Food Chemistry 132 (2012) 567–573

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Food Chemistry
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Analytical Methods

Feasibility study for producing a carrot/potato matrix reference material for 11

selected pesticides at EU MRL level: Material processing, homogeneity and
stability assessment
Helena Saldanha, Berit Sejerøe-Olsen, Franz Ulberth, Hendrik Emons, Reinhard Zeleny ⇑
European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Retieseweg 111, 2440 Geel, Belgium

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

Article history: The feasibility for producing a matrix reference material for selected pesticides in a carrot/potato matrix
Received 15 July 2011 was investigated. A commercially available baby food (carrot/potato-based mash) was spiked with 11
Received in revised form 9 August 2011 pesticides at the respective EU maximum residue limits (MRLs), and further processed by either freezing
Accepted 22 October 2011
or freeze-drying. Batches of some 150 units were produced per material type. First, the materials were
Available online 30 October 2011
assessed for the relative amount of pesticide recovered after processing (ratio of pesticide concentration
in the processed material to the initially spiked pesticide concentration). In addition, the materials’
Keywords:
homogeneity (bottle-to-bottle variation), and the short-term (1 month) and mid-term (5 months) stabil-
Pesticides
Food
ity at different temperatures were assessed. For this, an in-house validated GC–EI-MS method operated in
Matrix reference material the SIM mode with a sample preparation procedure based on the QuEChERS (‘‘quick, easy, cheap, effec-
Feasibility study tive, rugged, and safe’’) principle was applied. Measurements on the frozen material provided the most
Processing promising results (smallest analyte losses during production), and also freeze-drying proved to be a suit-
Homogeneity able alternative processing technique for most of the investigated pesticides. Both the frozen and the
Stability freeze-dried material showed to be sufficiently homogeneous for the intended use, and storage at
GC–MS 20 °C for 5 months did not reveal any detectable material degradation. The results constitute an impor-
tant step towards the development of a pesticide matrix reference material.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction
Pesticides are used to protect crops and plants from pests and
diseases (Tomlin, 2003). In the European Union, several hundred
pesticides from different chemical classes (e.g. pyrethroids, organo-
phosphates, dicarboximides) are currently authorised for the use of
plant protection (Council Directive 91/414/EC, 1991; Regulation
(EC) No. 396/2005 of the European Parliament and of the Council,
2005). Due to their potential acute and chronic toxicity however, res-
idues of pesticides in the food chain can constitute a health risk to
consumers. Maximum residue limits (MRLs) have therefore been
fixed for a large number of pesticides (Council Directive 76/895/EC,
1976; Council Directive 90/642/EC, 1990). To ensure effective con-
sumer protection, reliable analytical methods for the detection, iden-
tification and quantification of pesticide residues in food are required.
At present, mostly liquid chromatography and gas chromatography
coupled to mass spectrometry (GC–MS; GC–MS/MS, LC–MS/MS) is
applied for pesticide residue analysis (Alder, Greulich, Kempe, &
Vieth, 2006; Soler & Picó, 2007; Štajnbaher & Zupančič-Kralj, 2008).
⇑ Corresponding author. Tel.: +32 14 571615; fax: +32 14 571548.
E-mail address: reinhard.zeleny@ec.europa.eu (R. Zeleny).
Pesticides are usually extracted using the QuEChERS (‘‘quick, easy,
cheap, effective, rugged, and safe’’) approach (Anastassiades, Lehotay,
Štajnbaher, & Schenck, 2003; Wilkowska & Biziuk, 2011), although
other extraction techniques such as matrix solid-phase dispersion
and pressurized liquid extraction are also applied (Blasco, Font, &
Picó, 2005; Navarro, Picó, Marín, & Mañes, 2002). Laboratories require
suitable quality control tools to deliver accurate results. Certified ref-
erence materials (CRMs) are key tools to establish and safeguard reli-
able measurements, as they can be used for method validation (e.g.
trueness determination) and method performance verification. In
the field of pesticide analysis however, few CRMs are available at
present, and the majority of those are either pure substances certified
for purity, or calibration solutions. As concerns available pesticide ref-
erence materials in food matrices, very few currently exist, among
those BCR-187 and BCR-188 (organochlorinepesticides (OCPs) in
milk), BCR-430 (OCPs in pork fat), BCR-598 (OCPs in cod liver oil),
and NMIJ 7401-a (OCPs in fish oil). Recently, NMIJ has released CRM
7504-a (etofenprox (a pyrethroid) and fenitrothion (an organophos-
phate) in rice), and CRM 7404-a (PCBs and 5 OCPs in fish) (Otake,
Aoyagi, Yarita, & Numata, 2010; Otake et al., 2009). Seen the lack of
CRMs in many cases, PT samples are sometimes used as quality con-
trol tools (EU Reference Laboratory proficiency testing activities;
FAPAS programme 2011–2012).

