Journal of Environmental Science and Health, Part B

Pesticides, Food Contaminants, and Agricultural Wastes

ISSN: 0360-1234 (Print) 1532-4109 (Online) Journal homepage: http://www.tandfonline.com/loi/lesb20

Determination of acidic herbicides in cereals by

QuEChERS extraction and LC/MS/MS

Angela Santilio , Patrizia Stefanelli , Silvana Girolimetti & Roberto

Dommarco

To cite this article: Angela Santilio , Patrizia Stefanelli , Silvana Girolimetti & Roberto Dommarco
(2011) Determination of acidic herbicides in cereals by QuEChERS extraction and LC/MS/MS,
Journal of Environmental Science and Health, Part B, 46:6, 535-543

To link to this article: http://dx.doi.org/10.1080/03601234.2011.583876

Published online: 04 Jul 2011.

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 Journal of Environmental Science and Health, Part B (2011) 46, 535–543
Copyright
C Istituto Superiore di Sanità

ISSN: 0360-1234 (Print); 1532-4109 (Online)

DOI: 10.1080/03601234.2011.583876

Determination of acidic herbicides in cereals by QuEChERS

extraction and LC/MS/MS
ANGELA SANTILIO, PATRIZIA STEFANELLI, SILVANA GIROLIMETTI and ROBERTO DOMMARCO
Department of Environmental and Primary Prevention, National Institute of Health, Rome, Italy
Downloaded by [Florida International University] at 21:50 20 September 2017

A simple and rapid method has been studied for the determination of acidic herbicides (2,4-D, Dichlorprop, Dichlorprop-p, Fluazifop,
Fluroxypyr, MCPA, Mecoprop and Mecoprop-p) on cereals (rye). The method involves an alkaline hydrolysis with sodium hydroxide
in order to release covalently bound compounds, prior to QuEChERS extraction, followed by neutralization and analysis via liquid
chromatography-double mass spectrometry LC/MS/MS. The performance of the method either with or without alkaline hydrolysis
was studied in terms of recovery rates and limit of quantification (LOQ). In either case, recoveries were determined at four spiking
levels (0.02 mg/kg, 0.05 mg/kg, 0.1 mg/kg and 0.5 mg/kg) with 5 replicates for each level. Mean recoveries ranged from 90 to 120
%, whereas relative standard deviations (RSD %) proved to be less than 20 %. Quantitative analysis was carried out by the internal
standard (Nicarbazin) and the LC/MS/MS analysis was performed in electrospray ionisation (ESI) negative mode using a Zorbax
XCB Eclipse column. The developed method was applied to the analysis of several cereals commercially available like as rye flour,
oat meal, oat flakes and dehusked oat. Residue levels were found below the limit of quantification (LOQ) of the method. The method
has been tested in EU Proficiency Tests for cereals with good results.
Keyword: QuEChERS, cereals, phenoxy acid, herbicides, LC/MS/MS.

