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Determination of pesticide residues in coconut (Cocos nucifera Linn.) tree trunks by modified QuEChERS method and Ultra-High-Performance Liquid Chromatography coupled to Triple Quadrupole Tandem Mass Spectrometry

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A rapid and reliable method for the determination of 10 pesticide residues in coconut (Cocos nucifera L.) tree trunks after endotherapy treatments has been established. A modified QuEChERS (quick, easy, cheap, effective, rugged and safe) method, using an homogeneous sample slurry and acetate buffer, followed by Ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) was developed and validated. Under the best extraction conditions, the average recoveries for all pesticides spiked at 40, 80 and 200 µg kg−1 ranged from 70 to 93%, with RSD <10%. Intermediate precision, expressed as RSD, ranged between 3 and 6% for all compounds. Calibration curves showed a wide linear range between 10.0 and 1000.0 µg kg-1 for all compounds studied. Limit of quantification was established as 40.0 µg kg-1. The developed procedure was employed in the analysis of real coconut tree trunk samples obtained 45 h after pesticides application using endotherapy treatment. Concentrations of pesticides were between 44.7±5 and 938.3±20 µg kg-1. These results prove the translocation of pesticides in different heights, in the coconut tree trunk, from the application point. Imidacloprid presented the highest acropetal translocation and was found near the leaves at 61±6 µg kg-1.
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Determination of pesticide residues in coconut tree
trunks by modied QuEChERS method and ultra-
high-performance liquid chromatography coupled
to triple quadrupole tandem mass spectrometry
J. A. Ferreira,
a
V. Talamine,
b
J. F. Facco,
c
T. M. Rizzetti,
c
J. M. S. Ferreira,
b
F. A. Oliveira,
b
O. D. Prestes,
c
R. Zanella,
c
M. L. Martins,
c
M. B. Adaime,
c
S. Navickiene
d
and C. B. G. Bottoli*
a
A rapid and reliable method for the determination of 10 pesticide residues in coconut (Cocos nucifera L.)
tree trunks after endotherapy treatments has been established. A modied QuEChERS (quick, easy,
cheap, eective, rugged and safe) method, using an homogeneous sample slurry and acetate buer,
followed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)
was developed and validated. Under the best extraction conditions, the average recoveries for all
pesticides spiked at 40, 80 and 200 mgkg
1
ranged from 70 to 93%, with RSD <10%. Intermediate
precision expressed as RSD, ranged between 3 and 6% for all compounds. Calibration curves showed a
wide linear range between 10.0 and 1000.0 mgkg
1
for all compounds studied. Limit of quantication
was established as 40.0 mgkg
1
. The developed procedure was employed in the analysis of real coconut
tree trunk samples obtained 45 h after pesticides application using endotherapy treatment.
Concentrations of pesticides were between 44.7 5 and 938.3 20 mgkg
1
. These results prove the
translocation of pesticides in dierent heights, in the coconut tree trunk, from the application point.
Imidacloprid presented the highest acropetal translocation and was found near the leaves at 61 6mgkg
1
.
1. Introduction
For millions of people living in coastal regions (tropical and
subtropical), the coconut tree has a large economical, social and
environmental importance, and it is classied as one of the
most important oil seed crops in the world. The coconut tree
(Cocos nucifera Linn.) is subject to attack by pests and diseases
causing production losses and aecting its quality. The appli-
cation of pesticides is still one of the most used practices to
control and prevent pests and diseases.
1,2
Traditional methods
such as spraying have been substituted by modern techniques
such as endotherapy by infusion or injection of pesticides into
the tree trunks, with such environment advantages as: (a) no
dispersion of plant protection products in the environment and
(b) safety for the public during and aer treatment. Endother-
apy is a pest control method for trees, which operates by xylem
or systemic injection of the protection products into the trunk.
It exploits the movement of pesticides applied in the tree trunk
so that the protection products can translocate into all the parts
of the plant.
3,4
Currently, many studies using endotherapy
techniques have been undertaken with pesticides and applied
in the plants.
5
In agriculture, endotherapy has been used
successfully and studied in tree species such as apple, pear,
cherry, avocado, palm (Elaeis guineensis), coconut, and even in
grape vines, becoming a new trend in modern
arboriculture.
2,3,69
In the northeastern part of Brazil, dierent pesticides such
as 3-hydroxy-carbofuran, carbendazim, carbofuran, carbo-
sulfan, cyproconazole, difenoconazole, imidacloprid, thiaben-
dazole, thiamethoxam, thiophanate-methyl and spirodiclofen
are mixed and used in order to control the pests and diseases in
the coconut crop.
9,10
On the other hand, there is no information
if endotherapic treatments are eective as a method of pest and
disease control in coconut trees or if the residues could
contaminate the products and consumers above the limits
established by sanitary agencies in the European Union (EU)
and Brazil.
