Foodstuff - Determination of pesticide residues by ethyl acetate extraction using GC- and LC-MS/MS (SweEt)

This draft European Standard describes a method for the analysis of pesticide residues in foods of plant and of animal origin by ethyl acetate extraction using GC- and LC-MS/MS (SweEt).

Lebensmittel - Bestimmung von Pestizidrückständen mit Ethylacetatextraktion durch GC- und LC-MS/MS (SweEt)

Dieses Dokument legt ein Verfahren zur Analyse von Pestizidrückständen in Lebensmitteln pflanzlichen und tierischen Ursprungs mittels Ethylacetatextraktion unter Verwendung von GC- und LC-MS/MS (SweEt) fest.

Produits alimentaires - Dosage des résidus de pesticides par extraction à l’acétate d’éthyle par GC- et LC-MS/MS (SweEt)

Ce document spécifie une méthode d’analyse des résidus de pesticides dans les aliments d’origine végétale et les aliments d’origine animale par extraction à l’acétate d’éthyle par GC- et LC-MS/MS (SweEt).

Živila - Določevanje ostankov pesticidov z ekstrakcijo z etil acetatom z uporabo GC- in LC-MS/MS (SweEt)

General Information

Status
Published
Public Enquiry End Date
19-Dec-2021
Publication Date
13-Apr-2022
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
01-Apr-2022
Due Date
06-Jun-2022
Completion Date
14-Apr-2022

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SLOVENSKI STANDARD
SIST-TS CEN/TS 17743:2022
01-maj-2022
Živila - Določevanje ostankov pesticidov z ekstrakcijo z etil acetatom z uporabo
GC- in LC-MS/MS (SweEt)

Foodstuff - Determination of pesticide residues by ethyl acetate extraction using GC- and

LC-MS/MS (SweEt)

Lebensmittel - Bestimmung von Pestizidrückständen mit Ethylacetatextraktion durch GC-

und LC-MS/MS (SweEt)

Produits alimentaires - Dosage des résidus de pesticides par extraction à l’acétate

d’éthyle par GC- et LC-MS/MS (SweEt)
Ta slovenski standard je istoveten z: CEN/TS 17743:2022
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST-TS CEN/TS 17743:2022 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST-TS CEN/TS 17743:2022
---------------------- Page: 2 ----------------------
SIST-TS CEN/TS 17743:2022
CEN/TS 17743
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
March 2022
TECHNISCHE SPEZIFIKATION
ICS 67.050
English Version
Foodstuff - Determination of pesticide residues by ethyl
acetate extraction using GC- and LC-MS/MS (SweEt)

Produits alimentaires - Dosage des résidus de Lebensmittel - Bestimmung von Pestizidrückständen

pesticides par extraction à l'acétate d'éthyle par GC- et mit Ethylacetatextraktion durch GC- und LC-MS/MS

LC-MS/MS (SweEt) (SweEt)

This Technical Specification (CEN/TS) was approved by CEN on 7 February 2022 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to

submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS

available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in

parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

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, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17743:2022 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST-TS CEN/TS 17743:2022
CEN/TS 17743:2022 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

1 Scope .................................................................................................................................................................... 4

2 Normative references .................................................................................................................................... 4

3 Terms and Definitions ................................................................................................................................... 4

4 Principle .............................................................................................................................................................. 4

5 Reagents .............................................................................................................................................................. 5

6 Apparatus ........................................................................................................................................................... 7

7 Procedure ........................................................................................................................................................... 8

8 Evaluation of results .................................................................................................................................... 10

9 Precision .......................................................................................................................................................... 11

10 Test report ...................................................................................................................................................... 12

Annex A (informative) Experimental conditions ........................................................................................... 13

A.1 Operating conditions for analysis with LC-MS/MS (ESI+/ESI-) .................................................... 13

A.2 Operating conditions for analysis using GC-MS/MS ......................................................................... 14

Bibliography ................................................................................................................................................................. 17

---------------------- Page: 4 ----------------------
SIST-TS CEN/TS 17743:2022
CEN/TS 17743:2022 (E)
European foreword

This document (CEN/TS 17743:2022) has been prepared by Technical Committee CEN/TC 275

“Food analysis - Horizontal methods”, the secretariat of which is held by DIN.