Introduction large co-extracted components that may adversely affect

Acidic pesticides containing a carboxylic group, such as
phenoxy, benzoic and aryloxyalkanoic acids, are widely em-
ployed in the agricultural practice as herbicides and growth
regulators. They are usually applied, either as free acids, or
salts and esters linked to a variety of alcoholic groups, on
cereals such as wheat, rice, rye and barley.
The European Union, to assure the safety of food for
consumers and to regulate the European and Interna-
tional trade, has established the maximum residue lev-
els (MRLs) for pesticides in/on foodstuffs by European
Regulation n.396/2005[1] and its amending.[2–4] The max-
imum residue levels (MRLs) for each acidic herbicide in
combination with a specific cereal are regulated by the
residue definition, which includes free acids, esters and
conjugates. In Table 1 are reported the residue defini-
tion for each studied herbicide with corresponding MRL
values.
The polar nature and high water solubility of acidic pes-
ticides make their specific extraction very difficult in the
case of complicated matrices such as cereals because of the
Address correspondence to Angela Santilio, Department of
Environmental and Primary Prevention, National Institute of
Health, Viale Regina Elena, 299-00161, Rome, Italy; E-mail:
angela.santilio@iss.it
the extraction efficiency and the quantitative determina-
tion. For this reason, the analysis of the residues for these
pesticides required a specific sample preparation in order to
release the corresponding acids and provide a high extrac-
tion efficiency. Usually, the analyses of the acidic herbicide
residues involve several steps of extraction and cleanup[5–7]
that entail the use of a large amount of organic solvents,
more time-consuming and also a lack of selectivity and sen-
sitivity to reach the low levels of detection required by EU
regulations. Methods reported for the analysis of acidic
herbicides are mostly focused on environmental samples
such as water[5–10] and soil[11] and only a few applications
are described on fruits and vegetables.[12–14] A number of
studies deal with cereals as matrix[15–19] a few cover the
phenoxy esters[19,20] and some deal with phenoxy acid and
esters together.[11,13]
The QuEChERS method, introduced by Anastassiades
et al.[21,22], coupled with LC/MS and GC/MS has be-
came an important methodology for the analysis of pes-
ticide residues because of its simplicity, the use of low
quantities of acetonitrile, the possibility to analyse a large
number of pesticides with few steps and high efficiency. Sev-
eral authors,[23–24] have studied and developed with mod-
ification the QuEChERS method applied to the analysis
of pesticide residues in several crops and they obtained
excellent results for monitoring purposes. But-only a few
authors[24] applied the QuEChERS method to the cereals.
 536
Table 1. Maximum Residue Level (MRL) definition and corresponding values for oat and rye.
Pesticides MRL definition Oat MRL (mg/kg)
2,4-D sum of 2,4-D and its esters
expressed as 2,4-D
Dichlorprop Dichlorprop including
Dichlorprop-p
Fluazifop Fluazifop acid (free and
conjugate
Fluroxypyr Fluroxypyr including its
esters expressed as
Fluroxypyr
MCPA MCPA, MCPB including
their salts, esters and
conjugate expressed as
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MCPA
Mecoprop Sum of Mecoprop-p and
Mecoprop expressed as
Mecoprop
∗
indicates the limit of quantification (LOQ)
Different chromatographic techniques dedicated to the
analysis of phenoxy acid residues such as gas chro-
matography (GC)[5,8,16–20,22] and liquid chromatography
[7,11,12,18,22,24]
have been applied. Gas chromatography
is a conventional analysis method for these herbicides
but derivation of the carboxyl group prior to analy-
sis is necessary to increase its stability during GC sep-
aration. The toxicity of the GC derivative agents has
induced analysts to limit the use of the gas chromato-
graphic method in favour of the liquid chromatographic
techniques.
The liquid chromatography has become an excellent
alternative for the analyses of phenoxy acid herbicides be-
cause this technique allows direct determination of phe-
noxy acid and their esters without previous derivation.[24]
The liquid chromatography coupled with different detec-
tors UV, diode array detector (DAD), and MS can be con-
sidered as an useful tool for the pesticide residue analysis.
In particular, the LC/MS/MS [16,18,22,24,25] is an effective
technique that can discriminate the residue at ultra trace