The literature reports the use of radioactively labeled pesti-
cides for studies of translocation. However, research involving
radiolabeled pesticides involves bureaucratic issues and it is
a
Instituto de Qu´
ımica, Universidade Estadual de Campinas, POB 6154, 13083-970
Campinas, SP, Brazil. E-mail: carlab@iqm.unicamp.br
b
Departamento de Engenharia Agronˆ
omica, Embrapa Tabuleiros Costeiros, Av. Beira
Mar, no 3250, 49025-040 Aracaju, SE, Brazil
c
Departamento de Qu´
ımica, Universidade Federal de Santa Maria, 97105-900 Santa
Maria, RS, Brazil
d
Departamento de Qu´
ımica, Universidade Federal de Sergipe, Av. Marechal Rondon
s/n, 49100-000 S˜
ao Crist´
ov˜
ao, SE, Brazil
Cite this: DOI: 10.1039/c5ay00323g
Received 5th February 2015
Accepted 8th April 2015
DOI: 10.1039/c5ay00323g
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more dicult to perform, because it is necessary to isolate the
work area (environmental problem), the number of available
radiolabeled pesticides is limited, trained professionals are
required, and it is an expensive work due to the controlled
acquisition and discarding of the radiolabeled pesticides.
1113
In this work, unlabeled pesticides were injected in coconut
tree trunk in order to know if pesticides translocate in the
xylem. For this a new method of sample preparation for the
coconut tree trunk was developed using the Quick, Easy,
Cheap, Eective, Rugged and Safe(QuEChERS) method for
extraction, rst introduced by Anastassiades et al. in 2003.
14
QuEChERS is a well-established method for the analysis of
multi-class and multi-residue analysis of pesticides in dierent
matrices such as fruits and vegetables, emerging contaminants,
oils, meats and soils using chromatographic techniques.
1521
Thus, QuEChERS was used for the rst time to determine
pesticides injected into tree trunks by endotherapy and for
quantifying the pesticides translocated for dierent distances
from the point of application. This method has many advan-
tages over the method reported by Ferreira et al.,
8
which
developed the rst methodology for determining ve pesticides
applied in treatment of coconut palm trunks using matrix solid-
phase dispersion (MSPD) with determination by liquid chro-
matography with an diode array detection (HPLC-DAD). The
main advantages of QuEChERS over MSPD are high recovery for
pesticides having a wide range of polarities, faster analysis and
use of smaller amounts of organic solvent.
The extracts were analyzed by ultra high performance liquid
chromatography-tandem mass spectrometry (UHPLC-MS/MS)
to guarantee better selectivity and detectivity.
2. Experimental
2.1. Chemicals and solvents
The certied standards employed for pesticide analysis were
carbendazim, carbofuran, 3-hydroxy-carbofuran (3-OH-carbo-
furan), carbosulfan, cyproconazole, difenoconazole, spi-
rodiclofen, imidacloprid, thiabendazole, thiamethoxam and
thiophanate-methyl, all acquired from Dr Ehrenstorfer (Augs-
burg, Germany). 3-OH carbofuran is a metabolite of carbofuran.
Due to the toxicity of this compound, we considered relevant the
evaluation of consumer exposure and risk.
22
The standards were
of at least 95% purity. The class, chemical group, toxicological
class and chemical structures of the pesticides used in this
study are shown in Table 1.
The HPLC grade solvents acetonitrile and methanol were
from Mallinckrodt (Phillipsburg, USA) and glacial acetic acid
was from J. T. Baker (Pennsylvania, USA). Ultrapure grade LC
water was obtained by purication of distilled water through a
Direct UV3® gradient system from Millipore (Molsheim, USA)
and used for the preparation of buers, mobile phase and other
reagents. Anhydrous sodium acetate (NaOAc), anhydrous
magnesium sulphate (MgSO
4
) and sodium chloride (NaCl), all
reagent grade, were purchased from Merck (Darmstadt,
Germany).
Primary secundary amine (PSA) and Bondesil C
18
adsorvent
(particles of 40 mm) were obtained from Agilent Technologies
(Wilmington, USA). Polypropylene centrifuge tubes were from
Sarstedt (N¨
umbrecht, Germany), using 50 mL ones for initial
extractions and 15 mL ones for the dispersive solid-phase
extraction (d-SPE) step.
2.2. Preparation of standard solutions
Individual stock solutions (1000 mg L
1
) were prepared in
dierent solvents (ethyl acetate for carbendazim and spi-
rodiclofen; methanol for thiophanate-methyl, carbofuran,
cyproconazole, carbosulfan and thiabendazole; acetone for
difenoconazole, thiamethoxam and 3-OH-carbofuran;
dichloromethane for imidacloprid). A mixture with all 11
pesticides was prepared in acetonitrile at 10 mg L
1
of each
pesticide. The stock and working solutions were stored at
18 C until needed. The working solutions were used for the
preparation of matrix-matched solution within the concentra-
tion range of 10.01000.0 mgkg
1
and for recovery studies,
which used samples spiked before the corresponding extraction
procedure at concentrations of 40.0; 80.0 and 200.0 mgkg
1
with
the studied pesticides.