0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.10.071
568 H. Saldanha et al. / Food Chemistry 132 (2012) 567–573
For the preparation and certification of a CRM, the following
main challenges are faced and have to be tackled accordingly:
(a) Material type. Although incurred materials would be gener-
ally preferred (similarity to real samples), they are difficult
to prepare. Spiked materials can be a suitable alternative.
However, the analyte extractability might be different
between the two types of material, and a commutability
study would be needed to demonstrate equivalence of the
materials.
(b) Choice of a suitable and representative analyte/matrix com-
bination. It is evident that no RM can be made available for
all possible food matrix/pesticide analyte combinations.
Instead, the aim is to provide a CRM with a representative
matrix, certified for a number of pesticides from different
chemical classes, depending on legal priorities, chemical
representativeness, and analytical capabilities.
(c) Minimum analyte and matrix disruption during processing
of a material. Modern pesticides are rather instable to avoid
their accumulation in the environment, and great care has to
be taken to find suitable processing and preservation proce-
dures. Moreover, a substantial disruption of the material
during processing might impede the commutability of such
as material when used as quality control for routine mea-
surements of real samples.
(d) Suitable homogeneity and stability of the material. These are
key properties for any kind of RM. Comprehensive studies
have to be performed, employing methods with a high
repeatability in order to detect possible inhomogeneity
and/or instability of the material.
(e) Metrological traceability. Ideally, the certified value(s)
should be independent of the methods used in the frame
of the certification exercise. Moreover, all property values
in a CRM should preferably be traceable to the International
System of Units (SI). The possibility of achieving this
depends on the availability of suitable calibrants, and of
comprehensively validated methods (performed ideally by
accredited laboratories), which deliver accurate and reliable
results.
In this paper we report on a feasibility study for producing a
matrix RM for 11 selected pesticides at the MRL level in a model
food matrix, i.e. potato/carrot puree (commercially available baby
food). We evaluated different processing procedures with the aim
to obtain materials with minimal disruption and pesticides degra-
dation and at the same time assuring suitable material homogene-
ity and stability. The presented results represent an important step
towards developing a CRM for pesticides in a plant matter matrix.
2. Materials and methods
2.1. Chemicals and consumables
The pesticides in this study (see Table 1) were obtained as ana-
lytical standards with stated purity from Dr. Ehrenstorfer (Augs-
burg, Germany). Individual stock solutions, a mixed stock solution
containing all pesticides, and subsequent working solutions were
gravimetrically prepared, using toluene as solvent. The isotopically
labelled standards phorate (diethoxy-13C4), parathion-ethyl-D10,
and pirimiphos-methyl-D6 were from Cambridge Isotope Laborato-
ries (Apeldoorn, The Netherlands). A mixed solution (0.5 lg/g of
each isotopically-labelled internal standard in acetonitrile) was pre-
pared. Toluene and acetonitrile (SupraSolv grade) were obtained
from Merck (Darmstadt, Germany). Dispersive SPE extraction tubes
Supelco 55234-U and Supelco 55230-U were purchased from
Sigma–Aldrich (Bornem, Belgium). Fluoroethylenepropylene FEP
centrifuge tubes (50 mL, NalgeneÒ) were from Sigma–Aldrich, Bor-
nem, Belgium. Ultrapure water from a MilliQ water purification sys-
tem (Millipore, Brussels, Belgium) was used. The vegetable matrix
used for preparation of the study materials was heat-treated
homogenized baby food based on carrots and potatoes (Olvarit
brand, Nutricia B.V., Bornem, Belgium).
2.2. Preparation of the study materials
The baby food matrix (ca. 35 kg) was placed in a stainless steel
mixing vessel of a paste mixer (IKA-Janke Kunkel, Staufen, Ger-
many) and mixed at full speed for 4 h. A spiking solution, consist-
ing of the 11 pesticides in acetonitrile, was added to the slurry
(target concentration was the respective MRL of each pesticide),
and the mixture was stirred for another 3 h.
2.2.1. Frozen material
Seventy grams portions of the homogenised material were
manually filled into 210 mL glass jars, closed with a screw cap,
and stored at 20 °C.
2.2.2. Freeze-dried (FD) material
Twenty kilograms of the homogenised material were diluted
with 8 L of distilled water and thoroughly mixed for 30 min. The
resulting slurry was frozen and freeze-dried in an Epsilon 2-85D
freeze-dryer (Martin Christ, Osterode, Germany). The freeze-dried
material was manually crushed with a PFTE pestle and then milled
with a heavy duty mill (Retsch, Haan, Germany) with 1.0, 0.5, and
0.25 mm sieve inserts. 2.0 kg of powder were obtained after mill-
ing. Finally, the powder was homogenised in a Dyna-MIX CM200
mixer (WAB, Basel, Switzerland). 12.5 g portions were filled into
100 mL amber glass vials and closed with screw caps. The material
was stored at 20 °C until analysis.
2.3. Characterisation of the FD material
The water content of the obtained powder was determined by vol-
umetric Karl-Fischer-titration on five randomly chosen bottles as de-
scribed elsewhere (Kestens, Charoud-Got, Bau’, Bernreuther, &
Emteborg, 2008). Moreover, particle size analysis was performed using
laser diffraction spectrometry. Samples were analysed over a range of
0.5–1750 lm using a Helos laser light scattering instrument (Sympa-
tec GmbH System-Partikel-Technik, Clausthal-Zellerfeld, DE).
2.4. Characterisation of the frozen material
The water content was determined by volumetric Karl-Fischer-
titration on five randomly chosen bottles as described elsewhere
(Kestens et al., 2008). In addition, five randomly chosen bottles
were subjected to an air oven method for mass loss determination
(water and volatile compounds): samples were dried until con-
stant weight at 103 ± 2 °C.
2.5. Analytical methodology for pesticide quantification
The description of calibration, sample preparation, GC–MS anal-
ysis, and method validation can be found in the Supplementary file.
2.6. Homogeneity studies
For each material, 10 units were chosen using a random strati-
fied sample selection scheme, and three sub-samples from each
unit were analysed for their pesticides content with the in-house
validated GC–MS method described earlier. Measurements were
carried out in one large series (repeatability conditions). Samples
were measured in a random order to allow distinction between
 H. Saldanha et al. / Food Chemistry 132 (2012) 567–573 569