Attention is drawn to the possibility that some of the elements of this document may be the

subject of patent rights. CEN shall not be held responsible for identifying any or all such patent

rights.
WARNING — The application of this Technical Specification can involve hazardous

materials, operations and equipment. This Technical Specification does not claim to

address all the safety problems associated with its use. It is the responsibility of the user of

this Technical Specification to establish appropriate safety and health practices and to

determine the applicability of regulatory limitations prior to use.

Any feedback and questions on this document should be directed to the users’ national standards

body. A complete listing of these bodies can be found on the CEN website.

According to the CEN/CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to announce this Technical Specification: 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, Turkey and the United Kingdom.
---------------------- Page: 5 ----------------------
SIST-TS CEN/TS 17743:2022
CEN/TS 17743:2022 (E)
1 Scope

This document specifies a method for the analysis of pesticide residues in foods of plant and of

animal origin by ethyl acetate extraction using GC- and LC-MS/MS (SweEt).
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 17061:2019, Foodstuffs - Guideline for the calibration and quantitative determination of

chromatographic methods for the determination of pesticide residues and organic contaminants

3 Terms and Definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological 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

The method is based on extraction with ethyl acetate followed by determination with liquid

chromatography (LC) and gas chromatography (GC). No clean-up is needed for low fat/oil fruit,

vegetables and cereals or honey. However, for commodities with high fat/oil content, ethyl

acetate/cyclohexane is used for extraction. Furthermore, a clean-up step using gel permeation

chromatography (GPC) is needed. The animal products with low fat/oil content are extracted with

as sorbents.
ethyl acetate followed by clean-up using Primary Secondary Amine (PSA) and C18

For extraction of cereals, acidified ethyl acetate is used, which makes it possible to analyze acidic

pesticides such as phenoxy acids.

Low fat/oil fruits, vegetables and honey: The homogenous sample is extracted with ethyl

acetate after addition of NaHCO . After extraction, Na SO is added to remove water. The sample

3 2 4
extract is centrifuged and filtered prior to injection to GC-MS/MS and LC-MS/MS.

Cereals: The grinded sample is extracted after water addition with acidified ethyl acetate (5.21).

After extraction, Na SO is added to remove water. The sample extract is centrifuged and filtered

2 4
prior to injection to GC-MS/MS and LC-MS/MS.
Animal origin: The homogenous sample is extracted using ethyl acetate or ethyl

acetate + cyclohexane (V1 + V2, 1 + 1). After extraction, Na SO is added to remove water. The

2 4

choice of extraction solvent depends on the fat/oil content of the sample. Samples with a fat/oil

content of a mass fraction ≤ 10 % are extracted with ethyl acetate and purified with the sorbents

PSA and C18 whereas for samples with higher fat/oil content ethyl acetate + cyclohexane (V + V ,

1 2

1 + 1) followed by GPC is used. The sample extract is finally filtered prior to injection to GC-

MS/MS and LC-MS/MS.
Octadecylsilyl chemically bonded silica gel.
---------------------- Page: 6 ----------------------
SIST-TS CEN/TS 17743:2022
CEN/TS 17743:2022 (E)
5 Reagents

Unless otherwise specified, use reagents of recognized analytical grade. Take every precaution to

avoid possible contamination of water, solvents, sorbents, inorganic salts, etc.
5.1 Water, HPLC quality.
5.2 Methanol, HPLC quality.
5.3 Acetonitrile, HPLC quality.
5.4 Ethyl acetate, pesticide residue grade.
5.5 Cyclohexane, pesticide residue grade.
5.6 Toluene, pesticide residue grade.
5.7 Acetic acid, purity greater than mass fraction ≥ 98 %.
5.8 Ammonia, mass fraction of 25 % NH (approximately 13,4 mol/l).
5.9 Formic acid, mass fraction of 98 % to 100 %.
5.10 Sulphuric acid, mass fraction of 95 % to 97 %.
5.11 Sodium hydroxide.
5.12 Sodium sulphate, Na SO , water free, p.a.
2 4
5.13 Sodium hydrogen monocarbonate, NaHCO , water free, p.a.
5.14 BioBeads S-X3 resin, 38 µm to 75 µm (200 to 400 mesh) .
5.15 Primary secondary amino sorbent (PSA).
® 3

EXAMPLE Bondesil-PSA 40 µm Agilent No 12213023 . Other amino sorbents may be used, but

investigations may be necessary to prove equivalency especially regarding analyte losses and pH of the final

extracts.
5.16 C18 sorbent
® 3
EXAMPLE Bondesil-C18 40 µm, Agilent .