2.3. Instrumentation and apparatus
QL-901 and NT 825 centrifuge vortex mixers were from Nova
T´
ecnica (S˜
ao Paulo, Brazil). A Sartorius CP-225 balance
(G¨
ottingen, Germany), a Rotox 46 centrifuge from Hettich
(Tuttlingen, Germany), a PT 3100 Polytron Ultra Turrax (Luzern,
Switzerland), and aIKA® A11 basic, analytical mill (Staufen,
Germany) were used.
Chromatographic analyses were performed in an Acquity
UPLCsystem from Waters (Milford, USA) equipped with a
binary solvent delivery system, degasser, autosampler and
column heater. Chromatographic separations were performed
using an Acquity BEH C18 UPLC column (100 mm 2.1 mm),
with 1.7 mm particle size from Waters. MS/MS detection was
performed using a Xevo TQD tandem quadrupole mass spec-
trometer from Waters (Manchester, UK), coupled with an elec-
trospray ionization interface (ESI) operating in the positive ion
mode. The source parameters were: capillary voltage: 2.5 kV;
source temperature: 150 C; and desolvation gas temperature:
500 C, with nitrogen ow rates of 600 and 80 L h
1
for the cone
and desolvation gases, respectively. Analytical instrument
control, data acquisition and data treatment were performed by
the soware MassLynx (Micromass, Manchester, UK), version
4.1. Mobile phase components were: eluent A: ultrapure
water : methanol (98 : 2, v/v) containing 0.1% formic acid and 5
mmol L
1
ammonium formate and eluent B: methanol with
0.1% formic acid and 5 mmol L
1
ammonium formate. The
gradient was: B from 5% at 0 min changing linearly to 100% at
8.50 min returning to 5% at 8.51 min and maintained until
10.00 min when a new injection was made. The ow rate was
0.225 mL min
1
and the injection volume was 10 mL. Selected
reaction monitoring (SRM) experiments were conducted with a
dwell time of 10 ms for all pesticides. The transition of higher
intensity was used for quantication and the second most
intense was used for conrmation. Collision induced dissocia-
tion (CID) was performed using argon as the collision gas at a
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pressure of 4 10
3
mbar and ow-rate of 0.15 mL min
1
.
Optimization of the collision energy for each individual pesti-
cide was done by direct-infusion into in the MS. The precursor
and product ions that were monitored by retention time, and
the molar masses, cone voltage and collision energies in posi-
tive ESI mode used for quantication and conrmation of each
analyte studied are shown in Table 2.
2.4. Sample
The coconut-tree variety selected for development of the
method in this study was hybrid coconut Port-Bouet-121 (PB-
121) acquired by the Brazilian Enterprise for Agricultural
Research (EMBRAPA) and planted in the experimental eld in
Itaporangad'Ajuda, Sergipe, Brazil. A real PB-121 tree was used
to study the translocation of pesticides by endotherapy in the
experimental eld of the Sococo Agroind´
ustria da Amazˆ
onia,
Moj´
u, Par´
a, Brazil. Both samples for the development of the
method and for analysis of real samples were without the use
of pesticides in the selected coconut trees. A manual drill with
0.8 cm diameter needle was used at a depth of 15 cm to open
the trunk of the coconut tree for pesticide injection. Coconut
bers (wood shavings) were collected in sterile plastic bags
and identied under the recommended conditions prior to
use. The samples were stored in a freezer at 17 Cuntil
needed.
Table 1 Description of the pesticides
Pesticides Class Chemical group Toxicological class Chemical structure
3-OH-carbofuran ——
Carbendazim Fungicide Benzimidazole Unlikely to present acute
hazard in normal use
Carbofuran
Insecticide,
nematicide,
acaricide
Carbamate Highly hazardous
Carbosulfan
Insecticide,
nematicide,
acaricide
Carbamate Moderately hazardous
Cyproconazole Fungicide Triazole Moderately hazardous
Difenoconazole Fungicide Triazole Moderately hazardous
Imidacloprid Insecticide Neonicotinoid Moderately hazardous
Thiabendazole Fungicide Benzimidazole Slightly hazardous
Thiamethoxam Insecticide Neonicotinoid No information
Thiophanate-methyl Fungicide Benzimidazole Unlikely to present acute
hazard in normal use
Spirodiclofen Acaricide, insecticide Tetronic acid No information
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2.5. Preparation of sample
The samples were homogenized in a ceramic mortar with dry
ice to reduce oxidation. Aer, the crushed samples were dried in
an oven for 6 h at 45 C. This temperature ensures that pesti-
cides do not degradate. This step facilitate the grinding in an
analytical mill in order for the sample to become a powder. The
powder was then stored in asks at 17 C in a freezer until the
sample preparation step. A slurry was prepared by weighing 10 g
of powdered trunk and adding 30 mL of puried water in a
proportion 1 : 3 (m/v), with posterior homogenization in an
Ultra-Turrax processor.