Table 1
Definition of the pesticide analytes in the feasibility study.

Trivial name and MRLa Chemical class IUPAC name CAS Chemical Molar mass Purityb
abbreviation (mg/kg) number formula (g/mol)
Phorate 0.05 Organophosphate O,O-Diethyl S-ethylthiomethyl phosphorodithioate 298-02-2 C7H17O2PS3 260.38 94.5
Propyzamide 0.02 Amide 3,5-Dichloro-N-(1,1-dimethylpropynyl)benzamide 23950-58-5 C12H11Cl2NO 256.13 98.1
Diazinon 0.01 Organophosphate O,O-Diethyl O-[2-isopropyl-6-methyl-4-pyrimidinyl] 333-41-5 C12H21N2O3PS 304.35 99.9
phosphorothioate
Vinclozolin 0.05 Dicarboximide 3-(3,5-Dichlorophenyl)-5-methyl-5-vinyloxazolidine- 83792-61-4 C12H9Cl2NO3 286.11 99.6
2,4-dione
Chlorpyrifos-methyl 0.05 Organophosphate O,O-Dimethyl O-3,5,6-trichloro-2-pyridyl 5598-13-0 C7H7Cl3NO3PS 322.53 99.9
phosphorothioate
Metalaxyl 0.05 Phenylamide N-(2,6-Dimethylphenyl)-N-(methoxy-acetyl)-alanine 57837-19-1 C15H21NO4 279.33 98.5
methyl ester
Pirimifos-methyl 0.05 Organophosphate O-[2-(Diethylamino)-6-methylpyrimidin-4-yl] O,O- 29232-93-7 C11H20N3O3PS 305.33 99.9
dimethyl phosphorothioate
Chlorpyrifos 0.05 Organophosphate O,O-Diethyl O-3,5,6-trichloropyridin-2-yl 2921-88-2 C9H11Cl3NO3PS 350.59 99.2
phosphorothioate
Parathion 0.05 Organophosphate O,O-Diethyl O-(4-nitrophenyl) phosphorothioate 56-38-2 C10H14NO5PS 291.26 98.8
Bromopropylate 0.05 Bridged diphenyl Propan-2-yl 2,2-bis(4-bromophenyl)-2-hydroxyacetate 18181-80-1 C17H16Br2O3 428.12 99.2
Permethrinc 0.05 Pyrethroid Bis-(4-bromo-phenyl)-hydroxy-acetic acid isopropyl 52645-53-1 C21H20Cl2O3 391.28 94.5
ester
a
Potato.
b
Purity of analytical standard as stated by the provider.
c
Sum of cis and trans isomer.

an analytical drift and a possible trend in the filling sequence. The
data were investigated for outliers using the Grubbs test, and linear
regression analysis was performed to check for possible trends in
the filling sequence. The distribution of results was checked using
normal probability plots and histograms. Finally, analysis of vari-
ance (ANOVA) was performed to quantify the within-bottle stan-
dard deviation (swb) and the between-bottle standard deviation
(sbb). Moreover, the uncertainty due to possible inhomogeneity
that can be hidden by the method repeatability (ubb ) was calcu-
lated as described elsewhere (Linsinger, Pauwels, van der Veen,
Schimmel & Lamberty, 2001b).
2.7. Stability studies
Studies were conduced as isochronous stability studies (Lam-
berty, Schimmel, & Pauwels, 1998). First, a short-term stability
study for evaluating suitable dispatch conditions was carried out.
Secondly, a mid-term stability study was performed to get infor-
mation about possible storage temperatures for the candidate ref-
erence materials. For both studies, six independent sub-samples
were analysed for each time/temperature point. Storage tempera-
tures in both studies were 20 °C (frozen batch), and 20 °C,
+4 °C, and +18 °C (freeze-dried batch); the reference temperature
was 70 °C. The storage times were 0, 1, 2, and 4 weeks (short-
term stability study), and 0, 3, 4, and 5 months (mid-term stability
study). After the indicated storage periods, samples were trans-
ferred to storage at 70 °C until analysis. Measurements were car-
ried out under repeatability conditions with the in-house validated
GC–MS method described earlier. Data were checked for outliers
