Food Control 115 (2020) 107289

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Analysis of highly polar pesticides and their main metabolites in animal T

origin matrices by hydrophilic interaction liquid chromatography and mass
spectrometry
Sonia Herrera López∗, Jonatan Dias, André de Kok
WFSR – Wageningen Food Safety Research, National Reference Laboratory (NRL) for Pesticide Residues in Food and Feed, Akkermaalsbos 2, 6708, WB Wageningen, the
Netherlands

ARTICLE INFO ABSTRACT

Keywords: In this work, a method for the analysis of 14 highly polar pesticides in animal origin matrices by hydrophilic
Polar pesticides interaction liquid chromatography and mass spectrometry has been described. Three methanol-based extraction
Glyphosate procedures with slight modifications, depending on the matrix, have been used. The method has been validated
Isotopically labelled internal standards according to the performance criteria from the SANTE guideline for pesticide residues analysis in food and feed.
Animal origin matrices
Most of the compounds showed a limit of quantitation at the lowest spike levels tested (0.01–0.02 mg kg−1) in
Hydrophilic interaction liquid chromatography
the matrices evaluated. A SPE clean-up step with mixed mode cation exchange sorbent has been used with some
matrices, like chicken meat, eggs, liver and kidney, in order to improve the limits of quantification. Glufosinate,
glyphosate and HEPA were the compounds which presented more difficulties during the study, due to the lack of
sensitivity of the qualifier ions and/or because of the interference from the matrices.

1. Introduction their physical-chemical properties. They are mainly analysed by single

Glyphosate is the active ingredient in the popular herbicide
Roundup. Since its introduction in the market approximately 50 years
ago, glyphosate has become one of the world's most widely used her-
bicides due to its relatively low cost and high efficiency (Zhang, Rana,
Shaffer, Taioli, & Sheppard, 2019). In the United States, the major in-
crease occurred after the introduction of genetically modified glypho-
sate-resistant, “Roundup-ready” crops in 1996 (Thelin & Stone, 2013).
Before the use of “Roundup-ready” crops, the pesticide was applied
after harvest to kill all the plants and prepare the field for the new crop.
The application of glyphosate on the “Roundup-ready crops” shortly
before harvest, so-called “green burndown,” to speed up their drying,
causes crops to have higher levels of glyphosate residues (Benbrook,
2016). Due to this “green burndown” procedure, the application rate
and residue concentrations of glyphosate have increased and resistant
weeds have emerged in Argentina and Brazil, but also in other parts of
world. (Cerdeira et al., 2012; Vila-Aiub, Yu, & Powles, 2019).
Glyphosate is the most known pesticide from the so-called “highly
polar pesticides”, due to the controversy about its toxicity in the last
years. However, besides glyphosate, there are some other highly polar
pesticides, as glufosinate, fosetyl, ethephon and their metabolites which
form an extremely challenging group of pesticides to be analysed due to
residue methods, special methods to analyse them individually or as a
small group of compounds with similar properties (Rajski, Díaz
Galiano, Cutillas, & Férnandez-Alba, 2018; Vass et al., 2016; Goscinny,
Unterluggauer, Aldrian, Hanot, & Masselter, 2012), using LC methods
and a derivatization step (Guo, Riter, Wujcik, & Armstrong, 2016;
Hanke, Singer, & Hollender, 2008). In a previous publication of our
group (Herrera López, Scholten, Kiedrowska, & de Kok, 2019), a
method for the direct analysis of these pesticides and their metabolites
(Dutch Polar Pesticides method, NL-PP) in fruit, vegetables and cereals
was developed, using hydrophilic interaction chromatography (HILIC)
and mass spectrometry (MS). The method showed good robustness and
it was successfully implemented in routine laboratories.
For the majority of these polar pesticides, maximum residue levels
(MRLs) have already been established with metabolites included in the
residue definition, while for glyphosate MRLs are under review, con-
sidering inclusion of the metabolites (EFSA 2018; EFSA 2019). For
products of animal origin, the lack of information about the presence of
glyphosate and its metabolites causes the MRLs still to be considered as
tentative. In the last report from EFSA (EFSA 2019), it has been men-
tioned that confirmatory methods for glyphosate, AMPA and N-acetyl-
glyphosate in fat, liver and kidney are still missing.
Most of the pesticides included in our previous publication (Herrera

∗
Corresponding author.
E-mail addresses: sonia.herreralopez@wur.nl (S.H. López), jonatan.dias@wur.nl (J. Dias), andre.dekok@wur.nl (A. de Kok).