2.6. Modied QuEChERS method
The extraction method comprised the following steps: a
representative 10 g slurry portion was weighed in a 50 mL PTFE
centrifuge tube and then 10 mL of acetonitrile with 1% (v/v) of
acetic acid was added. The tube was shaken in a vortex for 1
min. Then, 1.7 g of sodium acetate and 4 g of anhydrous
magnesiumsulphatewereaddedandimmediatelythetube
was vigorously shaken using a vortex mixer for 1 min. The tube
was then centrifuged (3400 rpm) for 8 min at 20 C. Four mL of
the supernatant (acetonitrile phase) were transferred to a 15
mL centrifuge tube containing 100 mg PSA, 500 mg C
18
and 600
mg MgSO
4
, and the tube was vortexed for 1 min. Aer that, the
extract was centrifuged again (3400 rpm) for 8 min at 20 C. The
extract was ltered through a 0.22 mmPTFElter and trans-
ferred into a vial and then diluted 1 : 4 (v/v) with ultrapure
water for the injection of 10 mL into the UHPLC-MS/MS system.
2.7. Trunk injection procedure
Pesticides were applied to a coconut tree trunk in the winter
of July 2014, with constant rainfall, temperature of approxi-
mately 25 C and humidity above 80%. Three samples of
coconut tree were collected in dierent height 15, 30, 50 and 60
cm above the injection points 45 h aer injection of pesticides.
To prepare the injection solution, 10 mL of each active ingre-
dients of the commercial pesticide formulations (3-OH-carbo-
sulfan, carbosulfan, carbendazim, carbofuran, cyproconazole,
difenoconazole, imidacloprid, spirodiclofen, thiamethoxam,
thiabendazole) were mixed with 10 g of thiophanate-methyl
diluted in 10 mL of water. The experiment was performed with
the hole made by the automatic drill in two opposite positions
of the stem. Using 10 mL of this mixture was injected in each
hole. Posteriorly, for each hole 20 mL of water was injected to
wash the syringe. Aer this procedure, all the holes were closed
with billets of green wood and to covered with natural tar to
avoid entrance of insects.
2.8. Method validation
In this study, the parameters limit of detection (LOD), limit of
quantication (LOQ), accuracy, precision, specicity and matrix
eect were considered. The analytical method validation was
carried out using SANCO guidelines (SANCO/12571/2013).
23
3. Results and discussion
For this study, pesticides that are widely used on coconut trees,
including insecticides, acaricides, nematicides, fungicides and
herbicides were selected.
For the analysis of components present in plant matrixes,
the most common modes of ionization in LC-MS/MS include
electrospray ionization with mass analyzers such as a triple-
quadruple. This technique permits additional structural infor-
mation and is a strategic approach to nd known constituents,
as well as to isolate the target compounds.
23
The optimization of the precursor and product ions were
carried out by the injection of 10 mL of the individual pesticide
solution directly into the mass spectrometer by infusion.
Dierent fragmentation voltages were applied and the optimal
voltages were between 19 V for thiamethoxam and 37 V for
difenoconazole. Collision energies were investigated and
ranged from 10 eV for 3-OH-carbofuran to 60 eV for difenoco-
nazole. The most intense transition was used as for quanti-
cation while the second most intense transition was used for
conrmation. These parameters are presented in Table 2.
Compared to conventional HPLC, UHPLC systems operate at
higher pressures and use sub-2 mm packings in the columns,
Table 2 Mass spectrometer parameters for pesticides in the positive ESI mode
Pesticides
t
R
(min)
Molar mass
(g mol
1
)
Quantication transition
b
(m/z)
Voltage cone
(V)
Conrmation transition
b
(m/z)
3-OH-carbofuran 3.94 237.2 238.0 > 163.0 (16) 25 238.0 > 181.0 (10)
Carbendazim 3.11 191.2 192.1 > 132.1 (28) 24 192.1 > 160.1 (18)
Carbofuran 5.14 221.3 222.1 > 123.0 (16) 25 222.1 > 165.1 (16)
Cyproconazole
a
6.49 291.8 292.2 > 70.2 (18) 27 292.2 > 125.1 (24)
6.64
Difenoconazole 7.31 406.3 406.0 > 111.1 (60) 37 406.0 > 251.1 (25)
Imidacloprid 3.62 255.7 256.1 > 175.1 (20) 23 256.1 > 209.1 (15)
Thiabendazole 3.45 201.2 202.0 > 175.2 (25) 42 202.0 > 209.1 (30)
Thiamethoxan 3.14 291.7 292.2 > 132.0 (22) 19 292.2 > 211.2 (12)
Thiophanate-methyl 5.04 342.4 343.2 > 151.0 (21) 23 343.2 > 311.1 (11)
Spirodiclofen 7.98 411.2 411.2 > 71.2 (13) 22 411.2 > 313.0 (13)
a
Cyproconazole isomers.
b
Collision energy (eV) is given in parentheses.
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which decrease analysis time and increase peak capacity,
repeatability and detectability. Due to this, reversed phase
UHPLC-MS/MS was the method of choice for this work.
Although all pesticides used in this study were ionized in ESI
(+), the option of maintaining the mobile phase to detect ana-
lytes in both ESI (+) and ESI () was chosen in case of the need
to insert and validate other pesticides with this methodology.