using the Grubbs test, and linear regression analysis as a function
of time was performed. Slopes were tested for significance using
a t-test. Finally, the uncertainty of stability usts (short-term stabil-
ity) and umts (mid-term stability) was calculated as described else-
where (Linsinger et al., 2001a) and indicated for a period of 1 week
(short-term study) and 5 months (mid-term study).
3. Results and discussion
3.1. Performance of the in-house QuEChERS GC–EI-MS method
Results on the method performance of the QuEChERS GC–EI-MS
method employed for assessing a suitable processing procedure as
well as homogeneity stability of the processed materials can be
found in the electronic Supplementary file.
3.2. Processing of materials
A commercially available carrot/potato mash was spiked with a
mixture of the 11 selected pesticides in acetonitrile. The spiking le-
vel was the respective MRL of each pesticide. The spiked mash was
then further processed by either freezing or freeze-drying. The fro-
zen material was tested for its water and moisture content by
applying vKFT and oven drying to five randomly picked samples.
Results were 87.8 ± 2.3 g/100 g and 89.2 ± 0.5 g/100 g with vKFT
and oven drying, respectively. The non-significant difference be-
tween the water-selective Karl-Fischer results and the oven-drying
results (water plus volatile components) indicate that the amount
of acetonitrile still present in the material after extensive stirring
and processing is negligible. Freeze-drying resulted in a mass loss
of about 90% of the starting material. The freeze-dried material
contained 2.3 ± 0.3 g/100 g residual water as determined by Karl-
Fischer titration. Particle size analysis by laser diffraction spec-
trometry yielded an average particle size of 128 lm, with 80% of
the particles having sizes between 20 and 300 lm.
The feasibility and suitability of the processing procedures was
evaluated by comparing the level of pesticide found before process-
ing (spiking level, 100%) and after the respective processing (Fig. 1).
In addition, the recovery data from validation is depicted to allow
comparison to non-processed samples (blank matrix, spiked and
analysed). For the processed materials, the data from the respective
homogeneity studies were taken (mean and standard deviation of
30 samples, comparable number of samples and analyses to that
of validation study). It can be seen that without processing, and esti-
mating the immediate recovery (blank spiked and analysed), analyte
losses cannot be detected. In the frozen batch, a significant
decrease in the measured level could be found for some pesticides
(permethrin, bromopropylate, chlorpyrifos-methyl). For some, a
slight analyte decrease was observed (diazinon, chlorpyrifos, vinc-
lozolin), and for the other five pesticides, no changes were observed.
Generally, losses were more pronounced in the freeze-dried mate-
rial: for all 11 pesticides, analyte levels were substantially smaller
after the processing, but differences were obtained nevertheless:
whereas e.g. phorate was presented only at about 25%, typical
analyte levels of some 55–75% of the added amount were found back
after processing for most of the pesticides.
570 H. Saldanha et al. / Food Chemistry 132 (2012) 567–573