https://doi.org/10.1016/j.foodcont.2020.107289
Received 12 February 2020; Received in revised form 28 March 2020; Accepted 30 March 2020
Available online 06 April 2020
0956-7135/ © 2020 Elsevier Ltd. All rights reserved.
S.H. López, et al.
López et al., 2019) showed a strong matrix effect. Fruit, vegetables and
cereals were evaluated and it was already observed that the matrix
effects were depending on the individual matrix and not on a com-
modity group. There are different quantitation approaches to com-
pensate for matrix effect and recovery losses according to the SANTE
guideline (European Commission, 2017), matrix-matched calibration
(Reichert et al., 2018; Song et al., 2019), procedural standard calibra-
tion (Martins et al., 2016), standard addition (Machado, Gérez, Pistón,
Heinzen, & Cesio, 2017) and isotopically labelled internal standards
(Herrera López et al., 2019; Zhao, Zhao, Lei, Guo, & Zhao, 2018). Iso-
topically labelled internal standards (ILIS) is the most effective ap-
proach to correct for matrix effect and recovery losses but the cost of
the analysis is increasing when the ILIS are used, because they use to be
expensive. For some compounds, an ILIS is not commercially available
and then the other alternatives have to be used to correct for matrix
effects.
In the present study, our developed Dutch Polar Pesticides (NL-PP)
method (Herrera López et al., 2019) has been used for the analysis of
products of animal origin: liver, kidney, fat, chicken meat, chicken eggs
and cow milk. Slight modifications in the procedure have been in-
cluded, in order to improve the results for some compounds in some of
the matrices. Freezing out at −80 °C, a clean-up step with Mixed mode
Cation Exchanged (MCX) sorbent or addition of EDTA solution before
the extraction are some of the changes included in the procedure. The
method has been validated for 14 highly polar pesticides (parents and
metabolites) according to the European Union (EU) SANTE Guideline
(European Commission, 2017). Different quantitation approaches, iso-
topically labelled internal standards, procedure standard calibration
and matrix matched calibration, have been tested in order to find the
best way to quantify the analytes in these type of matrices.
2. Experimental
2.1. Chemicals and reagents
High purity pesticide (> 98%) standards of ethephon, glufosinate,
fosetyl, phosphonic acid, glyphosate and HEPA were purchased from
LGC-Dr. Ehrenstorfer (Augsburg, Germany), N-acetyl-AMPA, N-acetyl-
glufosinate, N-acetyl-glyphosate and MPPA from Toronto Research
Chemicals, TRC (North York, Canada), AMPA was purchased from
Sigma–Aldrich (Steinheim, Germany) and bromide, chlorate and per-
chlorate from Inorganic Ventures (Christiansburg, Virginia, USA).
Isotopically labelled internal standards, AMPA 13C15N, ethephon D4,
fosetyl–aluminium D15, HEPA D4, glufosinate D3 hydrochloride, N-
acetyl-glufosinate D3, MPPA D3, glyphosate 1,2–13C2, 15N, N-acetyl-
glyphosate D3, were purchased from LGC-Dr. Ehrenstorfer and from
Toronto Research Chemicals. Phosphonic acid- 18O3, 18O3-chlorate,
18
O4-perchlorate were supplied by the EURL-SRM in Stuttgart,
Germany. Ethylenediaminetetraacetic acid (EDTA) disodium salt de-
hydrate, Titriplex, was obtained from Merck (Darmstadt, Germany).
HPLC-grade water from a Water Purification System of Millipore
(Burlington, MA, United States) was used. Formic acid was purchased
from VWR (Lutterworth, United Kingdom) and trifluoroacetic acid from
Merck (Darmstadt, Germany). For the optimisation of experimental
conditions (ion-source-dependent parameters, and for MS/MS opera-
tion), working solutions of individual standards were prepared in me-
thanol or water at 100 ng mL−1. For the linearity study, working so-
lutions of a mixture of the standards were prepared at different
concentration levels in ACN:H2O (60:40, 0.2% TFA) and these were
kept at 4 °C. UPLC-grade acetonitrile and LC-grade methanol were
supplied by Biosolve (Dieuze, France) and Merck (Darmstadt,
Germany), respectively.
2.2. Sample preparation and extraction procedure
This study has been carried out with different kind of samples of
2
Food Control 115 (2020) 107289
animal origin: cow milk, swine fat, liver (bovine, swine and poultry),
kidney (bovine and swine), chicken meat and chicken eggs. The sample
types have been selected for the study because they are included in the
EU pesticide residues monitoring programme for samples of animal
origin for 2020 to 2022 (European Commission, 2019). Cow milk, fat,
liver and kidney samples have been collected directly from a slaugh-
terhouse in the Netherlands where samples are taken for our residue
monitoring programs. Chicken meat and eggs were purchased in a local
store for biological products, in Wageningen, The Netherlands. The
samples were homogenised and then stored at −20 °C until spiking and
sample extraction. Samples were prepared and processed according to
the SANTE document for pesticide residues analysis in food and feed
(European Commission, 2017). For quantitation purposes, internal
standards were used for all compounds. For N-acetyl-AMPA and bro-
mide, isotopically-labelled standards were not commercially available,
therefore, the same internal standards as for ethephon and chlorate
were used, respectively.
2.2.1. Extraction procedure for liver, kidney, chicken meat and chicken eggs
2.0 g of sample were weighed in a 50 mL centrifuge tube and spiked
with isotopically labelled internal standard (ILIS) solution at
250 μg kg−1. Then, 8 mL of ultra-pure water were added and the
samples were vortexed during 30 s. Then, 10 mL of MeOH with 1% of
formic acid were added. The tubes were shaken in an automatic axial
extractor (AGYTAX®, Cirta Lab. S.L., Spain) during 5 min and they were
placed in the freezer at −80 °C for 15 min. Thereafter, the extracts were
centrifuged at 4150 R.C.F. for 10 min, at 10 °C, and passed through an
Oasis® (Mixed mode cation exchange) MCX cartridge from Waters
(Milford, New Zealand) for the clean-up procedure. Finally, they were
diluted 10 times extra with dilution solvent (mixture of ACN/H2O
(60:40) and 0.2% TFA), prior to injection into the LC–MS/MS system.
Thereto, a 50 μL aliquot of the extract was diluted with 450 μL of in-
jection solvent. The final matrix equivalent concentration in the diluted
extract in the vial was 0.01 g mL−1, which corresponds with a dilution
factor of 100. In the case of chicken eggs, the method without MCX was
validated.
2.2.2. Extraction procedure for cow milk
2.0 g of sample were weighed in a 50 mL centrifuge tube and spiked
with isotopically labelled internal standard (ILIS) solution at
250 μg kg−1. Then, 2 mL of EDTA solution (5 mM) and 6 mL of ultra-
pure water were added and the samples were vortexed during 30 s.
Then, 10 mL of MeOH with 1% of formic acid were added. The tubes
were shaken in an automatic axial extractor (AGYTAX®, Cirta Lab. S.L.,
Spain) during 5 min and they were placed in the freezer at −80 °C for
