FprEN 18082

SLOVENSKI STANDARD
oSIST prEN 18082:2024
01-julij-2024
Živila živalskega izvora - Multimetoda za določanje ostankov pesticidov z uporabo
LC-analize po acetonitrilni ekstrakciji/delitvi in čiščenju z disperzijsko SPE
Foods of animal origin - Multimethod for the determination of pesticide residues using
LC-based analysis following acetonitrile extraction/partitioning and clean-up by
dispersive SPE
Tierische Lebensmittel - Multiverfahren zur Bestimmung von Pestizidrückständen mit LC
nach Acetonitril-Extraktion/Verteilung und Reinigung mit dispersiver SPE
Ta slovenski standard je istoveten z: prEN 18082
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
67.120.01 Živila živalskega izvora na Animal produce in general
splošno
oSIST prEN 18082:2024 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN 18082:2024
oSIST prEN 18082:2024
DRAFT
EUROPEAN STANDARD
prEN 18082
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2024
ICS 67.050; 67.120.01
English Version
Foods of animal origin - Multimethod for the
determination of pesticide residues using LC-based
analysis following acetonitrile extraction/partitioning and
clean-up by dispersive SPE
Tierische Lebensmittel - Multiverfahren zur
Bestimmung von Pestizidrückständen mit LC nach
Acetonitril-Extraktion/Verteilung und Reinigung mit
dispersiver SPE
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 275.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 18082:2024 E
worldwide for CEN national Members.

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prEN 18082:2024(E)
Contents Page
European foreword . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
5 Preparation and storage of the samples . 5
5.1 General. 5
5.2 Laboratory sample . 6
5.3 Partly-prepared test sample . 6
5.4 Test sample . 6
5.5 Test portion . 6
6 Procedure . 6
7 Evaluation of results . 7
7.1 Identification and quantification . 7
7.2 Calibration . 8
7.3 Calculation of residue concentration. 8
7.4 Validity of the method . 9
8 Precision . 11
9 Test report . 11
Annex A (informative) Description of modules . 12
A.1 Reagents used in extraction (E) and clean-up (C) modules . 12
A.2 Apparatus used in extraction (E) and clean-up (C) modules . 13
A.3 Description of extraction (E) module . 14
A.3.1 Module E: Extraction of a 5 g test portion with additional water using acetonitrile . 14
A.4 Description of clean-up (C) modules . 16
A.4.1 Module C1: Clean-up by freezing-out of co-extracted fat . 16
A.4.2 Module C2: Clean-up by dispersive SPE with amine sorbent (PSA) . 17
A.5 Description of detection (D) modules . 18
A.5.1 Module D: Determination using liquid chromatography with mass spectrometric detection
(LC-MS) . 18
A.6 Description of quantification (Q) options . 22
A.6.1 Option Q1: Quantification using external standards in solvent . 22
A.6.2 Option Q2: Quantification using external standards in matrix . 22
A.6.3 Option Q3: Quantification using a procedural internal standard and standards in solvent
............................................................................................................................................................................. 23
A.6.4 Option Q4: Quantification using standard addition to the final extract . 23
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A.6.5 Option Q5: Quantification using a procedural internal standard and standards in matrix or
isotope-labelled internal standards . 24
A.6.6 Option Q6: Quantification using standard addition to the sample . 25
A.6.7 Option Q7: Quantification by calibration of the entire procedure . 25
Annex B (informative) Complementary information . 26
B.1 General . 26
B.2 Preparation of the buffer-salt mixture (A.1.8) . 26
B.3 Reagents for clean-up. 26
B.4 Prolongation of extraction time . 26
B.5 Working with internal standards . 26
B.6 Scaling . 26
B.7 Validation data . 26
Annex C (informative) Abbreviations and symbols. 42
Bibliography . 44
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European foreword
This document (prEN 18082:2024) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
WARNING — The use of this document can involve hazardous materials, operations and equipment. This
document does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this document to establish appropriate safety and health practices and
determine the applicability of regulatory limitations prior to use.
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prEN 18082:2024(E)
1 Scope
This document specifies a method for the analysis of pesticide residues in foods of animal origin with a
fat content of up to approximatively 10 %, such as meat, egg or milk by LC-MS/MS. Because of the low
material requirements for miniaturized processing and the few work steps, the process is particularly
time and cost-saving with high reliability and effectiveness. The method has been collaboratively studied
on a number of commodity/pesticide combinations. Precision data are summarized in Table B.1.