levels together with a high sensitivity and selectivity.
This paper describes a rapid and effective method for the
analysis of low levels of phenoxy acid herbicides residues in
rye. The obtained results proved that the applied method-
ology is useful for the analysis of the herbicides of interest
on rye and able to combine the advantages of both QuECh-
ERS and LC/MS/MS, such as high sensitivity, short anal-
ysis time and cost reduction.
The method was applied to the analysis of several
kind of cereals (rye flour, oat meal, oat flakes and de-
husked oat) and for all samples the levels of residues
were found below the limit of quantification (LOQ) of the
method.
Santilio et al.
Rye MRL (mg/kg) European Regulation
0.05∗ 0.05∗ 149/2008
0.2 0.2 149/2008
0.1 0.1 839/2008
0.1 0.1 822/2009
0.05∗ 0.05∗ 149/2008
0.05∗ 0.05∗ 149/2008
Materials and methods
Reagents
The pesticides studied were: 2,4-D, Dichlorprop,
Dichlorprop-p, Fluazifop, Fluroxypyr, MCPA, Meco-
prop (MCPP), Mecoprop-p, The reference standards of
these pesticides were purchased from ChemService (West
Chester, PA USA) and were from 98 % to 99.9 % certified
purity. Individual stock standard solutions were prepared
at 1 mg/mL in acetonitrile and stored in the darkness at
+4◦ C. The mixture working solutions were prepared at con-
centration of 100 µg/mL and stored at +4◦ C. N,N’-bis(4-
Nitrophenyl)urea (Nicarbazin) was obtained from Sigma
Aldrich (Steinheim, Germany), due to its limited solubility
a stock solution at 20 µg/mL in acetonitrile was prepared.
Acetonitrile (HPLC grade) was obtained from Carlo
Erba (Milan, Italy). Water purified through Elix/Milli-Q
system (Millipore, Bedford, MA, USA) was used. Sulphuric
acid (96 %, pure for analysis) was provided from Carlo
Erba (Milano, IT). A solution of sulphuric acid at 5N was
prepared by dilution of sulfuric acid. Sodium hydroxide
(analytical grade) was purchased from Carlo Erba (Mi-
lano, IT). A solution 5N was prepared by dissolution of
20 g of sodium hydroxide in 100 mL of water. Anhydrous
magnesium sulphate (97 % pure) was supplied from.
Across Organics (New Jersey, USA). Sodium Chloride
was obtained from Merck (Darmstadt, Germany) and pu-
rified from interfering substances by 3 h heating at 600◦ C
in a muffle furnace and then for 12 h at 120◦ C in an oven.
Sodium citrate dibasic sesquihydrate (> 99 %) was supplied
from Fluka (Steinheim, Germany). Sodium citrate tribasic
dehydrate was obtained from sigma-Aldrich (Steinheim,
 Determination of acidic herbicides in cereals
Germany) Anotop 25 LC (0.2 µm pore size) were pur-
chased from Whatman (Maidstone, England).
Apparatus
Electric Rotator STR 4/3 from Bibby, Stuart Scientific
(Staffordshire, UK)
A Robot Coupe R10 chopper (Grossi Impianti, Italy)
was used to chop up the laboratory samples.
A vortex mixer IKA MS 3 basics (Wilmington, USA)
was used to mix the samples for the extraction.
A centrifuge ALC 4237R (Milan, Italy) was used to cen-
trifuge the sample after extraction.
A Varian System ProStar HPLC (Walnut Creek, CA),
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equipped with a Varian 1200L Quadrupole MS/MS detec-
tor was employed. The liquid chromatograph was equipped
with a Varian 410 autosampler (Walnut Creek, CA). A
Valco valve of injection with a loop of 100 µl was used.
The injection mode was partial loopfill. The injection vol-
ume was 5 µL, and to avoid carry-over, the autosampler
was flushed with methanol before sample injection.
Chromatographic separation was performed on 150 ×
2.1 mm Zorbax XDB Eclipse column (Agilent Technolo-
gies, USA) with a 3.5 µm pore size. Acetonitrile and water
acidified with acetic acid (0.1 %) were used as mobile phase
at the flow rate of 0.3 ml/min. The linear gradient started
at 20 % acetonitrile and was raised to 100 % within 20 min,
the column was eluted 2 min with acetonitrile and then the
content of acetonitrile was lowered to 20 % in 8 min. This
composition was held for a further 2 min before returning
to the initial conditions. The column was re-equilibrated
for 10 min at the initial mobile phase composition. The
total run-time was 32 min.
The detection was achieved by mass spectrometry
equipped with electrospray ionization interface (ESI) oper-
ating in negative ion mode. Typical source parameters were
as follow: ionization voltage was 1400 V; capillary voltage
was -40 V; the nebulizer gas was synthetic air at 30 psi. The
solvent evaporation in the source was assisted by a drying
gas (heater synthetic at 200 ◦ C at 30 psi). Data analyses
were assured by software Varian MS workstation vers. 6.8