15 min. Thereafter, the extracts were centrifuged at 4150 R.C.F. for
10 min, at 10 °C and filtered using a 0.2 μm PVDF syringe filter from
Pall corporation (New York, United States). Finally, they were diluted 5
times extra with dilution solvent (mixture of ACN/H2O (60:40) and
0.2% TFA), prior to injection into the LC–MS/MS system. Thereto, a100
μL aliquot of the extract was diluted with 400 μL of injection solvent.
The final matrix equivalent concentration in the diluted extract in the
vial was 0.02 g mL−1, which corresponds with a dilution factor of 50.
2.2.3. Extraction procedure for fat
Fat samples were melted in a water bath at 60 °C. Then, 2.0 g of
sample were weighed in a 50 mL centrifuge tube and spiked with iso-
topically labelled internal standard (ILIS) solution at 250 μg kg−1.
Then, 10 mL of ultra-pure hot water (60 °C) were added and the sam-
ples were vortexed during 30 s. Then, 10 mL of MeOH with 1% of
formic acid were added. The tubes were shaken in an automatic axial
extractor (AGYTAX®, Cirta Lab. S.L., Spain) during 5 min and they were
placed in the freezer at −80 °C for 15 min. Thereafter, the extracts were
centrifuged at 4150 R.C.F. for 10 min, at 10 °C and filtered, using a
0.2 μm PVDF syringe filter from Pall corporation (New York, United
States). Finally, they were diluted 5 times extra with dilution solvent
S.H. López, et al.
(mixture of ACN/H2O (60:40) and 0.2% TFA), prior to injection into the
LC–MS/MS system. Thereto, a100 μL aliquot of the extract was diluted
with 400 μL of injection solvent. The final matrix equivalent con-
centration in the diluted extract in the vial was 0.02 g mL−1, which
corresponds with a dilution factor of 50.
2.3. LC-MS/MS system
A hybrid quadrupole/linear ion trap mass spectrometer system
(6500+ QTRAP®, Sciex Instruments, Concord, Ontario, Canada) with
an electrospray ion (ESI) source, coupled to a Shimadzu LC-system
consisting of two Nexera X2 LC-30AD LC-pumps and a SIL-30AC au-
tosampler (Shimadzu, Kyoto, Japan) has been used for method devel-
opment and validation. The LC analysis was performed with a HILIC-
column, Obelisc-N (5 μm, 100 Å), L = 150 mm, ID = 2,1 mm (SIELC,
Wheeling, IL, USA). The LC system operated with mobile phase A
(water with 1% formic acid) and mobile phase B (ACN). The LC-ESI-TQ-
MS system was operated in the MRM (multiple reaction monitoring)
and negative ionization mode with a unit mass resolution set for Q1 and
Q3. Declustering potential (DP), entrance potential (EP), collision en-
ergy (CE) and collision cell exit potential (CXP), were optimized using
flow injection analysis (FIA). The acquisition method used for the
analysis has been described in our previous publication (Herrera López
et al., 2019).
2.4. Different quantitation approaches
Due to the variable and strong matrix effects observed in a previous
evaluation of this group of polar pesticides (Herrera López et al., 2019)
and after observing inconclusive results for absolute recoveries of
AMPA in liver samples during the recovery study, an extra evaluation of
different quantitation approaches has been carried out. Isotopically
labelled internal standards calibration, matrix-matched calibration and
procedural standard calibration have been compared for quantitation. It
is well known, that the most effective way to compensate for matrix
effects, is the use of standard addition or isotopically labelled internal
standards. When isotopically labelled standards are not available or are
too costly, the use of procedural standards is often considered as the
best alternative to compensate for matrix effects and low recoveries,
Fig. 1. Extracted ion chromatograms of problematic compounds, (a) glufosinate, m/z: 180.0 → 95.0 (blue line), 180.0 → 85.0 (pink line in the back), (b) glyphosate,
m/z: 168.0 → 63.0 (blue line), 168.0 → 81.0 (pink line in the back) and (c) HEPA, m/z: 125.0 → 95.0 (blue line), 125.0 → 107.0 (pink line in the back). (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3
Food Control 115 (2020) 107289
especially for some pesticides/commodity combinations.
The test has been carried out for two animal product matrices, but
from different individual animals: liver (bovine, swine and poultry) and
kidney (bovine and swine). The different approaches have been tested
in order to evaluate the difference in the quantitation for this group of
polar pesticides in matrices from the same commodity group and the
same type of matrix, but from different animal origin.
3. Results and discussion
3.1. Clean-up
Different SPE clean-up sorbents were tested in our previous study
for fruits, vegetables and cereals, in order to improve the LOQs for some
compounds in some of the matrices evaluated (Herrera López et al.,
2019). No significant improvement was observed in that study for any
pesticide/matrix combination. Considering the difference between
products of animal origin and those of plant origin, the effect of clean-
up sorbents was also studied for difficult matrices as chicken eggs, liver
and kidney. Four different clean-up methods were tested: dispersive
SPE with Enhanced Matrix Removal for lipid (EMR-lipid) and Primary-
Secondary Amine (PSA) sorbent, Mixed-mode Cation-eXchange (MCX)
SPE cartridges, and freezing out at −80 °C. During all the experiments,
visually cleaner extracts were obtained after the clean-up step. As with
the products of plant origin, for most of the compounds, also no sig-
nificant differences were observed now between the various clean-up
steps tested. However, it was beneficial in order to reduce the instru-
ment maintenance time and to increase the robustness of the final
method. There were three compounds, glufosinate, glyphosate and
HEPA, which presented more difficulties. Significantly different results
were obtained when a clean-up was applied. In Fig. 1, chromatograms
for these compounds in two of the six matrices evaluated (liver and
chicken eggs) are shown. For liver, slightly better results were observed
for glyphosate in terms of detection of the qualifier ion, using MCX in
the clean-up step (see Fig. 1b). However, due to some remaining in-
terference from the matrix, the ion ratio did still not meet the criterion
from the SANTE guideline ( ± 30%).
HEPA showed also better results when MCX sorbent was used (see
Fig. 1c). For liver, the positive effect on the response of both the
S.H. López, et al.
Fig. 1. (continued)
quantifier and qualifier ions allowed to reach a lower LOQ and for
chicken egg, the use of MCX sorbent was essential to detect the com-
pound in this matrix (see Fig. 1c). However, as a disadvantage, MCX
clean-up caused the partial loss of glufosinate for liver, chicken meat
and other validated matrices, and even the complete loss of glufosinate
for chicken eggs (see Fig. 1S). These losses are likely to be related with
the interactions between the sulphite functional group of the sorbent
(MCX) and the amino group in the glufosinate structure. The interaction