24
3.1. Sample preparation and optimization of the extraction
procedure
Normally, for plant tissues, during the step of sample prepara-
tion for posterior chromatographic analysis, dissolution of the
analytes in a suitable solvent occurs as well as removal from the
solution of interfering compounds derived from biochemical
properties of the plant. Dissolution ruptures the cells within the
tissue matrix, increasing the surface area and the interactions
between solvent and matrix in the extraction process.
25
Generally, for vegetables and fruits, cryogenic milling with
dry ice is used in order to minimize degradation and improve
homogeneity.
26
The coconut tree trunk has high oxidative
instability and the ber of the coconut is very dense, making the
process of milling more dicult. To facilitate ber disruption,
the sample must be completely dry, to eliminate water and
increase the surface area. Therefore, dry ice was homogenized
with the sample, which was then submitted to drying in an oven
at 45 C for 6 h, followed by grinding until the sample became a
powder.
Aer the sample was dried, a volume of water was added to
the sample to form a slurry in a proportion 1 : 3 (w/w), in order
to improve the extraction step of samples with low water
content. The advantage of this slurry is the standardization and
homogenization of the sample, the swelling of the stem and
better interaction of the sample with the solvent in the extrac-
tion step. In recent years, acetonitrile is the most widely used
extraction solvent for pesticide residues analysis. The main
advantages of using acidied acetonitrile as extraction solvent
are the lack of emulsions and no phase separation step.
The extraction procedure is oen the most critical step of a
method due to the diversity of substances that may be extracted
simultaneously.
19
In this work, two procedures for pesticides
extraction were tested: the original QuEChERS method of
Anastassiades et al.
14
and the acetate buered QuEChERS
method modied by Prestes.
27
The original acetate buered
QuEChERS method was developed by Lehotay et al.,
28
proposed
extraction of the sample with acetonitrile containing 1% (v/v)
acetic acid and partitioning by adding MgSO
4
plus sodium
acetate (NaOAc) followed by a clean-up step using dispersive
solid phase extraction (d-SPE) with PSA and MgSO
4
. However,
Prestes
27
proposed the following alterations of the Lehotay
et al.
28
method: (a) weight of the samples (from 15 g to 10 g); (b)
the volume of extraction solvent (from 15 mL to 10 mL); (c) mass
of salts and reagents in the partition step (from 6 g MgSO
4
plus
1.5 g NaOAc to 4 g MgSO
4
plus 1.7 g NaOAc), and clean-up step
(from 50 mg PSA plus 150 mg MgSO
4
to 100 mg PSA plus 600 mg
MgSO
4
); (d) change of the weight of C
18
adsorbent to 500 mg in
the clean-up step.
In this work, nal extracts from the original QuEChERS
method and the modied acetate buered QuEChERS method
were evaluated by drying the samples in a nitrogen gas ow (N
2
)
and verifying the formation of residues in the vial through
weighing. It was found that the amount of residue (co-extrac-
tives) in the vial formed by the original QuEChERS method was
higher than with the modied other method. Therefore, the
best alternative was to use the modied QuEChERS method.
Probably, the modications of the original procedure can be
more ecient for stems due to chemical characteristics of the
trunk tree, where the pesticides to be extracted are located
inside the bers and translocated through the xylem together
with transport of nutrients from the soil.
29
The trunk tree is
basically composed of holocellulose, pentosans, lignin, and
extractives as the minority substances: aromatic, aliphatic,
nitrogen containing compounds, glycosides, terpenes, steroids
and carbohydrates, among others, and can be removed by
solvents during the extraction procedure.
30
Thus, they can store
starches, oils, resins and crystals useful in any stress period.
31
The extraction method for pesticides used in this work becomes
suitable to extract pesticides from the bers of the tree trunk
due to some factors such as: (i) use of sodium acetate buer and
acetonitrile acidied with acetic acid to a pH of approximately 6
Table 3 Linear range, coecients of determination (r
2
) for analytical curves in solvent (acetonitrile), in the matrix extract (coconut tree trunk) and
the analytical curve equation of pesticides prepared from solutions spiked in blank coconut trunk
Pesticides Linear range (mgL
1
)
r
2
Equation y¼ax +bAcetonitrile Trunk coconut
3-OH-carbofuran 10 to 1000 0.9937 0.9984 12 424.9x+ 418.393
Carbendazim 0.9934 0.9979 10 891x+ 677.346
Carbofuran 0.9934 0.9982 26 973.6x1481.15
Cyproconazole 0.9965 0.9990 12 927.7x+ 560.555
Difenoconazole 0.9917 0.9976 12 976.1x+ 2016.34
Imidacloprid 0.9978 0.9989 3367.69x71.2287
Thiabendazole 0.9950 0.9972 16 979.2x+ 4129.62
Thiamethoxam 0.9953 0.9987 3232.65x182.052
Thiophanate-methyl 0.9957 0.9989 62.2367x11.0491
Spirodiclofen 0.9924 0.9920 2661.42x+ 224.469
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to prevent degradation of analytes sensitive to pH such as car-
bosulfan, carbendazim and thiabendazole; (ii) use of larger
amounts of PSA removal of sugars and fatty acids, smaller
organic acids, lipids and some pigments, (iii) use of C
18
to
remove lipids, sterols, long chain fatty compounds and other
non-polar interferences. Furthermore, the increase in MgSO
4
removes water from the organic phase, and forms a visible layer
of extractives in the bottom of the tube aer
centrifugation.