Guidelines for calibration are outlined in CEN/TS 17061.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
CEN/TS 17061, Foodstuffs — Guidelines for the calibration and quantitative determination of pesticide
residues and organic contaminants using chromatographic methods
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
Water is added to the homogeneous sample and is extracted with the help of acetonitrile. After addition
of magnesium sulfate, sodium chloride and buffering citrate salts, the mixture is shaken intensively and
centrifuged for phase separation. An aliquot of the organic phase is cleaned-up by dispersive solid phase
extraction (D-SPE) with amino-sorbents (e.g. primary secondary amine sorbent, PSA) as well as
magnesium sulfate for the removal of residual water. Following clean-up extracts are centrifuged. Fat
contained in the sample is removed by freezing out by storing the purified acetonitrile extract in the
freezer. The pesticides contained in the fat-free solution can be determined directly by LC-based analysis.
For the analysis with LC hyphenations with tandem mass-spectrometry (LC-MS/MS) or high resolution
mass-spectrometry (LC-HR-MS) are particularly suitable. Quantification may be performed using an
internal standard, which is added to the test portion before the first extraction, but this is not mandatory.
Details for calibration, see 7.2.
5 Preparation and storage of the samples
5.1 General
Sample processing and storage procedures should be demonstrated to have no significant effect on the
residues present in the test sample (sometimes also called “analytical sample”). Processing should also
ensure that the test sample is homogeneous enough so that portion to portion (sub-sampling) variability
is acceptable. If a single analytical portion is unlikely to be representative of the test sample, larger or
replicate portions shall be analysed, to provide a better estimate of the true value. The degree of
comminution should support a quantitative residue extraction. Otherwise, the extraction shall be carried
out with the aid of a mechanical shredding device (e.g. a homogenizing rod).
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5.2 Laboratory sample
A laboratory sample that is wholly or extensively spoiled or degraded should not be analysed. When
possible, prepare laboratory samples immediately after arrival and in any event, before any significant
physical or chemical changes have taken place. If a laboratory sample cannot be prepared without delay,
it should be stored under appropriate conditions to keep it fresh and to avoid deterioration. In general,
laboratory samples should not be stored for more than 3 days in total, with the exception of dried or
processed samples that are to be analysed within the specified minimum shelf life.
5.3 Partly-prepared test sample
For preparation of the partly-prepared test sample take only the portion of the laboratory sample to
which the maximum residue level applies, e.g. bones or shells should be removed.
5.4 Test sample
Where the homogeneity of the test sample is not sufficient or the extraction of residues could be
significantly compromised due to large particle sizes, intensive comminution should be performed using
appropriate means. This is possible at ambient temperature, if separation of flesh and juice or
degradation of target pesticides does not occur to a significant extent. Comminution of samples in a frozen
state can significantly reduce losses of chemically labile pesticides and usually results in smaller particle
sizes and a higher degree of homogeneity. Cutting the samples coarsely (e.g. 3 cm × 3 cm) with a knife
and putting them into the freezer (e.g. −18 °C overnight) prior to comminution facilitates processing.
Processing can be also assisted and improved by cryogenic milling (using dry ice or liquid nitrogen) by
keeping the temperature below 0 °C. When processing test samples at low temperatures, condensation
caused by high humidity should be avoided. Residual carbon dioxide should be allowed to sufficiently
dissipate so that its contribution to weigh of the sample will be negligible.
5.5 Test portion
Individual test portions each sufficient for one analysis should be taken from the comminuted test
sample. These test portions should be analysed immediately. If test portions cannot be analysed directly,
the test sample or the test portions shall be frozen until required. If it is noted that homogeneity of the
test sample has been compromised during storage, the test sample shall be mixed before taking test
portions to ensure that homogeneity has been re-established.
6 Procedure
Extraction of samples is specified in module E. Extraction is usually followed by a clean-up of the obtained
raw extracts (Freezing out of the fat module C1 and /or cleaning with PSA module C2) and analysed by
module D. All modules are described in detail in Annex A. Complementary information is given in
Annex B.
Table 1 contains brief descriptions of the modules as well as application notes and examples of use. For
the calculation of residue concentrations in the sample extracts all of the calibration procedures and
quantification methods described in options Q1 to Q7 are applicable.