rec. from validation

120
frozen -20 ºC
110 freeze-dried

100

90

80
relative recovery (spiked = 100%)

70

60

50

40

30

20

10

l
e

in
te
yl
on

hy
e

lin

s
hy
id

io
at

ifo

hr
ax

la
et
zo
zin
am

th
et
or

py

et
r
al

py

ra
lo

m
Ph

rm
ia

et
yz

ro
s-
nc

s-

Pa
or
D

op
ho
op

Pe
ifo
Vi

hl

om
ip

C
Pr

r
py

im

Br
or

Pr
hl

pesticide
C

Fig. 1. Influence of processing type on level of pesticides in the three candidate reference materials. For each pesticide, the relative recovery (spiked = 100%) is shown. From
left to right: recovery from validation (no processing, only sample preparation and analysis of samples spiked and immediately analysed); recovery after freezing, and
recovery after freeze-drying. For the two processing types, the data from the respective homogeneity studies were taken. Error bars are standard deviations from 30 samples
(2 levels, 5 days, three replicates per day).

Each of those preservation and stabilisation techniques has for diazinon, metalaxyl, parathion, pirimifos-methyl and chlorpyri-
advantages and disadvantages; freezing of a homogeneous slurry fos. Bottle mean outliers were detected for chlorpyrifos-methyl,
is easy to achieve and usually yields a product of minimal material pirimifos-methyl and parathion. As no technical reason for the
disruption and therefore closeness to real-world (‘‘fresh’’) samples. occurrence of these outliers could be found, all data were retained
However, storage (cooling capacities, surveillance of freezing sta- for statistical analysis. Regression analysis indicated no trend in
tus) is expensive, and samples have to be shipped to the customer the filling sequence for neither pesticide in the frozen batch. In
in the frozen state (e.g. on dry ice), which, again, is costly, and the freeze-dried batch, a significant slope of the regression lines
poses some logistic challenges (packaging, overseas shipment). was detected for diazinon and metalaxyl, which can be attributed
Freeze-dried samples (when confirmed to be stable at room tem-
perature or at least under cooled conditions) can be shipped more
easily and with less costs; additionally, storage is cheaper, and as –
Table 2
in this case – about 90% of the weight (water, volatiles) are re- Homogeneity study results for the frozen material (left side) and freeze-dried
moved by freeze-drying, the volume for one unit is substantially material (right side). n.c. cannot be calculated as ANOVA result reveals that
decreased, resulting in less storage space needed. The most impor- MSbetween < MSwithin; swb, within-bottle standard deviation.
tant concern for freeze-dried reference materials however relates Pesticide Frozen batch Freeze-dried batch
to the question whether they behave similarly compared to real-
swba sbb b
ubb c uoutl d
swba sbbb ubb c uoutld
world (‘‘fresh’’) samples; material properties might result in differ-
[%] [%] [%] [%] [%] [%] [%] [%]
ent analyte extractability for the two materials and therefore a