14,15,28
3.2. Validation of the method
Validation of the method developed was according to the
procedure described in item 2.6. For all analytes in both curves,
detector response was linear with coecient of determination
(r
2
) higher than 0.99 and residuals lower than 20% (Table 3).
In the stem matrix, detector saturation was not a problem
because samples were diluted ve times. Fig. 1 shows a
representative chromatogram obtained aer injection of
blank coconut trunk spiked at 200 mgkg
1
with the selected
pesticides. Analyte selectivity was ensured because the blank
extracts presented no interferences for the quantication and
conrmation transitions at the retention time of each
analyte.
In this work, the limits of quantication and detection of the
method was dened as the analyte quantity for which the
relative standard deviation and recoveries of the analyses
reached a preestablished level. The values of LOD and LOQ of
the method were 12.1 and 40.0 mgkg
1
, respectively, for all
compounds. These values were obtained experimentally when
the pesticides were spiked into blank matrices at the 40 mgkg
1
level for all pesticides. This provided recoveries between 70 and
120% with RSD 20%. The choice of representative spiked levels
of pesticides were carried out considering the values found in
real samples, the physicalchemical characteristics of the
pesticides and the matrix (coconut tree trunk), which is a
complex matrix dicult to analyze.
The results showed recovery values between 70 to 93%, with
RSD #10% for three concentration levels. In the evaluation of
intermediate precision, values of recovery ranged from 86 to
113% and RSD
i.p.
values were less than 6%, which are lower
than the recommended values (RSD #20%). These values can
be observed in Table 4. Therefore, the developed method is
suitable for quantifying pesticides in coconut tree trunks.
However, the experiments showed that was not possible to
analyse/validate carbosulfan in coconut tree trunk samples.
The sample processing step and extraction by the new buered
QuEChERS method were not sucient for carbosulfan anal-
ysis. Some studies suggest that this compound is easily
hydrolyzed to carbofuran and 3-OH-carbofuran under acidic
conditions.
32
In complex matrices, such as plants, fruits and vegetables,
the number of ions formed in MS can be lower due to ion
suppression, or increased, resulting in a corresponding nega-
tive or positive matrix eect, respectively. In LC-MS/MS, the
matrix eect (ME) is usually caused by interferences of the
Table 4 Recovery, relative standard deviation (RSD) and matrix eect of pesticides prepared from solutions in acetonitrile (n¼3) and the
coconut tree trunk matrix eect (n¼3)
Pesticides
Spiked level (mgkg
1
)
40 80 80 200
Matrix eect (%)RRSD (%) RRSD (%) RRSD
i.p.a
(%) RRSD (%)
3-OH-carbofuran 80 3913 104 690326%
Carbendazim 73 785687680755%
Carbofuran 72 481592477622%
Cyproconazole 85 38759638827%
Difenoconazole 91 2905916894 +25%
Imidacloprid 79 1914 102 58845%
Thiabendazole 70 675387473653%
Thiamethoxam 76 2927 102 490950%
Thiophanate-methyl 82 7909 113 48410 47%
Spirodiclofen 91 9935865895 +36%
a
i.p. ¼intermediate precision.
Fig. 1 UHPLC-MS/MS chromatogram for the extract of coconut tree
trunk pesticides spiked at 200 mgkg
1
with the following compounds:
(1) carbendazim; (2) thiamethoxam (3) thiabendazole (4) imidacloprid
(5) 3-OH carbofuran (6) thiophanate-methyl (7), carbofuran (8)
cyproconazole (9) difenoconazole (10) spirodiclofen.
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matrix and components in the extract that compete in the
ionization process at the ion source when eluting at the same
retention time as the analyte.
33
The matrix eects can be
considered low (020%), medium (2050%) and strong
($50). Above 20%, it is necessary to use certain methods to
overcome the inuence of the matrix on the analytes.
34,35
The ME varied from 55% (strong signal suppression) to
+36% (increase of average signal), demonstrating the impor-
tance of use matrix-matched calibration. The dilution associ-
ated to the clean-up process is one of the ways to reduce matrix
eects. The assessment of values of the matrix eects showed
that some analytes, such as carbendazim, thiabendazole, thia-
methoxam and thiophanate-methyl, showed strong signal
suppression while spirodiclofen a showed strong signal
increase. Values of ME are presented in Table 4.
3.3. Application to real samples
Aer validation of the analytical methodology, the method was
applied to analyze samples from coconut tree trunks. In order to
check if the UHPLC-MS/MS was in the same conditions as
during the validation step, a matrix-matched calibration, a
reagent blank, a matrix blank and a spiked blank sample at 80
mgkg
1
were evaluated before starting the analyse.