Samples
The samples of rye flour, oat meal, oat flakes and dehusked
oat were collected from local markets in Rome.
For recovery tests the sample of rye flour was obtained
from the Community Reference Laboratory for cereal
(CRL Cereal) during the participation to the annual Profi-
ciency tests. The sample was tested as blank before analysis
for recovery assay.
Preparation of samples and extraction procedure with al-
kaline hydrolysis. The samples of rye flour and oat meal
were mechanically homogenized by using an electronic ro-
tator while the samples of oat flakes and dehusked oat were
537
chopped, blended at high speed for 2–3 minutes to achieve
the best sample homogeneity.
A portion of 5 g (± 0.01) of each homogenized crop was
weighted into a 50 mL PTFE centrifuge tube. The sample
was added with 10 mL of cold water (+4◦ C) and mixed by
vortex for few seconds.
After, 300 µL of 5N NaOH solution were added, the tube
was closed and shaken vigorously for 1 minute by hand. The
mixture was left for 30 minutes and occasionally (e.g. every
10 minutes) the tube was shaken. After 30 minutes, 300 µL
of a 5N H2 SO4 solution was added to the tube and it
was shaken. Then 10 mL of CH3 CN were added followed
by 100 µL of Internal Standard solution (Nicarbazin) at
20 µg/mL. The tube was closed and shaken vigorously by
vortex mixer for 1 minute.
After, a mixture of 4 g of anhydrous magnesium sulphate,
1 g of sodium chloride, 1 g of tri-sodium citrate dehydrate,
0.5 g of disodium citrate sesquihydrate were added to the
sample. The tube was capped well and shaken vigorously by
vortex for 1 min. When running a series of samples, a short
shaking of each sample immediately after salt-addition
helped avoid the formation of big salt-conglomerates. Af-
terward, the homogenate was centrifuged at 3000 rpm
for 5 min. The acetonitrile phase was passed through a
0.2 µm filter and collected in a 15 mL tube. The final ex-
tract was filled up to 10 mL with acetonitrile and the tube
was placed in a freezer for over night (−20◦ C), this pro-
cedure removes most of the co-extracted lipophilic as well
as other components with limited solubility in acetonitrile.
Then, an aliquot of 1 mL was transferred into a vial for
analysis by LC/MS/MS system.
Preparation of samples and extraction procedure without
alkaline hydrolysis. The analysis of the samples without
alkaline hydrolysis was performed following the method
described in the previous section without the addition of
NaOH and H2 SO4 .
Recovery Tests
Rye flour which turned out to be free from the studied
pesticides were used for the fortification experiments. Stan-
dard mixtures of the studied pesticides at concentration of
1 µg/mL, 2.5 µg/mL and 100 µg/mL were used to fortify
the samples.
A homogenized sample measuring was added with an
adequate quantity of standard mixtures to give a con-
centration of 0.02 mg/kg, 0.05 mg/kg, 0.1 mg/kg and
0.5 mg/kg.
The spiked samples were mixed by vortex for few seconds
and left to stand for 20 minutes before extraction to favour
the diffusion of pesticides into the matrix. Then the sample
was prepared according to the determination procedure de-
scribed above. Moreover, the recovery tests were performed
without alkaline hydrolysis. Recoveries were determined in
 538
five replicates at four concentration levels for each studied
compound.
Preparation of calibration standard
To test the linearity of the detector 4 calibration solutions
at concentration of 0.01 µg/mL, 0.03 µg/mL, 0.05 µg/mL
and 0.25 µg/mL were prepared in acetonitrile. The linearity
was checked by plotting 4-point calibration curves. Each
point of the linearity curves was obtained as average of two
consecutive injections.
Validation experiments
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The validation of the method was carried out taking
into consideration the criteria of the document SANCO/
10684/.[26] The validation experiments covered the studied
compounds (2,4-D, Dichlorprop, Dichlorprop-p, Fluazi-
fop, Fluroxypyr, MCPA, Mecoprop and Mecoprop-p) that
were spiked onto the rye samples. The following param-
eters were determined during validation of the analytical
method: accuracy, precision, linear dynamic range, limit of
quantification (LOQ). The accuracy and precision of the
method was tested via recovery experiments. Recoveries
were determined for five replicates at four spiked levels (0.02
mg/kg, 0.05 mg/kg, 0.1 mg/kg and 0.5 mg/kg). To quan-
tify the recovery rates the use of matrix-matched calibration