seems to depend on the percentage of proteins in the matrix. Glufosi-
nate was completely lost during the clean-up step in matrices as oat and
egg, with a percentage of proteins between 11 and 17. However, in
matrices as chicken meat, kidney, liver and soya beans, with a per-
centage of proteins in the range 20–38, it was only partially lost. This
could be explained by the competition between the amino group in the
proteins and in the glufosinate molecule for the sulphite functional
group in the sorbent. Therefore, when the amount of proteins in the
matrix increases, the sulphite group is completely blocked by the amino
groups of the proteins and glufosinate will not be irreversibly bound to
the sorbent.
Fig. 1. (continued)
4
Food Control 115 (2020) 107289
3.2. Use of ethylenediaminetetraacetic acid (EDTA) disodium salt in the
extraction procedure
The use of EDTA in the extraction procedure has also been evaluated
in this work. It is well known that glyphosate can behave as a chelator
for metal ions due to its chemical structure. It may bind with metal ions
present in the samples. In our study, there are two specific matrices,
milk and liver, that may contain high concentrations of polyvalent ca-
tions, such as calcium and magnesium. The use of EDTA as chelating
agent in milk samples for the determination of veterinary drugs, like the
antibiotic tetracycline, has already been described in previous pub-
lications, because this antibiotic may also bind to the metal ions present
in the samples, as can happen with glyphosate (Bruno, Curini, Di
Corcia, Nazzari, & Pallagrosi, 2002; Pena, Lino, & Silveira, 1999). Also,
a previous application for anionic polar pesticides using EDTA in the
extraction solvent has been reported for milk samples (Chamkasem,
Morrisa, & Harmona, 2015). In our study, the addition of EDTA solution
before the extraction was tested together with the different clean-up
steps mentioned in the previous section. For liver samples, no
S.H. López, et al.
Fig. 2. Extracted ion chromatograms (quantifier and qualifier ions) for glyphosate in cow milk with and without addition of EDTA solution before the extraction.
improvements were observed in terms of recovery when EDTA was
used. However, for milk samples, the use of EDTA was critical to assure
the good recovery of glyphosate from the samples. Fig. 2 shows the
extracted ion chromatograms of the quantifier and qualifier ions for
glyphosate in cow milk extracts, using EDTA or not in the extraction
procedure. It is clear from this figure, that glyphosate is not recovered
from the sample when EDTA is not used. However, when EDTA is added
to the samples before extraction, EDTA competes for the binding to the
polyvalent cations and displaces glyphosate molecules attached to the
cations, resulting in the full recovery from the samples.
3.3. Validation
3.3.1. Linearity
In Table 1S, the results from the linearity study have been sum-
marized for the six matrices evaluated (cow milk, swine fat, liver,
kidney, chicken meat and chicken eggs). Calibration standards in sol-
vent at 5 concentration levels were injected 4 times in order to get the
results for linearity of the calibration curve, instrument detection limits,
estimated method detection and quantitation limits. The calibration
curve constructed from single injections of matrix-matched calibration
standards was also checked for linearity. For all pesticides, the criterion
for a linear calibration curve (r2 > 0.98) is met, both for standards in
solvent and in matrix extract (see Table 1S). The deviation of the back-
calculated concentrations of the calibration standards from the true
concentrations is always in the range of ± 20%, as required according
to the criterion in the European guideline for the analysis of pesticide
residues in food and feed (European Commission, 2017).
3.3.2. Matrix effect
The matrix effects have been calculated from the slope of the cali-
bration curves from standards in solvent and in matrix for the six
evaluated matrices. The results are summarized in Table 1.
Only fosetyl and chlorate showed consistently a significant positive
matrix effect (> 20%) in all the matrices. For chlorate, the matrix effect
ranged from 8% in kidney to 36% in fat, for fosetyl, from 17% in kidney
to 55% in swine fat.
In general, variable matrix effects within the range of −86 to
5
Food Control 115 (2020) 107289
+60% have been observed. Chicken eggs and swine fat showed a high
percentage of compounds, 86 and 79%, respectively, with an insignif-
icant matrix effect (< ± 20%). Cow milk and chicken meat showed the
highest number of compounds with strong negative matrix effect. HEPA
showed a strong negative matrix effect in both matrices (−86 and
−50%) and AMPA a stronger negative matrix effect in cow milk
(−58%) than in chicken meat (−35%). Glyphosate and N-acetyl-glu-
fosinate were also compounds with significant suppression of the re-
sponse in cow milk, −45 and −27% respectively. The other two
compounds with suppression in the chicken meat were N-acetyl-AMPA
and ethephon (−35 and −24%, respectively).
Liver and chicken meat showed the highest number of compounds
with a positive matrix effect. Fosetyl and MPPA had a matrix effect
around 45% in liver and 27 and 35%, in chicken meat, respectively.
Glyphosate had a matrix effect of 38% in chicken meat.
Glyphosate showed a negative matrix effect for swine fat and cow
milk. On the other hand, it showed a positive or non-significant matrix
effect in liver, kidney, chicken eggs and chicken meat. This positive
effect is a consequence of the clean-up used for these four matrices.
When using MCX as sorbent in the clean-up, some interfering ions from
the matrix were removed and the response of the target ion improved.
However, the lack of sensitivity of the qualifier ion made the identifi-
cation of glyphosate impossible at the lower levels evaluated. The same
effect was already observed before in our previous validation study for
oat and soy beans, when different clean-up approaches were tested to
improve the LOQs of the method (Herrera López et al., 2019). This is
the reason why glyphosate was not fully validated according to the
SANTE guideline criteria for these two matrices (kidney and liver) at
the lowest concentration level. Glyphosate showed the most extreme
and opposite matrix effect in cow milk (−45%) and in kidney (47%), as
is plotted in the graph in the supplementary material (Fig. 2S). Due to
the very variable results of the matrix effects, isotopically-labelled in-
ternal standards (ILIS) were required for the correction for matrix effect
and obtain a reliable quantitation in the final method.
3.3.3. Accuracy (trueness and precision) and method quantitation limits
The accuracy (trueness and precision) of the method has been de-
termined by analysing recovery samples at 4 different spike levels,
S.H. López, et al. Food Control 115 (2020) 107289