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Table 1 — Overview of possible modules
Module Description Preferred application
E After addition of water a test portion of Meat, milk and eggs with a fat content of up to
5 g is extracted with acetonitrile in the approx. 10 %
presence of buffering salts
C1 Freezing-out Removal of co-extracted fat (even in combination
with further clean-up steps, e.g. C2)
C2 Dispersive SPE with amino-sorbent Clean-up of raw-extracts prior to the
(PSA) determination of basic and neutral pesticides
D LC-MS Extracts from module E subsequently cleaned-up
with modules C1 to C2
Q1 Quantification using external standards Determinations where matrix-effects are
in solvent assumed to be negligible
Q2 Quantification using external standards Determinations where matrix-effects shall be
in matrix considered
Q3 Quantification using a procedural Determinations where matrix-effects are
internal standard and standards in assumed to be negligible
solvent
Q4 Quantification using standard addition Determinations where matrix effects shall be
to the final extract considered and suitable blank matrices are not
available
Q5 Quantification using a procedural Determinations where matrix-effects shall be
internal standard and standards in considered for compensation of low recovery
matrix or isotope-labelled internal
standards
Q6 Quantification using standard addition Determinations where matrix-effects shall be
to the sample considered without availability of blank (control)
samples or incomplete extractions of the analyte
occur
Q7 Quantification by calibration of the Determinations where matrix-effects shall be
entire procedure considered or incomplete extractions of the
analyte occur
7 Evaluation of results
7.1 Identification and quantification
A number of parameters can be employed to determine the identity of an analyte present in the sample
extract. This includes:
— retention time of the analyte in question (R ) or, even better, the retention time ratio against the ISTD
t
(R /R ) obtained from the same run;
t(A) t(ISTD)
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— in case of MS or MS/MS detection, the relative abundance of the recorded masses or transitions
respectively (in general 2 selected reaction monitoring (SRM) transitions are required in MS/MS and
3 ions in MS applications), see also [1], [2], [3], [4];
— characteristic peak shape/pattern of the analyte.
The parameters obtained for the analyte to be identified in the sample extract are compared with those
obtained for the pesticides in the calibration solution(s). Should a higher degree of certainty be required
for the confirmation of the analyte identity, additional measures may be necessary, such as the use of
different chromatographic separation conditions or the evaluation of additional m/z or SRM transitions.
For more information about the required confirmation criteria (e.g. the recommended maximum
tolerances for ion ratios using different MS techniques) see the guidelines described in EU Quality Control
Procedures [2]. Table A.1 gives a list of the ISTDs that can be employed. The use of more than one ISTD
will provide some backup information.
Use standard solutions to check linearity and to determine the calibration functions for each analyte. The
use of matrix-matched standards is to be preferred, however, for a first estimate of the residue level of
pesticides in the food or to show their absence, the standard solutions in pure solvent can be used. They
can be also used for quantification if preliminary experiments indicate that any suppression or
enhancement effects experienced do not significantly affect the results obtained. As soon as relevant
residue concentrations are detected (e.g. suspected maximum residue level (MRL) violations), a more
precise determination using matrix-matched standards or the standard addition method should be used.
NOTE Matrix effects influence the response of target analytes in sample extracts compared to the response of
standard solutions in pure solvent.
The calibration range should be appropriate to the residue concentrations to be quantified. Thus, it may
be necessary to construct more than one calibration graph from the results of calibration measurements.
This standard contains the option to use an internal standard for quantification and identification.
Nevertheless, it is still possible to quantify without ISTD. Without ISTD, the volume of the acetonitrile
phase is assumed to be identical to the volume of acetonitrile added to the sample (10 ml).
In this method, the internal standard is basically used for process control of sample preparation, but by
default it is not used for quantification. However, there is the option to use an internal standard for
quantification, for which the validity shall be ensured.
7.2 Calibration
The analytical method shall be calibrated according to CEN/TS 17061. In addition, it may be calibrated
according to the EU Quality Control Procedures [2]. A suitable calibration procedure should be selected
from one of the quantification options Q1 to Q7 in A.6.