Phorate 7.1 n.c. 2.3 7.7 n.c. 2.5
non-commutability of the FD material for the usual testing meth-
Propyzamide 6.1 n.c. 2.0 3.5 6.6 3.1 2.1
ods. For this, a dedicated commutability study would be required, Diazinon 4.7 n.c. 1.5 5.2 1.6 1.7 3.2
which compares fresh with processed materials, and employs at Vinclozolin 4.3 n.c. 1.4 3.3 1.8 1.1
least two different types of methods (Zeleny, Emteborg, & Chlorpyrifos- 6.3 n.c. 2.0 4.3 3.5 1.4 1.1 3.3
Schimmel, 2010). methyl
Metalaxyl 7.0 n.c. 2.3 15.2 6.3 4.9 9.9
In summary, both freezing and freeze-drying are suitable pro-
Pirimifos- 4.6 1.2 1.5 3.1 n.c. 1.0 1.5
cessing techniques for a candidate RM. When producing a large methyl
RM batch with pesticide target concentrations at the respective Chlorpyrifos 7.4 3.5 2.4 7.5 10.7 5.4 3.5
MRL levels, it has to be considered that spiking is performed at Parathion 4.3 0.8 1.4 3.1 2.2 1.0 4.1
Bromopropylate 7.1 2.2 2.3 6.6 4.5 n.c. 1.5
higher levels to compensate for losses during processing.
Permethrine 8.4 2.9 2.7 4.4 1.5 1.4
a
swb, within-bottle standard deviation.
3.3. Homogeneity b
sbb, between-bottle standard deviation.
c
ubb , defined as uncertainty due to possible inhomogeneity that can be hidden by
First, data were checked for the presence of outliers. For the fro- the method repeatability. ubb is an alternative estimate for the uncertainty
contribution due to possible inhomogeneity and is calculated when sbb cannot be
zen batch, no outlying individual results were found, whereas a calculated (MSbetween < MSwithin in ANOVA).
few outliers for bottle mean values were detected (1 each for pro- d
uoutl, alternative uncertainty estimation in case of outlying bottle means or a
pyzamide, chlorpyrifos-methyl, chlorpyrifos and bromopropylate). trend in the filling sequence (Linsinger et al., 2009).
e
For the freeze-dried batch, individual outliers (1 each) were found Sum of cis and trans isomer.
 H. Saldanha et al. / Food Chemistry 132 (2012) 567–573 571

Table 3
Short-term stability results (left side) and mid-term stability results (right side) for the frozen batch.

Pesticide Outlier (95% level Slope Significance of slope(95% ustsa [%/ Outlier (95% level Slope [%/ Significance of slope (95% umtsb [%/
of confidence) [%/ level of confidence) week] of confidence) month] level of confidence) 5 month]c
week]
Phorate No 1.13 No 0.9 2 0.8 No 2.2
Propyzamide No 2.15 No 1.4 No 0.3 No 2.1
Diazinon No 0.09 No 0.9 No 2.0 No 5.6
Vinclozolin No 0.75 No 0.9 No 1.4 No 4.4
Chlorpyrifos-methyl No 1.07 No 1.1 No 1.6 No 4.2
Metalaxyl No 2.18 No 1.1 1 0.0 No 5.0
Pirimifos-methyl No 0.91 No 0.7 1 0.8 No 3.9
Chlorpyrifos No 1.94 No 1.3 1 1.3 No 3.7
Parathion No 1.08 No 0.7 1 0.9 No 2.7
Bromopropylate No 4.23 Yes 2.7 No 2.2 No 5.4
Permethrind No 3.40 Yes 2.3 No 2.0 No 6.5
a
usts, uncertainty of short-term stability.
b
umts, uncertainty of mid-term stability.
c
Study period, taken as shelf-life.
d
Sum of cis and trans isomer.

