−1 BR buffer pH 2.0
Work potential +1900 mV
Dispensing rate 13.3 mL min−1
Injection volume 50 μL
3.2.3. Analytical figures of merit for the BIA-amperometric method
The amperometric responses obtained under optimized conditions
for increasing (from 10 to 100 μmol L− 1) and for decreasing (from
100 to 10 μmol L−1) picoxystrobin concentrations (triplicate injections)
and the respective calibration curves (insert B and C) are shown in Fig. 7.
Linear responses (R2 N 0.999) were observed in such concentration
range for both the analytical curves and the sensitivities were: 2.65 ×
10−1 + 2.63 × 10−3 μA μmol L−1 (for the curve made with increasing
concentrations) and − 2.61 × 10−1 + 3.44 × 10−3 μA μmol L−1 (for
the curve made with decreasing concentrations). This shows that
trations even without agitating the solutions between additions of
standards, thus the measurements were made without agitating the
based on amperometric recording of Fig. 7, the analytical frequency
was calculated as 108 h− 1 injection. The LOD and LOQ were 1.6 and
5.3 μmol L−1, respectively, using the same criteria for the voltammetric
method: 3sb/m (for LOD) and 10sb/m for (LOQ).
3.2.4. Application of the BIA-amperometric method
The BIA-amperometric method was applied for the picoxystrobin
determination in natural water samples. In Table 4 the results of
the analysis are listed, indicating the expected analyte concentrations
in two different samples fortified at two concentration levels:
30 μmol L−1 (11 mg L−1) and 50 μmol L−1 (18 mg L−1), analyzed in
triplicate on three different days. The recovery values were within 93%
and 105%, which is considered acceptable for such a low concentration
of picoxystrobin. Results also suggest that the method is not affected
by interferences from other components present in the natural water
matrix.
However, when potential interferents such as other strobilurins or
other organic substances are present in the sample, interference may
occur on the determination of picoxystrobin, because of the electro-
chemical oxidation processes occurring near the oxidation potential
region of picoxystrobin. In order to detect the presence of these possible
interfering electroactive molecules in water samples, a simple strategy
can be adopted based on the peculiar electrochemical behavior of
picoxystrobin using amperometry of multiple pulses. As two indepen-
dent electrochemical processes were observed for picoxystrobin at
the BDD, one can calculate an amperometric factor by monitoring the
intensity of these two different electrochemical events related to
picoxystrobin in a sequential manner. Thus, a characteristic factor for
picoxystrobin is obtained as the ratio IE2/IE1, where IE2 is the most
intense current pulse generated at the potential for the second oxida-
tion peak and IE1 is the less intense current generated at the potential
of the first oxidation peak resulting in a factor value always greater
than unity. This factor remains constant when only picoxystrobin is
present in the sample as electroactive species. The presence of other
electroactive species in the same sample should change the factor
value, increasing or decreasing this value, mainly due to two factors:
(i) adsorption of interfering molecules or their oxidation products on
the electrode surface may increase or decrease the IE2/IE1 value; (ii) ox-
idation of these molecules producing currents that add up to the analyte
current changing the IE2/IE1 value [20]. Even when other strobilurins,
which also present two electrochemical processes, are present in the
sample, the IE2/IE1 ratio for picoxystrobin is altered because the other
strobilurins undergo electrochemical oxidation processes at different
maximum potential values and of different current intensities due to
the individual characteristics of each of the strobilurin molecules
(e.g., diffusion coefficient and formal potential).
Following this idea, the factor value (IE2/IE1) was calculated from
data collected by multiple pulse amperometry at +1600 mV (E1) and
+ 1900 mV (E2) for picoxystrobin in the absence and presence of
potential interfering molecules. The value obtained for picoxystrobin
factor in the absence of interfering chemical species (2.96 ± 0.18) was
altered in the presence of pyraclostrobin (1.81 ± 0.17), trifloxystrobin
 R.M. Dornellas et al. / Microchemical Journal 123 (2015) 1–8 7

A 3.5 f

3.0 e e'

2.5 d d'

I (µA)
2.0 c c'

1.5 b b'
a a'
1.0

0.5
0 250 500 750 1000 1250
B Time (s) C
30 30

25 25

20 20

Ip (µA)
Ip (µA)

15 15

10 10

5 5

0 0

0 20 40 60 80 100 0 20 40 60 80 100
Concentration Concentration

Fig. 7. (1) BIA amperometric responses of the BDD electrode for triplicate injections of (a) 10; (b) 20; (c) 40; (d) 60; (e) 80; and (f) 100 μmol L−1 picoxystrobin standard solutions.
(B) Calibration curve of picoxystrobin increasing concentrations. (C) Calibration curve of picoxystrobin decreasing concentrations. Electrolyte: 0.04 mol L−1 Britton–Robinson buffer
solution pH 2; dispensing rate: 13.3 mL min−1; injected volume: 50 μL.