solutions with IS were employed to minimize errors related
to matrix-induced signal enhancement effects. In regard to
Dichlorprop/Dichlorprop-p and Mecoprop/Mecoprop-p,
the recoveries were determined according to the residue
definition reported in the EU Regulation 149/2008.
Results and discussion
A new approach for the analysis of acidic herbicides in
cereals (rye) is described, based on modified QuEChERS
methodology followed by high performance liquid chro-
matography tandem mass spectrometry (HPLC/MS/MS).
The acidic herbicides are mostly applied as esters or as
their salts, the ester formulations hydrolyze rapidly to the
free acid form and the free acid can bind the matrix and
form conjugates. Due to the existence of different esters
for one parent acid, a step for the formation of the corre-
sponding acid is necessary prior to analyse the compounds
by HPLC/MS/MS. Moreover, the alkaline hydrolysis is ap-
plied to the sample to break-up any covalent bond between
acidic pesticides and the matrix components.
For the analyses of dry products, such as cereals, the
addition of water before the extraction steps is necessary.
The addition of water weakens the interaction of pesticides
with the matrix, ensures a sufficient partitioning, and it is
necessary for the mechanism of alkaline hydrolysis. The al-
kaline hydrolysis is easy to be controlled and is irreversible.
Santilio et al.
In addition it is necessary for the determination of ary-
loxyphenoxy herbicides as Fluroxypyr which are not stable
in acidic media but require controlled conditions.
After alkaline hydrolysis, a neutralization step is neces-
sary before proceeding with the QuEChERS methodology.
Following extraction, the extract is placed into a freezer
for over night to remove most of the co-extracted fatty
substances as well as other components with limited solu-
bility in acetonitrile. Then the purified extract is ready to
be analysed by HPLC/MS/MS.
Five recoveries for all fortification levels (0.02 mg/kg,
0.05 mg/kg and 0.1 mg/kg 0.5 mg/kg) were performed on
rye with and without hydrolyses. The rye samples used to
test the recoveries were obtained from the blank sample
obtained during the participation to the EUPT – C4 in
2010 on cereals. The blank was tested to be free from the
studied compounds.
The instrumental conditions were studied with the op-
timization of the LC/MS/MS acquisition parameters. In
Table 2 the condition used to analyse the herbicides by
the multiple reaction monitoring (MRM) are reported.
The general criteria required by European Guidelines
(SANCO/10684/2009) for a reliable identification and
quantification of pesticide residues were met and two tran-
sitions were used for any target analyte, however, the quan-
tification was always obtained using one transition while
the second transition served for the confirmation of the
detected herbicides.
Recoveries experiments were carried out at four differ-
ent spiking levels ranged from 0.02 mg/kg to 0.5 mg/kg,
the tests were performed both with and without alkaline
hydrolysis and the results are reported in Tables 3 and
4, respectively. In the Tables 3 and 4, for all tested com-
pounds at levels of 0.05 mg/kg – 0.5 mg/kg any differ-
ence between alkali treated and non-treated samples could
not be evidenced in terms of recoveries and accuracy The
results at level of 0.05 mg/kg show good recoveries for
both treated and non-treated samples in terms of precision
and accuracy. Also the lower spiked level of 0.02 mg/kg
gives good recoveries for both with and without alkaline
hydrolysis.
The Zorbax Eclipse XDB C18 column was used to
achieve the chromatographic separation yielded good
resolution. A typical chromatogram in MRM of a for-
tified sample of rye at 0.05 mg/kg level and the chro-
matogram of matched-matrix standard solution at the
same fortification level are showed in Figures 1 and 2,
respectively.
The quantitative results of the recoveries were calculated
using matrix–matched standard prepared by spiking ex-
tracts of the rye blank at corresponding spiked levels. The
use of the standard matched-matrix minimizes the matrix
effect related errors and permits to obtain accurate re-
sults. The quantitative determination of each compound
has been performed by the quantification ions listed in
Table 3.
 Determination of acidic herbicides in cereals 539
Table 2. Molecular weights, ions and quantification transitions in bold used to determine the studied pesticides by LC/MS/MS.
Transition
Compounds Molecular weight (m/z) Precursor ion (m/z) Product ion (m/z) Collision Energy (eV)