Table 4
Short-term stability results (left side) and mid-term stability results (right side) for the freeze-dried batch.

Pesticide Outlier (95% level Slope Significance of slope (95% ustsa Outlier (95% level Slope Significance of slope (95% umtsb
of confidence) [%/week] level of confidence) [%/week] of confidence) [%/month] level of confidence) [%/5 month]
Storage temperature 20 °C Storage temperature 20 °C
Phorate No 1.47 No 1.44 No 0.51 No 11.8
Propyzamide No 1.40 No 0.96 No 3.17 No 12.3
Diazinon No 2.29 No 1.35 No 1.91 No 4.4
Vinclozolin No 3.10 No 1.76 No 4.26 No 10.1
Chlorpyrifos-methyl No 1.95 No 1.48 No 2.96 No 7.9
Metalaxyl No 1.09 No 1.06 No 2.18 No 6.6
Pirimifos-methyl No 1.94 No 1.43 2 4.50 No 7.3
Chlorpyrifos 1 0.88 No 1.83 No 0.07 No 3.1
Parathion No 1.98 No 1.15 3 3.62 No 6.6
Bromopropylate No 1.31 No 2.59 No 0.42 No 5.9
Permethrind No 5.72 No 2.58 No 0.23 No 2.4
Storage temperature 4 °C Storage temperature 4 °C
Phorate No Values below working rangec Values below working rangec
Propyzamide 2 0.16 No 0.76 No 5.10 Yes
Diazinon No 0.81 No 1.25 No 8.92 Yes
Vinclozolin No 0.91 No 1.45 No 7.60 Yes
Chlorpyrifos-methyl No 1.22 No 1.26 No Values below working rangec
Metalaxyl No 0.13 No 0.82 No 3.42 No 15.8
Pirimifos-methyl 1 1.73 No 2.10 No 4.85 Yes
Chlorpyrifos No 0.19 No 1.29 No 0.85 No 9.6
Parathion 1 0.24 No 1.17 No 5.56 Yes
Bromopropylate 2 0.64 No 2.07 No 0.37 No 14.5
Permethrind No Values below working rangec No Values below working rangec
Storage temperature 18 °C Storage temperature 18 °C
Phorate Values below working rangec Values below working rangec
Propyzamide No 2.86 Yes No 4.30 Yes
Diazinon No 7.99 Yes No 6.93 Yes
Vinclozolin No 4.61 Yes No 5.31 Yes
Chlorpyrifos-methyl No 8.03 Yes No Values below working rangea
Metalaxyl 1 0.57 No 1.16 No 0.37 No 15.9
Pirimifos-methyl No 5.12 Yes No Values below working rangec
Chlorpyrifos No 2.00 No 3.98 No Values below working rangec
Parathion No 5.53 Yes No Values below working rangec
Bromopropylate No Values below working rangec No Values below working rangec
Permethrind No Values below working rangec No Values below working rangec
a
usts, uncertainty of short-term stability.
b
umts, uncertainty of mid-term stability.
c
Result below 0.25  MRL (outside method working range) for one or more time points, which points at substantial degradation of analyte.
d
Sum of cis and trans isomer.