In samples collected aer application by trunk injection,
the pesticide levels found in the coconut tree trunk 15 cm
above the injection point had values for thiamethoxam, car-
bendazim, thiabendazole and imidacloprid above the linear
working range (1000 mgkg
1
). For carbofuran, difenoconazole,
spirodiclofen, 3-OH-carbofuran and cyproconazole, the values
were 343.9 10; 253.2 20; 434 20; 72.3 10 and 938.3 20
mgkg
1
, respectively. The pesticide levels found in the coconut
tree trunk 30 cm above injection point had values for thiame-
thoxam of 554.6 8mgkg
1
, for carbofuran of 62.3 3mgkg
1
,
imidacloprid of 267.8 16 mgkg
1
and cyproconazole of 131.6
9mgkg
1
. 3-OH-carbofuran, spirodiclofen, difenoconazole,
thiabendazole, carbendazim were detected, but not quantied,
because they were below the limit of quantication. The
pesticide levels found in the coconut tree trunk 60 cm above
the injection point had values for thiamethoxam of 127.8 0.5
mgkg
1
and for imidacloprid of 44.7 5mgkg
1
. Cyprocona-
zole was detected, but below the LOQ. The only pesticide found
near of leaves 45 hours aer injection was imidacloprid. It has
demonstrated good acropetal translocation in 45 h in the
coconut tree trunk had value for 61 6mgkg
1
. The chro-
matogram obtained for the extract of coconut tree trunk
pesticides in samples 15 cm above point application can be
seen in Fig. 2 and the results of acropetal translocation of
pesticides at all heights 45 h aer injection of pesticides are
shown in Fig. 3.
According to the octanolwater partition coecient (K
ow
)of
the pesticides studied, the results showed the acropetal
translocation of the majority of pesticides, except for thio-
phanate-methyl.
32
The lipophilic ones with log K
ow
3to>6,
such as cyproconazole, difenoconazole and spirodiclofen,
presented high concentrations. Pesticides with log K
ow
between 0 to 3, such as 3-OH-carbofuran, carbendazim, car-
bofuran, thiabendazole and imidacloprid were found/quanti-
ed. Thiophanate-methyl was not found at any height in the
coconut tree trunk, probably because, in plants and in the
environment, thiophanate-methyl was metabolized to carben-
dazim. Thiamethoxan was the only hydrophilic pesticides
(log K
ow
0.13) used that showed mobility in the phloem/
xylem. Spirodiclofen was the only non-systemic pesticides
applied and even then showed the translocation of 15 cm above
the injection point. These results suggest two main possibili-
ties: pesticides need more than 45 h to translocate or, alter-
natively, the pesticides require an adjuvant to potentiate the
move/translocation.
29
In contrast, despite all the benets of endotherapy treat-
ment, little is known regarding the distribution of liquids
within woody plants and more research is needed to explore
their benecial eects.
3,9,35
As pesticide uptake research
requires both chemical and biological expertise, a close co-
operation between chemists and plant scientists, combined
with modern analytical techniques, is necessary to account for
the complex interactions between pesticides and the uptake
process in plants.
36,37
Fig. 3 Results of acropetal translocation of pesticides at dierent
heights (15, 30, 60 and 50 cm) in coconut tree trunks above the
injection points 45 h after injection of pesticides. The working range
used was 40 to 1000 mgkg
1
.
Fig. 2 UHPLC-MS/MS chromatogram for the extract of coconut tree
trunk pesticides in samples 15 cm above point application compounds:
(1) thiamethoxam; (2) carbendazim (3) thiabendazole, (4) imidacloprid,
(5) 3-OH-carbofuran, (6) carbofuran, (7) cyproconazole, (8) difeno-
conazole, (9) spirodiclofen.
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4. Conclusions
The modied QuEChERS method has been evaluated as the
extraction method for the determination of pesticide residues
applied by endotherapy in the coconut tree trunk with deter-
mination of the nal extract using UHPLC-MS/MS. The results
showed quite good analytical performance in terms of precision
and recovery, showed adequate linearity and low LOQ. The
present work could be used as an add on-method,i.e.,
allowing new pesticides to be constantly included without
needing to modify the method or to optimize for analysis of
pesticide residues in other monocot plants (from the same
biological family).
This study becomes important because it allows under-
standing if the pesticides can translocate to products derived
from coconut tree which have signicant nutritional value as
foods, such as fruit and sap. Furthermore, this method may be
used to evaluate whether it is possible to use another applica-
tion procedure as a secure alternative to endotherapy treatment
for the coconut tree trunk, to eliminate the pests and diseases of
coconuts.
This study provided innovative results showing the eciency
of the extraction method and analysis that contribute to the
knowledge of the process of translocation of pesticides aer
endotherapy treatment. This technique can become an impor-
tant tool for the application of pesticides, minimizing envi-
ronmental and farmer exposure, as well as substituting
techniques as use of radioactively labeled pesticides, to follow
ecacy as well as substituting the spraying procedure. It should
also be noted that the endotherapy treatment is protected from
the sun and rain.