Table 1
Matrix effects calculated from the slopes of calibration curves from standards in solvent and in matrix.
Pesticides Matrix effect (%)

Swine fat Cow milk Liver Chicken eggs Kidney Chicken meat

AMPA −2 −58 −77 −6 −45 −35

Bromide 10 −1 4 −1 −13 7
Chlorate 36 33 19 10 8 8
Ethephon −6 −12 −1 −4 −3 −24
Fosetyl 55 48 44 27 17 27
Glufosinate −5 1 6 1 4 8
Glyphosate −13 −45 23 −10 47 38
HEPA −25 −86 −47 −13 −40 −50
MPPA −4 59 48 2 60 35
N-acetyl-AMPA −1 −13 3 −1 −7 −35
N-acetyl-glufosinate 2 −27 1 3 −7 1
N-acetyl-glyphosate 20 5 14 36 1 −5
Perchlorate 0 −11 2 −1 −6 −4
Phosphonic acid 5 0 9 2 −1 0

depending on the matrix evaluated, and with 6 replicates (n = 6) for
each spike level. The trueness (as % recovery, n = 6) and precision (as
% RSDr, n = 6) of the method were determined. The results for “ab-
solute” and ILIS-corrected recoveries are summarized in table 2S, 3S,
4S, 5S, 6S, 7S and 8S. Absolute recoveries have been calculated without
using internal standards and with matrix-matched calibration stan-
dards. The corrected recoveries were calculated using the standards in
solvent and isotopically labelled internal standards.
Some compounds could not be validated at the lower concentration
level 0.02 mg kg−1, fulfilling all the requirements from the SANTE
document, due to the lack of sensitivity of the qualifier ion or because of
the interferences from the matrix, also mainly for the qualifier ion.
However, they could easily be detected at that concentration with one
ion and perfect matching with the target ion from the isotopically la-
belled internal standards. That concentration was then set as screening
detection limit (SDL).
Blank samples of all matrices to be used in the validation study have
been analysed to determine the possible background contamination for
the target pesticides. Bromide was present at different concentrations in
milk, eggs, chicken meat and kidney within the range of 0.8–3 mg kg−1.
The least contaminated samples were used for the validation and the
background concentrations present in the samples were subtracted to

Fig. 3. Extracted ion chromatograms (quantifier and qualifier ions) and ion ratios for glyphosate in bovine and swine kidney, at different spike levels, (a) glyphosate
(0.02 mg kg-1) - Kidney, (b) glyphosate (0.2 mg kg-1) - Kidney and (c) glyphosate (0.5 mg kg-1) – Kidney.

6
S.H. López, et al.
Fig. 3. (continued)
calculate the real recoveries. When the concentration in the sample was
very high, it was not possible to validate this compound at the lowest
levels, even though detection and correct identification at these levels
was not a problem.
3.3.3.1. Milk. From the 14 evaluated compounds, 12 showed
recoveries between 70 and 120% and RSD ≤20% for all the
concentrations (0.01, 0.02, 0.05 and 0.1 mg kg−1), as shown in
Table 2S. For bromide, an ILIS was not commercially available and
the recoveries were calculated using the ILIS for chlorate (18O3-
chlorate). Because the unlabelled chlorate and bromide showed
different matrix effects (33 and −1%, respectively), the calculated
bromide recoveries obtained were thus lower than they should be, that
is around 70%. HEPA was only validated at the highest level,
0.1 mg kg−1, due to the high suppression of the response and the
lack of sensitivity of the qualifier ion. But its SDL could be set as
0.02 mg kg−1.
3.3.3.2. Fat. For swine fat, with the exception of N-acetyl-glyphosate,
all the compounds showed recoveries and RSDs within the acceptable
range for all concentration levels, 0.01, 0.02 and 0.05 mg kg−1 (see
Table 3S), and could be fully validated.
3.3.3.3. Chicken eggs. For chicken eggs, the validation was carried out
with and without clean-up with MCX sorbent (see results in Tables 4S
and 5S). The first validation was performed without clean-up and most
of the compounds met the criteria of the SANTE document at the lowest
validated level (0.02 mg kg−1). However, considering the results for
7
Food Control 115 (2020) 107289
glyphosate, validated at 0.05 mg kg−1 (SDL 0.01 mg kg−1) and HEPA,
which could not be validated at any concentration (see Table 4S), it was
decided to employ a clean-up step in order to improve the results for
these compounds. Fig. 3Sa and 3Sb show the chromatograms at 3
concentration levels (0.02, 0.05 and 0.1 mg kg−1) for both compounds
with and without MCX in the clean-up step.
The use of MCX clean-up improved the results for glyphosate,
especially for the response of the second ion, and also for HEPA that
could be detected at 0.02 mg kg−1 despite poor sensitivity for the
second ion. On the other hand, the use of MCX caused the loss of glu-
fosinate (see Fig. 3Sc).
In the case of eggs validation, the use of MCX clean-up or no the
clean-up affected the individual compounds differently. The final de-