to the two individual outliers detected for these pesticides. Individ- ance (ANOVA) as described by Linsinger et al. (2001b). In the cases
ual data and bottle means showed normal or at least unimodal dis- where either outlying bottle means were detected, or a trend in the
tribution in all cases. Therefore, the uncertainty contribution from filling sequence was found, a more conservative approach was
possible heterogeneity could be estimated by an analysis of vari- taken to estimate the uncertainty contribution, as described
572 H. Saldanha et al. / Food Chemistry 132 (2012) 567–573
elsewhere (Linsinger et al., 2009); results are summarized in
Table 2. The estimated uncertainty contributions of less than 7%
in all cases (sbb or ubb ) point at an acceptable level of inhomogene-
ity in both materials, which slightly lower values for the frozen
material. Some higher values (e.g. metalaxyl) are most likely to
come from the worse method repeatability compared to other
pesticides. The homogeneity established is suitable for the
intended use.
3.4. Stability
Isochronous studies were performed, whereby the materials
were stored at different temperature–time intervals and then kept
at the reference temperature until measurements took place. A few
outliers were found, but retained as no technical reason for their
occurrence could be found. Table 3 summarizes the results of the
short-term and mid-term stability study for the frozen material,
and Table 4 summarizes the results of the short-term and mid-
term stability study for the freeze-dried material. For the frozen
material, no pesticide showed an indication of instability when
stored at 20 °C for 4 weeks. In two cases (bromopropylate, per-
methrin), a positive trend was observed. This can be regarded as
an analytical artefact, as the content of the pesticides cannot in-
crease in the material. The results of the mid-term stability study
(i.e. no sign of instability for any pesticide in the material) con-
firmed those of the short-term stability. For the freeze-dried mate-
rial, some outliers were detected in the two studies, but retained as
no technical reason was found to exclude them. At 20 °C, both
short-term and long-term study indicated stability of the investi-
gated pesticides in the material. At 4 °C however, the short-term
stability study indicated stability of all pesticides except phorate
and permethrin. For these two pesticide, the obtained values were
below the low end of the working range of the method
(0.25  MRL), indicating substantial degradation of these com-
pounds. Consequently, no quantitative result could be indicated.
When the material is stored up to 5 months at this temperature
however, six more pesticides showed significant degradation, and
only three of the 11 pesticides (metalaxyl, chlorpyrifos, bromopro-
pylate) appeared to be stable. When stored at 18 °C, only metalaxyl
and chlorpyrifos did not show any measurable degradation after
4 weeks, and only metalaxyl appeared to be stable when stored
for 5 months. In conclusion, the freeze-dried material has to be
kept under frozen conditions for long-term storage, but dispatch
under cooled conditions to customers should be feasible without
impeding the integrity of the material.
The obtained results in the stability studies for the 11 pesticides
in the carrot/potato matrix basically confirm stability data re-
ported for the neat substances reported in the Pesticide Manual
(Tomlin, 2003). However, it has to be stressed that stability of pes-
ticides are depending on a number of parameters, including the
type of matrix and interactions of pesticides with the matrix, tem-
perature, pH value, water content of the matrix, processing type,
extent, etc. Consequently, extrapolation of existing stability data
to other, even similar matrices in terms of proximate composition
and/or to chemically similar pesticides is therefore not an option,
and dedicated stability studies for each pesticide/matrix combina-
tion are required.
4. Conclusion and outlook
This feasibility study towards the preparation of a matrix refer-
ence material for selected pesticides in a food matrix revealed that
two processing types (freezing, freeze-drying) are suitable. How-
ever, the abundance of analyte losses due to processing was typi-
cally higher in the freeze-dried material. In order to compensate
for the losses during processing and to achieve pesticide mass
fractions around the respective MRLs in the RM, a higher pesticide
amount has to be spiked into the matrix. Both the frozen and the
freeze-dried materials exhibit suitable homogeneity, and stability
studies demonstrated that no detectable instability for the pesti-
cides was found for a storage temperature of 20 °C. At 4 °C how-
ever, most of the investigated pesticides appeared to degrade in
the freeze-dried material. These findings constitute important
information for the aim to process and certify a reference material
for selected pesticides in a food matrix. Nevertheless, additional
work needs to be carried out before starting the processing and
certification of a RM: this involves (i) further improvement of the
GC–MS method performance, especially repeatability (this could
be accomplished by using isotopically-labelled internal standards
whenever available), (ii) to carry out a long-term stability study
(e.g. 24 months) with a larger number of samples and measure-
ments to allow a solid estimation of ults values, and (iii) the evalu-
ation of currently available validated methods for their suitability
in a characterisation intercomparison study. Moreover, as regards
the freeze-dried material, the freeze-drying process could be fur-
ther optimised concerning minimum analyte loss. Last but not
least, a commutability study should be performed to confirm that
the freeze-dried material does not behave substantially different
to the frozen material.
Acknowledgements
The authors gratefully acknowledge the contributions of Katha-
rina Teipel and Håkan Emteborg for guidance and assistance in
processing the materials.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.foodchem.2011.10.071.
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