BioBeads -S-X3 resin, 38 µm to 75 µm (200 to 400 mesh) is the trademark of a product supplied by Bio-

Rad. This information is given for the convenience of users of this document and does not constitute an

endorsement by CEN or CENELEC of the product named. Equivalent products may be used if they can be

shown to lead to the same results.
® ®

Bondesil-PSA 40 µm Agilent No 12213023 and Bondesil-C18 40 µm are the trademarks of products

supplied by Agilent. This information is given for the convenience of users of this document and does not

constitute an endorsement by CEN or CENELEC of the product named. Equivalent products may be used if

they can be shown to lead to the same results.
---------------------- Page: 7 ----------------------
SIST-TS CEN/TS 17743:2022
CEN/TS 17743:2022 (E)
5.17 Ammonium formate solution (50 mM), pH 4,1.

Add 1,92 ml ± 0,02 ml 98 % formic acid (26 mol/l; 5.9) to 900 ml ± 5 ml water. Adjust the pH

between 4,1 and 4,15 with ammonia (5.8). Dilute 9 parts buffer solution with 1 part water.

The buffer solution is stable up to 3 months at +4 °C.
5.18 Mobile phase A, 10 mmol/l ammonium formate, pH 4,0 to 4,2.

Add 200 ml ± 2 ml (volumetric glass) of 50 mmol/l buffer (5.17) to 800 ml ± 5 ml of water (5.1).

The buffer solution is stable up to 3 months at +4 °C.
5.19 Mobile phase B, Methanol (5.2).
5.20 Wash solution for HPLC, acetonitrile + water, V + V , 80 + 20.
1 2
Add 200 ml ± 2 ml water (5.1) to 800 ml ± 5 ml acetonitrile (5.3).
5.21 Acidified ethyl acetate for extraction, volume fraction of 1 % acetic acid.
Mix 495 ml of ethyl acetate (5.4) with 5 ml acetic acid (5.7).
5.22 Sodium hydroxide, 5 mol/l.

Dissolve 20 g of NaOH (5.11) in approximately 80 ml of water (5.1) and dilute to 100 ml.

5.23 Sodium chloride, NaCl.
5.24 Sulphuric acid, H SO , 2,5 mol/l.
2 4
Mix 7 ml of H SO (5.10) with 50 ml water (5.1).
2 4
5.25 Primary pesticide standards.
Primary standards are purchased with purity > 95 %.
5.26 Pesticide stock solutions.

Stock solutions are prepared in the most suitable solvent with regards to their solubility and

stability.
5.27 Intermediate pesticide working solutions.

Intermediate standard solutions of pesticide mixtures (40 µg/ml) are prepared in methanol,

acetonitrile or toluene.
5.28 Pesticide calibration solutions.

Working solutions used for calibration on LC-MS/MS in fruit and vegetables are prepared in blank

matrix (generic matrix) with low suppression, e.g. banana or carrot. By doing so the matrix effect

can be compensated and the stability of pesticides improved as well.

Working solutions for the calibration on GC-MS/MS are prepared in generic matrix, e.g. cucumber

with no or little enhancement of the signal. True matrix matched working solutions are always

used for the calibration on both types of analytical techniques for cereals and products of animal

origin.

The findings in fruit and vegetables above the maximum residue level (MRL) are always

confirmed against true matrix matched standards.
---------------------- Page: 8 ----------------------
SIST-TS CEN/TS 17743:2022
CEN/TS 17743:2022 (E)

5.29 GPC eluent and extraction solvent for commodities with high fat/oil content, ethyl

acetate + cyclohexane (V1 + V2, 1 + 1).
6 Apparatus
Usual laboratory apparatus and, in particular, the following:

6.1 Automatic pipettes, suitable for handling volumes of 10 µL to 100 µL, 200 µL to 1 000 µL

and 2 ml to 10 ml.
NOTE Instead of the latter, 10 ml graduated glass pipettes can be used.
6.2 Centrifuge tubes with screw caps, 114 mm × 28 mm, polypropylene (50 ml).
6.3 Volumetric cylinder for ethyl acetate (25 ml).
6.4 Syringes, e.g. 10 ml disposable syringes.

6.5 Syringe filters, 0,20 μm pore size, polytetrafluoroethylene (PTFE) for filtration of final

extracts.
6.6 Injection vials, 1,5 ml suitable for LC and GC auto-samplers.
6.7 Analytical balance, accuracy 0,1 mg.
6.8 Laboratory balance, accuracy 0,01 g.
6.9 Centrifuge.
6.10 Ultrasonic bath.