Acknowledgements
This research was supported by FAPESP 2012/18318-4, INCT
Bioanal´
ıtica-FAPESP 2008/57805-2 and CNPq 573672/2008-3.
The authors wish to thank Prof. Marco Aurelio De Paoli for
helpful comments, Prof. Carol H. Collins for language assis-
tance and Sococo Agroind´
ustria da Amazˆ
onia, mainly Dr Paulo
Lins for logistics and facilities.
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... Coconut palm (Cocos nucifera L.) is one of the major perennial crops with high commercial value [8]. In tropical countries, such as Brazil, this palm has an important role in culinary and industrial purposes [9]. However, several factors can reduce the crop yield, such as pest infestation [8]. ...
... Pesticide determination in wood and wood-based products is of considerable interest due to the problems associated with removal or recycling of wood waste, the contamination of indoor air, and manufacture of consumer products [11]. The few methods available for the determination of pesticide residues in wood matrices are mainly based on solid-liquid extraction [8][9][10]. Matrix solid-phase dispersion (MSPD) is a widely employed technique in trace contaminants preconcentration due to its simplicity, minimal cost, and low consumption of reagents [12][13][14]. ...
... In MSPD, the adsorbent material is very important because it determines the extraction efficiency and selectivity. To date, several commercial materials [9,10,14,15], as well as different MOFs [6,13,16,17] have been applied as MSPD sorbents for recovery of multiclass pesticides in various sample types. However, the recoveries of some analytes were not satisfactory in some cases. ...
... %ME values higher or lower than zero indicate the presence of signal enhancement or suppression in comparison with the instrumental response observed in procedural blank (Scordo et al., 2020). The matrix effects can be considered low (±0-20%), medium (±20-50%) and strong (≥±50%) (Ferreira et al., 2015). ...
... The highest matrix effects were found in white wine for carbendazim with − 47% and fluazifop p-butyl with 43%, while for red wine for carbendazim with − 48% and chlorpyriphos with − 45%. Previous study has reported strong signal suppression for carbendazim (-55%) (Ferreira et al., 2015). In conclusion, the matrix effect results, underline the importance of the use of matrix-matched calibration curves to overcome quantification accuracy problems in a single step. ...
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In this research, a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction procedure and Ultra-High Performance Liquid Chromatography-Orbitrap-Mass Spectrometry (UHPLC-Orbitrap-MS), were combined to obtain a sensitive and rapid method for the determination of multiclass pesticides in white and red wines. The optimization strategy involved the selection of buffering conditions, by applying different QuEChERS procedures and sorbents for the cleanup step in order to achieve acceptably high recoveries and low co-extractives in the final extracts. Identification was based on both accurate mass and retention time, while further confirmation was achieved by MS fragmentation. The method was evaluated in terms of linearity, recovery, precision, limit of detection (LOD) and quantification (LOQ), matrix effects (ME) and expanded uncertainty. The validated method was successfully applied to real samples (home-made and commercial) revealing the presence of two selected fungicides, in relatively low levels compared to the MRLs defined by the EU for vinification grapes.
... But more sensitive results can be obtained with LC-MS/MS in spite of strong chromatographic and mass spectrometric interferences caused by coeluting matrix-compounds, as tebuconazole is a highly polar compound. Ferreira et al. (2015) and Ferreira et al. (2016) developed a method for multi-residue determination of pesticides in coconut trunk and in coconut water and pulp following QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safety) extraction method (Anastassiades, Lehotay, Stajnbaher, & Schenck, 2003) and LC-MS/MS. However, no studies have been done on tebuconazole method validation in coconut matrix and the persistence of tebuconazole in coconut water, kernel and leaves after root feeding which is the preferred mode of pesticide application by coconut farmers. ...
... Residues were below quantifiable level in coconut kernel and water in 1st and 3rd day samples but was below quantification limit afterwards. Ferreira et al. (2015) studied recoveries from 40 μg kg − 1 of coconut tree trunk sample and reported concentrations of pesticides between 44.7 ± 5 and 938.3 ± 20 μg kg − 1 and proved the translocation of pesticides in different heights in the coconut tree trunk after trunk injection treatment. The researchers also reported acropetal movement of imidacloprid near the leaves at 61 ± 6 μg kg − 1 . ...
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... Despite this, SANTE has been the most employed guideline for pesticide residues analysis validation in food using QuEChERS and liquid chromatography determination. This can be evidenced since SANTE was used in most publications in this area in the recent years (Paz et al. 2015;Golge and Kabak 2015;Andrade et al. 2015;Christia et al. 2015;Abad-Fuentes et al. 2015;Lichtmannegger et al. 2015;Botero-Coy et al. 2015;Lopes et al. 2015;Ferreira et al. 2015;Martins et al. 2016;Bernardi et al. 2016;Vázquez et al. 2016;Mol et al. 2016). ...
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