(6.89 ± 0.50) and kresoxim-methyl (1.95 ± 0.18). Therefore, the use for the analysis of such a sample, but the square-wave voltammetric
of multiple pulse amperometry selecting the two potential pulses method, as seen earlier, may still be used to determine picoxystrobin
at picoxystrobin undergoes oxidation can be applied to detect the in the same sample.
presence of interfering strobilurin molecules. When such strobilurins
are found in a water sample, BIA-amperometry cannot be performed 4. Conclusion

Picoxystrobin was determined by two new electrochemical methods

Table 4
using SWV and BIA-multiple pulses amperometry employing a cathod-
Picooxystrobin concentration in natural water samples analyzed by the proposed BIA
method and recovery tests (n = 3). ically treated BDD electrode, which presented superior currents in com-
parison with the anodically treated surface. The SWV method presented
Sample Picoxystrobin Recovery (%)
narrower linear range (from 0.7 to 20.0 μmol L−1) in comparison with
(μmol L−1)
the BIA-amperometry method (from 5.3 to 100.0 μmol L−1) but higher
Added Found Day
sensitivity with lower LOD (0.2 μmol L− 1 or 0.07 mg L−1) and LOQ
Mineral water 0.0 bLOD 1 – (0.7 μmol L−1 or 0.25 mg L−1) values than the ones obtained by the am-
2 – perometric method (LOD of 1.6 μmol L−1 or 0.58 mg L−1 and LOQ of
3 –
30.0 27.9 ± 0.5 1 93.1 + 1.7
5.3 μmol L−1 or 1.95 mg L−1).
28.7 ± 1.0 2 95.6 ± 3.4 In terms of selectivity, the SWV method suffered interference at
29.6 ± 1.3 3 98.6 + 4.2 the 1:1 concentration ratio from three strobilurins (azoxystrobin,
50.0 49.5 ± 0.3 1 99.1 ± 0.6 kresoxim-methyl and pyraclostrobin). The other evaluated interfering
50.2 ± 1.2 2 100.3 ± 2.5
strobilurins did not show significant interference in mixtures containing
50.2 ± 1.5 3 100.4 ± 3.1
Rodrigo de Freitas Lagoon 0.0 bLOD 1 – either trifluoxystrobin or fluoxystrobin at concentrations up to three
2 – times higher than that of picoxystrobin. The BIA-amperometry method
3 – was not as selective as SWV; however, the use of multiple-pulse
30.0 30.7 ± 0.9 1 102.2 ± 3.0 amperometry can identify the presence of interfering strobilurin mole-
30.6 ± 0.9 2 102.1 ± 2.6
31.7 ± 0.9 3 105.3 ± 3.0
cules in the solution. When required, solid phase extraction using a C18
50.0 50.1 ± 1.5 1 100.3 ± 3.0 sorbent can be used to isolate picoxystrobin from polar organic constit-
50.4 ± 0.7 2 100.8 ± 1.4 uents in water as indicated in the literature [4,10].
50.9 ± 0.6 3 101.9 + 0.9 On the other hand, the frequency rate of the BIA-amperometry was
Electrolyte: 0.04 mol L− 1 Britton–Robinson buffer solution pH 2.0; dispensing rate: higher, with 108 injections per hour using one built external calibration
13.3 mL min−1; injected volume: 50 μL. curve (compared to about 10 samples per hour using SWV and analyte
8 R.M. Dornellas et al. / Microchemical Journal 123 (2015) 1–8
addition method for quantification). Hence, the most appropriate
method to determine picoxystrobin depends on the analyte concentra-
tion range in the sample, presence of potential interfering species, and
required time of analysis. BIA-amperometry is a simpler approach and
can be adapted to be used in the field to detect environment and
farmers degree of fungicide exposition after crop aerial pulverization.
Acknowledgments
The authors thank the Brazilian agencies FAPERJ, FAPEMIG, and
CNPq for scholarships and research funding. Grant numbers: E-26/
110.740/2012; E-26/201.406/2014; and 302888/20132-6.
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