7.3 Calculation of residue concentration
The mass fraction w of each identified analyte depends on the mass concentration of the sample in the
A
final extract
final extract ρ after application of clean-up (modules C1 to C2) and the concentration ρ of the
A
sample
substance in this extract (determined according to one of the options Q1 to Q7 given in A.6). It is
expressed in mg/kg and is calculated by Formula (1).
ρ
A
w = (1)
A
final extract
ρ
sample
where
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is the mass concentration of the analyte in the sample extract (option Q, see A.6.1.3,
ρ
A
A.6.2.3, A.6.3.3, A.6.5.3, and A.6.7.3) in µg/ml;
final extract
is the mass concentration of the sample in the final extract (modules C, see A.4.1.3,
ρ
sample
A.4.2.3), in g/ml.
7.4 Validity of the method
The recoveries obtained from experiments (spiking levels 0,01 mg/kg or 0,05 mg/kg to 0,1 mg/kg) were
usually between 70 % and 110 %.
Interlaboratory method validation studies covered a multitude of analytes using representative
commodities (meat, egg and milk). Furthermore extensive individual validation has been carried out. All
validation data provided by laboratories are published in the Data Pool of the European Union Reference
Laboratories [3].
Validity of the method is confirmed for any specific commodity/pesticide combination if at least four
laboratories conducted independently validation studies with the same matrix at two fortification levels
with at least five replicates per level and obtained a recovery between 70 % and 120 %. Furthermore, the
relative standard deviation had to be below or equal 20 % for both spiking levels in each laboratory.
For the respective calculation of the relative standard deviation, each laboratory had to have at least 5
additional tests for each concentration level. Based on these conditions, it was found that sufficient
validation data are available for 41 pesticides or pesticide metabolites. An overview is shown in Table 2.
In addition to the active substances listed in Table 2, fenpropimorph, the metabolites flusilazole-IN-
F7321 and ioxynil octanoate were also investigated. These substances could not be validly analysed with
the presented method.
Detailed validation results are given in Table B.1. This table also contains successful validations which
have not been carried out by at least four laboratories for the same analyte-matrix combination or for
which values are not available for at least two concentration levels. Table B.1 contains also validation
data for analyte-matrix combinations with average recoveries and/or RSD outside the acceptable range.
This data should be considered as supportive information.
The limit of quantification of an analyte depends on the performance of the analytical equipment used
and the sample matrix. With modern analytical equipment, residues at 0,01 mg/kg (in most cases the
lowest maximum residue level) can be typically analysed (see Table B.1).
Table 2 shows the confirmed validation data of the method for specific analytes and commodity groups.
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Table 2 — Validation data
No. Commodity group (see [2])
a
Meat Milk and Eggs
Analyte CAS-No.
(muscle) milk
b
and products
seafood
1 Azinphos-ethyl 2642–71–9 x x x
2 Bifenthrin 82657–04–3 x x
3 Bixafen 581809–46–3 x x x
4 Boscalid 188425–85–6 x x x
5 Boscalid-M 510F01 - x x x
6 Carbendazim 10605–21–7 x x x
7 Chlorpyrifos 2921–88–2 x x
8 Chlorpyrifos-methyl 5598–13–0 x x
9 Cyfluthrin 68359–37–5 x x
10 Cypermethrin 52315–07–8 x x x
11 Deltamethrin 52918–63–5 x x
12 Diazinon 333–41–5 x x x
13 Etofenprox 80844–07–1 x x
14 Famoxadone 131807–57–3 x x x
15 Fenthion 55–38–9 x x x
16 Fenthion-oxon 6592–12–1 x x x
17 Fenthion-oxon-sulfone 14086–35–2 x x x
18 Fenthion-oxon-sulfoxid 6552–13–2 x x x
19 Fenthion-sulfone 3761–42–0 x x x
20 Fenthion-sulfoxid 3761–41–9 x x x
21 Fenvalerate/Esfenvalerate 51630–58–1 x x
22 Fluopyram 658066–35–4 x x x
23 Fluquinconazole 136426–54–5 x x x
24 Flusilazole 85509–19–9 x x x
25 Indoxacarb 144171–61–9 x x x
26 Ioxynil 1689–83–4 x x x
27 Metaflumizone 139968–49–3 x x x
28 Methidathion 950–37–8 x x x
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No. Commodity group (see [2])
a
Meat Milk and Eggs
Analyte CAS-No.