cision about the use or deletion of MCX clean-up will depend on the
preferred scope of the target analytes for each analysis.
3.3.3.4. Chicken meat. In the case of chicken meat, as shown in
Table 6S, glufosinate and HEPA were validated from the level of
0.1 mg kg−1. However, the SDL for these compounds could be set at
0.02 and 0.05 mg kg−1, respectively. Glyphosate and MPPA complied
with the criteria for recovery and precision at 0.05 mg kg−1, but a SDL
could be set at 0.02 mg kg−1. N-acetyl-AMPA showed lower ILIS-
corrected recoveries than the absolute recoveries, but they were still
within the range 70–120%. This could be explained by the
unavailability of the corresponding ILIS for N-acetyl-AMPA and the
use of Ethephon-D4 as a substitute ILIS. The different matrix effects
shown for N-acetyl-AMPA and Ethephon-D4 is probably the reason why
the ILIS-corrected recoveries are lower than the uncorrected ones.
S.H. López, et al.
Fig. 3. (continued)
3.3.3.5. Kidney. Several samples of bovine and swine kidney were
tested to evaluate possible background levels of some of the target
compounds in the blank samples. As has been mentioned above, all the
samples tested were positive for bromide. The samples with the lower
level (3 mg kg−1) for bovine and swine kidney were chosen to use for
the validation study. Bromide could thus only be validated at
5 mg kg−1. HEPA was also detected in the blank samples of bovine
kidney at 0.07 mg kg−1. This concentration was subtracted from the
blank samples for the recovery calculation. As can been seen in
Table 7S, glufosinate was validated at 0.05, 0.2 and 0.5 mg kg−1 and
its SDL could be set at 0.02 mg kg−1.
In this matrix, glyphosate showed different results in the two dif-
ferent types of kidney evaluated. The interferences from the matrix
affected the qualifier ion in different ways depending on the type of
kidney (see Fig. 3). The recoveries for glyphosate in Table 7S at
0.2 mg kg−1 are only for swine kidney because the second ion could not
be resolved from the interferences from the bovine kidney matrix and
hence these data were not considered. However the results at
0.5 mg kg−1 are for swine and bovine kidney, because the ion ratio
criterion ( ± 30%) is met. See also Fig. 3b and c. Despite the validated
LOQs have been set at the two higher concentrations, glyphosate could
be detected (quantifier ion) at the lowest level evaluated for both types
of kidney (Fig. 3a). Therefore, its SDL could be set at 0.02 mg kg−1.
3.3.3.6. Liver. The validation for the matrix liver was performed using
3 different types of liver (bovine, swine and poultry, n = 2 for each)
(see results in Table 8S). Ten out of fourteen compounds were validated
from the lowest level tested (0.02 mg kg−1). During validation using
the clean-up with MCX, glyphosate showed a different behaviour in the
8
Food Control 115 (2020) 107289
three livers tested, as it was also observed during the evaluation of
kidney. The worst results were for swine liver. In Fig. 4, the extracted
ion chromatograms of the three livers, at three different spike levels,
are shown. At the lowest spike level, 0.02 mg kg−1, the full
identification was not possible in any type of liver evaluated. At the
two higher levels tested (0.2, 0.5 mg kg−1), the identification was
possible for bovine and poultry liver, but not for swine liver. For swine
liver, the full identification was only possible at the highest level for
glyphosate. However, the SDL for glyphosate could be set for all three
types of liver at 0.02 mg kg−1 (see Fig. 4a). In Table 8S the recovery
data at 0.2 mg kg−1 are for bovine and poultry liver and at 0.5 mg
kg−1for all three of types.
The LOQs and screening detection limits of the final method using
the ILIS for quantitation are shown in Table 2.
In summary, the following validation results have been obtained.
Most of the compounds have been validated at 0.01 mg kg−1 for cow
milk and swine fat. Only N-acetyl-glufosinate and N-acetyl-glyphosate
were validated at 0.02 mg kg−1 in swine fat and HEPA at 0.1 mg kg−1
in cow milk. In the case of kidney and liver 70 and 64% of the com-
pounds, respectively, were validated at 0.02 mg kg−1. Glufosinate was
validated in kidney at 0.05 mg kg−1 and HEPA at 0.2 mg kg−1 in liver.
Glyphosate showed LOQs of 0.5 mg kg−1 for these two matrices. In
chicken meat, 8 compounds were validated at 0.02 mg kg−1, two
compounds (glyphosate and MPPA) at 0.05 mg kg−1 and another two
(glufosinate and HEPA) at 0.1 mg kg−1. Bromide and phosphonic acid
were validated at 0.2 mg kg−1, the lowest spike levels tested for these
compounds. For chicken eggs, all the compounds were validated at the
lowest spike level tested. In order to be able to detect glufosinate, no
clean-up should be applied and in that case its LOQ is 0.01 mg kg−1.
S.H. López, et al. Food Control 115 (2020) 107289