2.4-D 221 219 161 16.0

219 125 36.0

Dichlorprop and 235 233 161 16.0

Dihlorprop-p
233 125 40.0

Fluazifop 383 326 254 20.0

326 226 20.0
326 108 20.0
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Fluroxypyr 255 253 195 18.0

253 233 10.0

MCPA 201 199 141 20.0

201 143 20.0

Mecoprop and 215 213 141 22.0

Mecoprop-p
213 105 44.0

Nicarbazin 301 301 137 20.0

The limit of quantification (LOQ) was defined for each
compound as the lowest level, for which the validation was
achieved; and it was determined on the accuracy and preci-
sion data obtained through the recoveries studies. As shown
in Tables 3 and 4, the recoveries at 0.02 mg/kg level are
above 120 % and the precision is about 20 % except for the
Dichlorprop/Dichlorprop-p and Mecoprop/Mecoprop-p
in alkaline hydrolysis for which the precision is around
10 %. Moreover, the lower residue level for cereals set
by the EU is 0.02 mg/kg. On the basis of this consid-
eration the LOQ of the method was set to 0.02 mg/kg
for all studied compounds. The calibration curves for
the acidic herbicides investigated were obtained by plot-
ting the analyte concentration against the peak area.
The curves were evaluated by duplicate injection at 5
concentration levels (0.02 – 0.5 µg/mL). For the tar-
get compounds, correlation coefficients (R2) above 0.99
were obtained and a linear relationship between the de-
tector response and the herbicide concentration can be
considered.
Application to analysis of real samples. In order to ver-
ify the effectiveness of the proposed approach, real sam-
ples were obtained from roman marked and analysed. Five
samples of different complex matrices including rye flour,
oat meal, oat flakes and oat dehusked were analysed with
and without alkaline hydrolysis. The identification of the

Table 3. Recoveries (mean ± S.D.; RSD %, n = 5) on rye by extraction with alkaline hydrolyses.
Rye spiked levels (mg/kg)
Compounds 0.02 0.05 0.1 0.5

2,4-D 127 ± 17; 13 87 ± 4; 5 143 ± 8; 5 98 ± 5; 5

Dichlorprop 76 ± 3; 5 104 ± 8; 11 157 ± 10; 4 98 ± 4; 4
Fluazifop 123 ± 14; 11 87 ± 10; 11 141 ± 6; 4 98 ± 4; 4
Fluroxypyr 120 ± 15; 12 115 ± 12; 10 138 ± 6; 4 100 ± 8; 8
MCPA 134 ± 12; 9 89 ± 10; 11 178 ± 7; 4 99 ± 4; 4
Mecoprop 123 ± 9; 7 93 ± 5; 5 163 ± 7; 4 102 ± 4; 4
 540 Santilio et al.
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Fig. 1. Multiple reaction monitory (MRM) chromatograms by LC/MS/MS of studied pesticides obtained from injection of rye
spiked sample at fortification level of 0.05 mg/kg. For each compound, the quantitative ion transition is reported (color figure
available online).

compounds was performed by retention time and ion tran- analyses of real samples do not show evidence of the each
sitions. Two MRM transitions were monitored for each target compounds at the lowest residue level studied of
compound, and the respective ion ratio were compared 0.02 mg/kg, which was also considered the limit of quan-
against those of matrix matched calibration standards. The tification (LOQ).