(muscle) milk
b
and products
seafood
29 Paraoxon-methyl 950–35–6 x x x
30 Parathion-methyl 298–00–0 x x x
31 Permethrin 52645–53–1 x x
32 Pirimiphos-methyl 29232–93–7 x x x
33 Prochloraz 67747–09–5 x x x
34 Profenofos 41198–08–7 x x x
35 Pyrazophos 13457–18–6 x x x
36 Resmethrin 10453–86–8 x x
37 Spiroxamine 118134–30–8 x x x
38 Tau-Fluvalinate 102851–06–9 x x x
39 Tetraconazole 112281–77–3 x x x
40 Thiophanate-methyl 23564–05–8 x x x
41 Triazophos 950–35–6 x x x
a
Pork with a fat content of 10 % was used for the validation.
b
Milk with a fat content of 3,8 % was used for the validation.
8 Precision
Details of the inter-laboratory test of the precision of the method are summarized in Table B.1. The values
derived from the inter-laboratory test are not be applicable to pesticide concentration ranges and
commodities other than given in Table B.1.
9 Test report
The test report shall contain at least the following:
— all information necessary for the identification of the sample;
— a reference to this European Standard (including its year of publication);
— the results and the units in which the results have been expressed;
— the date and type of sampling procedure (if possible);
— the date of receipt of sample in the laboratory;
— the date of test;
— any particular observations made in the course of the test;
— any operations not specified in the method or regarded as optional which might have affected the
results.
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Annex A
(informative)
Description of modules
A.1 Reagents used in extraction (E) and clean-up (C) modules
Unless otherwise specified, use reagents of recognized analytical grade. Take every precaution to avoid
possible contamination of water, solvents, sorbents, inorganic salts, etc.
A.1.1 Internal standard and quality control standard solutions in acetonitrile, ρ = 10 µg/ml to
50 µg/ml.
Table A.1 shows a list of potential internal standards (ISTDs) and quality control (QC) standard solutions
that may be used in this method. The suggested concentration values (C ) listed refer to the ISTD
ISTD
solutions that should be added at the first extraction step. An appropriate dilution of this solution
cal mix
( C ) should be prepared to be used for the preparation of the standard solutions.
ISTD
A.1.2 Acetonitrile, HPLC quality.
A.1.3 Magnesium sulfate, anhydrous, grit.
Phthalates may be removed in a muffle furnace by heating to 550 °C (e.g. overnight).
A.1.4 Sodium chloride.
A.1.5 Disodium hydrogencitrate sesquihydrate.
A.1.6 Trisodium citrate dihydrate.
A.1.7 Buffer-salt-mixture for second extraction and partitioning.
Weigh 4 g ± 0,2 g of anhydrous magnesium sulfate (A.1.3), 1 g ± 0,05 g of sodium chloride (A.1.4),
1 g ± 0,05 g of trisodium citrate dihydrate (A.1.6) and 0,5 g ± 0,03 g of disodium hydrogencitrate
sesquihydrate (A.1.5) into a centrifuge tube (A.2.4). This amount refers to approximately 10 ml water in
the sample. Commercially available salt mixtures of identical composition can be used.
NOTE It is advisable to prepare a sufficient number of buffer-salt-mixtures in advance so that extraction series
can be performed quickly without interruption.
A.1.8 Cold water.
A.1.9 Dry ice.
A.1.10 Liquid nitrogen.
A.1.11 Magnesium sulfate, anhydrous, fine powder, phthalates may be removed in a muffle furnace
by heating to 550 °C (e.g. overnight).
A.1.12 Primary secondary amine sorbent.
Other amine sorbents can be used, but investigations are necessary to prove equivalency especially
regarding analyte losses and pH value of the final extracts.
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Table A.1 — Potential internal standards (ISTDs) or quality control (QC) standards
b
LC
ρ
Chlorine
a
[µg/ml]
Compound Log P
MS/MS MS/MS
atoms
C
ISTD
ESI (+) ESI (-)
Potential internal standards
f d f
Triphenylphosphate 4,59 20
d e
Tris-(1,3-dichlorisopropyl)-phosphate 3,65 6 50
13 f d
2,4-D C6 (ring) depends 2 10
on pH
d f
Chlorpyrifos D10 (diethyl D10) 4,7 3 10
d f
Diuron D6 2,9 2 10
(dimethyl D6)
f d f
Diazinon D10 3,8 20
(diethyl D10)
d f
Metalaxyl D6 1,65 0 10
(dimethyl D6)
c f f d
N,N´-Bis-4-nitrophenyl) urea (BNPU) 3,76 10
Potential Quality Control Standards (may be contained in the same mixture as the other ISTDs used
or added at a different stage of analysis to detect and localize sources of error)
a
octanol-water partition coefficient.
b
proposed concentration; solvent: acetonitrile.
c
component of nicarbazin.
d
very good detectable.
e
poor detectable.
f
not applicable.