Fig. 4. Extracted ion chromatograms (quantifier and qualifier ions) and ion ratios for glyphosate in bovine, poultry and swine liver, at different spike levels, (a)
glyphosate (0.02 mg kg-1) - Liver, (b) glyphosate (0.2 mg kg-1) - Liver and (c) Glyphosate (0.5 mg kg-1) – Liver.

Fig. 4. (continued)

9
S.H. López, et al. Food Control 115 (2020) 107289

Fig. 4. (continued)

Table 2
Limits of quantification (LOQs) and screening detection limits (SDLs) of the final validated method for the six evaluated matrices.
Pesticide LOQs (mg kg−1)

Cow Milk Swine Fat Kidney Liver Chicken meat Chicken eggs

AMPA 0.01 0.01 0.02 0.02 0.02 0.02

Bromide 0.5 0.1 5 5 0.2 2
Chlorate 0.01 0.01 0.02 0.02 0.02 0.02
Ethephon 0.01 0.01 0.02 0.02 0.02 0.02
Fosetyl 0.01 0.01 0.02 0.02 0.02 0.02
Glufosinate 0.01 0.01 0.05 (0.02c) 0.2 0.1 (0.02c) 0.01d
Glyphosate 0.01 0.01 0.2 (0.02c)
a
0.2 (0.02c)
b
0.05 (0.02c) 0.02
HEPA 0.1 (0.02c) 0.01 0.02 0.2 0.1 (0.02c) 0.02
MPPA 0.01 0.01 0.02 0.02 0.05 (0.02c) 0.02
N-acetyl-AMPA 0.01 0.01 0.02 0.02 0.02 0.02
N-acetyl-glufosinate 0.01 0.01 0.02 0.02 0.02 0.02
N-acetyl-glyphosate 0.01 0.02 0.02 0.02 0.02 0.02
Perchlorate 0.01 0.01 0.02 0.02 0.02 0.02
Phosphonic acid 0.1 0.1 0.2 0.2 0.2 0.2

a
LOQ for swine kidney.
b
LOQ for bovine and poultry liver.
c
SDL= Screening detection limit.
d
LOQ for method without clean-up.