Table 4. Recoveries (mean ± S.D.; RSD %, n = 5) on rye by extraction without alkaline hydrolyses.
Rye spiked levels (mg/kg)
Pesticides 0.02 0.05 0.1 0.5

2,4-D 118 ± 19; 16 101 ± 13; 13 96 ± 12; 13 99 ± 5; 5

Dichlorprop 123 ± 10; 15 97 ± 12; 17 95 ± 19; 21 107 ± 7; 9
Fluazifop 120 ± 13; 11 95 ± 6; 6 96 ± 9; 10 100 ± 4; 4
Fluroxypyr 135 ± 28; 21 85 ± 17; 20 98 ± 10; 11 98 ± 6; 6
MCPA 133 ± 20; 15 89 ± 15; 17 98 ± 20; 21 104 ± 9; 9
Mecoprop 97 ± 15; 16 95 ± 9; 10 95 ± 21; 22 105 ± 5; 5
 Determination of acidic herbicides in cereals
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Fig. 2. Multiple reaction monitory (MRM) chromatograms by LC/MS/MS of studied pesticides obtained from injection of a
matrix-matched calibration solution corresponding to the fortification level of 0.05 mg/kg. For each compound, the quantitative ion
transition is reported (color figure available online).
Finally, an additional verification of the method perfor-
mance was made by participation to the EUPT- C3 SRM5
and EUPT-C4 respectively in 2009 and 2010, organized by
Community Reference Laboratory (CRL) for cereals and
for single residue methods. According to the specific proto-
col of the EUPTs, the target pesticides were analysed with
and without alkaline hydrolyses. For both EUPTs the list
of the target pesticides included the compounds that ave
the object of this paper.
The validity of the results was quantified by z-score. The
z-score value was calculated according to the CRLs proto-
col as follows:
zi = (xi − µi)/δi
where
541
xi is the value reported by the laboratory
µi is the assigned value for each compound calculated as
the median value obtained from the values presented from
all participant laboratories to EUPT.
δi is the standard deviation of the median. It is calcu-
lated using a Fit-For-Pourpose Relative Standard Devia-
tion (FFP-RSD) set at 25 % based on the experience from
the previous EUPTs.
The z-score value is interpreted in the following way:
/z/ ≤ 2 acceptable; 2 < /z/ ≤ 3 questionable; /z/ > 3
unacceptable.
As showed in the Table 5 for both EUPTs the 2,4-D
was correctly identified with good quantitative results. The
z-score values, calculated from CRL, were less than 2 and
indicating results acceptable when the studied methodology
is applied.
 542
Table 5. Results of the EUPTs for 2,4-D following alkaline hydrolysis and without alkaline hydrolysis and corresponding z-score
values.
EUPT 2,4-D (alkaline
C3/SRM2009 hydrolyses)
Median (mg/kg) 0.499
Result (mg/kg)
0.52
EUPT-C4 2010 2,4-D (alkaline
hydrolyses)
Median (mg/kg) 0.367
Result (mg/kg)
0.329
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Conclusion
In this work, the performance of QuEChERS methodology
with and without alkaline hydrolysis for the determination
of acidic herbicide residues in cereals (rye) has been stud-
ied. Satisfactory results were obtained for the herbicides
studied as regards recovery rates and data reproducibility.
The proposed method take the advantages of the selectivity
provided from Tandem Mass Spectrometry coupled to LC
and the versatility of the QuEChERS method. The appli-
cability of the method was demonstrated by analysis of real
samples and by the EUPT results, thus the method can be
considered suitable for the routine monitoring of residual
amounts of studied herbicides in cereals within the MRL
values of these compounds.
References
[1] European Council. Regulation (EC) No 396/2005 of the European
Parliament and of the Council of 23 February 2005 on maximum
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imal origin and amending Council Directive 91/414/EEC. Official
Journal of the European Union L70/1, 16.03.2005
[2] European Council. Regulation (EC) No 299/2008 of the European
Parliament and of the Council of 11 March 2008 amending regula-
tion (EC) No 396/2005 on maximum residue levels of pesticides in
or on food and feed of plant and animal origin as regards the im-
plementation powers coffered on the Commission. Official Journal
of the European Union L97/67, 09.04.2008.
[3] European Council. Commission Regulation (EC) No 839/2008 of
30 August 2008 amending Regulation (EC) No 396/2005 of the Eu-
ropean Parliament and of the Council as regards Annex II, III and
IV on maximum residue levels of pesticides in or on certain products.
Official Journal of the European Union L234/1, 30.08.2008.
[4] European Council. Commission Regulation (EC) No 149/2008 of
29 January 2008 amending Regulation (EC) No 396/2005 of the
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