More generic ISTDs can be used, however the lab has to show, that applied ISTDs are fit for purpose.
A.2 Apparatus used in extraction (E) and clean-up (C) modules
Usual laboratory apparatus and, in particular, the following.
A.2.1 Centrifuge tubes with screw caps, 50 ml.
For example 50 ml centrifuge tubes made of poly-tetrafluoroethylene with screw caps, or disposable
50 ml polypropylene centrifuge tubes with screw caps.
A.2.2 Solvent-dispenser for acetonitrile, capacity 10 ml, to be employed for dosing of extraction
solvent.
A.2.3 Automatic pipettes, suitable for handling volumes of 10 µl to 100 µl, 200 µl to 1 000 µl and 1 ml
to 10 ml, or 10 ml graduated glass pipettes.
A.2.4 Polypropylene-single use centrifuge tubes with screw caps, 10 ml or 12 ml.
−1
A.2.5 Shaker, horizontal, vertical or orbital shaker, at least 200 min
.
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A.2.6 Centrifuges, suitable for the centrifuge tubes given in (A.2.1, A.2.4) and capable of achieving at
least 3 000 g.
A.2.7 High speed dispersing device, the diameter of the dispersing elements fitting the openings of
the centrifuge tubes (A.2.1) used.
A.2.8 Grinding device.
A.2.9 Freezer, operating at −18 °C to −25 °C.
A.2.10 Injection vials, 1,5 ml, suitable for LC autosampler, if necessary with micro-inserts.
A.2.11 Screw capped glass vials, 10 ml or 20 ml, for the storage of excessive amounts of the final
extract, if necessary.
A.2.12 Vortex mixer.
A.3 Description of extraction (E) module
A.3.1 Module E: Extraction of a 5 g test portion with additional water using acetonitrile
A.3.1.1 Principle
After water addition the homogenized test material is extracted with acetonitrile. After adding
magnesium sulfate, sodium chloride and citrate salts for buffering, the mixture is shaken vigorously again
and then centrifuged. The acetonitrile phase is separated off and purified (Module C).
A.3.1.2 Procedure
A.3.1.2.1 Test portion
Transfer a representative test portion of 5 g ± 0,1 g (m ) of the comminuted homogenous sample into
sample
a 50 ml centrifuge tube (A.2.1). Add 5,0 ml of water (A.1.8).
A.3.1.2.2 ISTD addition
add
Add a defined small volume of the ISTD solution (A.1.1) ( , e.g. 100 µl) containing one or several of
V
ISTD
the compounds listed in Table A.1 at the concentrations exemplary given (C )
ISTD
A.3.1.2.3 First extraction
Add 10 ml of acetonitrile (A.1.2) (V ). Close the tube and shake samples having room temperature
Ex
vigorously for 1 min to 3 min or frozen samples for 15 min, using a shaker (A.2.5) if necessary.
Samples should be extracted frozen or while in the process of thawing. If samples are employed for
extraction at ambient temperature, it shall be ensured that no significant degradation of the target
pesticides occurs. Extended extraction time may be used if shown that it does not affect significantly the
recovery rate.
If the sample’s degree of comminution is insufficient, the extraction can be assisted by a high-speed
disperser (A.2.7). In this case the addition of an ISTD prior to the dispersing step is mandatory. The
dispersing element is immersed into the sample/acetonitrile mixture and comminution is performed for
about 2 min at high speed. The dispersing element shall be thoroughly cleaned before being used for the
next sample to avoid cross-contamination.
oSIST prEN 18082:2024
prEN 18082:2024(E)
A.3.1.2.4 Second extraction step and partitioning
Add the prepared buffer-salt mixture (A.1.7) to the suspension from A.3.1.2.3 Close the tube and
immediately shake vigorously for some seconds to avoid the formation of clumps. Thereafter, shake again
between 1 min and 20 min by a hand or shaker (A.2.5) and centrifuge for 5 min at > 3 000 g.