3.4. Different quantitation approaches
During the recovery study of liver matrix, the absolute recoveries
for all pesticides were also calculated using matrix-matched, external
calibration standards using bovine liver extracts. It was observed that,
for AMPA, at all four levels, absolute recoveries above 200% were
calculated (see Table 9S). This effect was not observed for all the pes-
ticides included in the method, as is shown in Table 9S for fosetyl, as an
example. The results for AMPA may be explained by different matrix
effects in the three types of liver tested. In order to prove that ILIS are
required for quantitation in routine batches of samples, where different
types of liver have to be analysed, different calibration curves were
constructed with external standards solutions prepared in solvent or
matrix-matched with poultry liver extract and procedural standards in
bovine, swine and poultry liver extract. The different quantitative re-
sults obtained using the different quantitation approaches were com-
pared. In Fig. 5, the five different calibration curves have been plotted
for (a) AMPA and (b) fosetyl. Fig. 5a shows the different response for
AMPA in solvent and in the three types of kidney. But it is also clear in
the graph, that the response for poultry liver is different than the re-
sponse for bovine and swine liver. Due to this difference, the absolute
recoveries calculated using matrix-matched standards using bovine
liver showed recoveries above 200% for poultry liver. These results
confirm that isotopically labelled internal standards are essential for the

10
S.H. López, et al.
Fig. 5. Five calibration curves constructed from different calibration approaches: standards in solvent, matrix-matched standards with poultry liver extract and
procedural standard calibration with poultry, bovine and swine liver. (a) AMPA and (b) fosetyl.
correct quantitation for some compound/matrix combinations, because
other approaches such as procedural standard calibration and matrix-
matched calibration would only work when the samples to be analysed
will be of exact the same type as the blank sample used for calibration
standards.
4. Conclusion
The method for the determination of fourteen polar pesticides and
their metabolites in cow milk, swine fat, chicken meat, chicken eggs,
liver and kidney has been validated according to the EU SANTE docu-
ment, using isotopically labelled internal standards for quantitation.
This is the first publication on the analysis of these fourteen anionic
polar pesticides in products of animal origin, using one extraction
method and a single, fast chromatographic run.
The main conclusion of this study is that the results are totally de-
pendent on the individual matrix types and not on the commodity
group. This was already observed in the previous validation for fruit,
vegetables, oat and soya, but in this study not only differences between
matrices belonging to the same commodity group as liver and kidney
were observed, but also differences between individual types of liver or
kidney.
The method quantitation limits are, for most of the compounds and
in most of the matrices evaluated, 0.01 or 0.02 mg kg−1. Some com-
pounds had higher LOQs, because they were intentionally validated at
higher levels (phosphonic acid and bromide), or because of their lower
sensitivity, especially for the qualifier ion (as for glufosinate, glyphosate
and HEPA). Bromide could not be validated in liver, kidney and chicken
11
Food Control 115 (2020) 107289
eggs because no blank samples were available.
In general, AMPA, glufosinate, glyphosate and HEPA were the
compounds which showed relatively more difficulties during the vali-
dation study. AMPA and HEPA showed the strongest matrix effects,
HEPA in cow milk, −86%, and AMPA in liver, −77%. The lack of
sensitivity of the qualifier ions of Glyphosate and HEPA did not allow
the full identification at the lower spike levels studied. The recoveries of
glufosinate were affected by the use of SPE cartridges with MCX as
clean-up. The compound was retained on the sorbent due to the in-
teraction between functional groups of the polymer and amino groups
in the glufosinate molecule. The retention was not the same for all
matrices evaluated. The compound was fully retained during the ex-
periments with chicken eggs, but for the other matrices glyphosate was
at least detectable at the higher concentrations evaluated.
Isotopically labelled internal standards are essential in terms of
quantitation for some compound/matrix combinations, because it has
been observed that other approaches such as procedural standard ca-
libration and matrix matched calibration did not work when the sample
matrices to be analysed are different from the type of matrix used to
prepare the calibration standard solutions.
The validated method appears to be suitable for the intended pur-
pose, that is the determination of 14 polar pesticides/metabolites, in the
six matrices mentioned above. The method can behave different for the
various individual matrices from the same SANTE commodity group to
which kidney and liver belong (group 7, offal). Therefore, the on-going
validation with different types of matrices should be carried out, to be
able to evaluate the target compounds in other matrices during the
application of the method in the routine analysis.
S.H. López, et al.
CRediT authorship contribution statement
Sonia Herrera López: Conceptualization, Data curation, Formal
analysis, Investigation, Methodology, Validation, Writing - original
draft, Writing - review & editing. Jonatan Dias: Data curation, Formal
analysis, Investigation, Methodology, Validation, Writing - review &
editing. André de Kok: Investigation, Project administration,
Supervision, Validation, Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgement
This work was funded by the Netherlands Food and Consumer
Product Safety Authority (NVWA).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodcont.2020.107289.
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