Without delay separate upper acetonitrile layer and pass the obtained raw extract to the clean-up
modules C.
In the presence of water, magnesium sulfate tends to form lumps, which can harden rapidly. This can be
avoided, if immediately after the addition of the salt mixture the centrifuge tube is shaken vigorously for
few seconds. The 1 min or 3 min extraction of the entire batch can be performed in parallel after the salts
were added to all the samples.
A.3.1.3 Calculation
raw extract
The mass concentration of the sample in the raw extract gives the ratio of the amount of the
ρ
sample
raw extract
sample, from which the residues are extracted, and the extraction volume. Usually ρ is given in
sample
g/ml and is calculated by Formula (A.1):
m
sample
raw extract
ρ = (A.1)
sample
V
Ex
where
is the mass of test portion, in g;
m
sample
is the volume of the organic phase after extraction (usually 10 ml), in ml.
V
Ex
raw extract
Calculate the mass concentration of ISTD in the raw extract ρ given in µg/ml with Formula (A.2):
sample
add add
ρ × V
raw extract ISTD ISTD
ρ = (A.2)
ISTD
V
Ex
where
add
is the mass concentration of added ISTD solution, in µg/ml;
ρ
ISTD
add
is the volume of ISTD added to the sample, in ml;
V
ISTD
is the volume of the organic phase after extraction (usually 10 ml), in ml.
V
Ex
A.3.1.4 Workflow of module E
Step 1:  Add 5 ml of water to the test portion (5 g ± 0,1 g)
Step 2:  Optional add ISTD to the test portion (e.g 100 µl)
Step 3:  Add 10 ml of acetonitrile to the test portion
Step 4:  Shake samples having room temperature vigorously for 1 min to 3 min or frozen samples
for 15 min
oSIST prEN 18082:2024
prEN 18082:2024(E)
Step 5:  Add buffer-salt mixture and immediately shake vigorously for some seconds to avoid the
formation of clumps. Thereafter, shake again between 1 min and 20 min by a hand or
shaker.
Step 6:  Centrifuge for 5 min at > 3 000 g.
Step 7:  Separate upper acetonitrile layer for use in modules C
A.4 Description of clean-up (C) modules
A.4.1 Module C1: Clean-up by freezing-out of co-extracted fat
A.4.1.1 Principle
This clean-up module C1 is used in combination with other clean-up modules (C2). The extract obtained
by the modules E is freed from fat by freezing-out.
A.4.1.2 Procedure
Transfer an aliquot of 6 ml of the acetonitrile phase obtained by modules E into a centrifuge tube (A.2.4)
and store at least 4 h in a freezer (A.2.9), where the major part of fat and waxes solidify and precipitate.
Following a short centrifugation (where necessary), 4 ml of the still cold extract is taken for
measurement.
NOTE Freezing out also helps to partly remove some additional sample co-extractives with limited solubility
in acetonitrile.
To reduce matrix effects an aliquot of the raw extract (V ) can be diluted with a defined volume of an
A
appropriate solvent (V ) to resulting total volume (V + V ) if the sensitivity of the used detection system
2 A B
is sufficient.
A.4.1.3 Calculation
final extract
The mass concentration of the final extract gives the ratio of the amount of the sample, from
ρ
sample
final extract
which the residues are extracted, and the extraction volume. Usually, ρ is given in g/ml and is
sample
calculated with Formula (A.21):
V
final extract raw extract
A
ρρ × (A.3)
sample sample
VV+
( )
AB
where
raw extract
mass concentration of the raw extract, in g/ml;
ρ
sample
is the used aliquot of the raw extract, in ml;
V
A
is the added volume of an appropriate solvent, in ml.
V
B
final extract
The mass concentration of ISTD in the final extract ρ given in µg/ml is calculated with
sample
Formula (A.22):
add add
ρ × V
V
final extract ISTD ISTD A
(A.4)
ρ ×
ISTD
V
VV+
( )
Ex
AB
=
=
oSIST prEN 18082:2024
prEN 18082:2024(E)
where
add
is the mass concentration of added ISTD solution, in µg/ml;
ρ
ISTD
add
is the volume of ISTD added to the sample, in ml;
V
ISTD
is the volume of the organic phase after extraction (usually 10 ml), in ml;
V
Ex
is the used aliquot of the raw extract, in ml;
V
A
is the added volume of an appropriate solvent, in ml.
V
B
A.4.1.4 Workflow of module C1
Step 1:  Transfer 6 ml of the raw extract into a centrifuge tube and store at least 4 h in a freezer
Step 2:  Centrifugation (where necessary) and take 4 ml of the still cold extract
Step 3:  Dilute (if applicable) and use in module C2 or D
A.4.2 Module C2: Clean-up by dispersive SPE with amine sorbent (PSA)
A.4.2.1 Principle
This clean-up module is used for extracts obtained by the module C1 for the determination of neutral and
alkaline pesticides in all commodities not mentioned further.
The extract obtained by the module C1 is cleaned by dispersive solid phase extraction (SPE) using
primary secondary amine sorbent (PSA).
A.4.2.2 Procedure
An aliquot of 4 ml of the acetonitrile phase obtained by the module C1 is transferred into a Polypropylene-
single use tube (A.2.4) already containing 100 mg PSA (A.1.12) and 600 mg of magnesium sulfate
(A.1.11). Close the tube, shake vigorously for 30 s using a shaker (A.2.5) if necessary and centrifuge for
5 min at > 3 000 g. Immediately isolate the clear extract.
For 1 ml of extract 150 mg magnesium sulfate and 25 mg PSA are necessary.
NOTE It is helpful to load the centrifuge tubes with the dispersive SPE sorbents before beginning the extraction
procedure needed for one batch of samples.
To reduce matrix effects an aliquot of the raw extract (V ) can be diluted with a defined volume of an
A
appropriate solvent (V ) to resulting total volume (V + V ) if the sensitivity of the used detection system
B A B
is sufficient.
A.4.2.3 Calculation
final extract
The mass concentration of the final extract ρ gives the ratio of the amount of the sample, from
sample
final extract
which the residues are extracted, and the extraction volume. Usually ρ is given in g/ml and is
sample
calculated with Formula (A.5):
V
final extract raw extract A
ρρ × (A.5)
sample sample
VV+
( )
AB
where
=
oSIST prEN 18082:2024
prEN 18082:2024(E)
raw extract
is the mass concentration of the raw extract, in g/ml;
ρ
sample
is the used aliquot of the raw extract, in ml;
V
A
is the added volume of an appropriate solvent, in ml
V
B
final extract
The mass concentration of ISTD in the final extract ρ given in µl/ml is calculated with
ISTD
Formula (A.6):
add add
ρ × V V
final extract ISTD ISTD A
(A.6)
ρ ×
ISTD
V
VV+
( )
Ex
AB
where
add
is the mass concentration of added ISTD solution, in µg/ml;
ρ
ISTD
add
is the volume of ISTD added to the sample, in ml;
V
ISTD
is the volume of the organic phase after extraction (usually 10 ml), in ml;
V
Ex
is the used aliquot of the raw extract, in ml;
V
A
is the added volume of an appropriate solvent, in ml.
V
B
A.4.2.4 Workflow of module C2
Step 1:  Transfer 4 ml of the extract into a centrifuge tube already containing 100 mg PSA and
600 mg of magnesium sulfate
Step 2:  Shake vigorously for 30 s and centrifuge for 5 min at > 3 000 g
Step 3:  Dilute (if applicable) and use in modules D
A.5 Description of detection (D) modules
A.5.1 Module D: Determination using liquid chromatography with mass spectrometric
detection (LC-MS)
A.5.1.1 Principle
The liquid chromatographic determination with mass spectrometric detection is suited for all analytes
extracted by module E, which are ionizable at atmospheric pressure. A clean-up using the modules C1 to
C2 is recommended, but not in every case necessary due to the high selectivity of the mass spectrometric
detection.
A.5.1.2 Procedure
Inject solutions from modules C1 or C2 into a liquid chromatographic system, which is hyphenated with
a tandem-mass spectrometer.
A.5.1.3 Apparatus
The measurement can be performed using various instruments, instrument parameters and columns.
Some instrument parameters and columns are listed in Table A.2 and Table A.3 in this module. The
following LC-MS operating conditions have been shown to be satisfactory, but they are only examples of
experimental conditions on the stated instruments. Variations of these conditions are
...