6.11 Instrument for GPC, equipped with a column, 25 mm internal diameter, 50 cm length, and

5 ml sample loop.

6.12 Mechanical shaker (300 r/min) or a high-speed mechanical shaker, for example from

Spex SamplePrep shaking ≥ 1 500 r/min.
6.13 Rotavapor or similar for evaporation.
6.14 Nitrogen evaporator with thermostated water bath.
6.15 Vibration device, e.g. Vortex.
6.16 LC-MS/MS system, see Annex A.
6.17 GC-MS/MS system, see Annex A.

A high-speed mechanical shaker from Spex SamplePrep is an example of a suitable product available

commercially. This information is given for the convenience of users of this document and does not

constitute an endorsement by CEN of those products.
---------------------- Page: 9 ----------------------
SIST-TS CEN/TS 17743:2022
CEN/TS 17743:2022 (E)
7 Procedure
7.1 Sample preparation
7.1.1 Fruit and vegetables

Weigh a test portion of 10,0 g ± 0,1 g (m ) of the homogenous laboratory sample into a 50 ml

sample

centrifuge tube (6.2). For a recovery test (QC sample), add 50 µl to 250 µl of standard solution

(5.27) at this stage. Wait for 5 min before adding 3,0 g ± 0,1 g of sodium hydrogen monocarbonate

(NaHCO , 5.13) and add 20,0 ml ± 0,1 ml ethyl acetate (V , 5.4). Shake vigorously by hand for

3 ex

approximately 30 s and thereafter place the tubes with a mechanical shaker at 300 r/min for

15 min, alternatively at 1 500 r/min for 3 min (6.12). Add 10,0 g ± 0,5 g of sodium sulphate

(Na SO , 5.12) and shake the sample for 1 min. Centrifuge the tubes for 3 min at 3 200 g (6.9).

2 4

Filter the crude extract using a 0,2 µm PTFE syringe filter (6.5). The final extract has a

concentration of 0,50 g/ml sample in ethyl acetate and is analyzed directly by GC- and LC-MS/MS

(6.16, 6.17).

For dried fruit (water content about 20 % or less) and honey, water should be added prior to the

extraction. Weigh e.g. 10 g of sample, add 15 ml ± 0,1 ml cold water, shake for 5 min and extract as

above.
NOTE: To improve phase separation for ex
...

SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17743:2021
01-december-2021
Živila - Določevanje ostankov pesticidov z ekstrakcijo z etil acetatom z uporabo
GC- in LC-MS/MS (SweEt)

Foodstuff - Determination of pesticide residues by ethyl acetate extraction using GC- and

LC-MS/MS (SweEt)

Lebensmittel - Bestimmung von Pestizidrückständen mit Ethylacetatextraktion durch GC-

und LC-MS/MS (SweEt)
Ta slovenski standard je istoveten z: FprCEN/TS 17743
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
kSIST-TS FprCEN/TS 17743:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
kSIST-TS FprCEN/TS 17743:2021
---------------------- Page: 2 ----------------------
kSIST-TS FprCEN/TS 17743:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17743
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
October 2021
ICS 67.050
English Version
Foodstuff - Determination of pesticide residues by ethyl
acetate extraction using GC- and LC-MS/MS (SweEt)
Lebensmittel - Bestimmung von Pestizidrückständen
mit Ethylacetatextraktion durch GC- und LC-MS/MS
(SweEt)

This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee

CEN/TC 275.

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, Turkey 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 Technical Specification. It is distributed for review and comments. It is subject to change

without notice and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17743:2021 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
kSIST-TS FprCEN/TS 17743:2021
FprCEN/TS 17743:2021 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

1 Scope .................................................................................................................................................................... 4

2 Normative references .................................................................................................................................... 4

3 Terms and Definitions ................................................................................................................................... 4

4 Principle .............................................................................................................................................................. 4

5 Reagents .............................................................................................................................................................. 5

6 Apparatus ........................................................................................................................................................... 7

7 Procedure ........................................................................................................................................................... 8

8 Evaluation of results .................................................................................................................................... 10

9 Precision .......................................................................................................................................................... 11

10 Test report ...................................................................................................................................................... 12

Annex A (informative) Experimental conditions ........................................................................................... 13

A.1 Operating conditions for analysis with LC-MS/MS (ESI+/ESI-) .................................................... 13

A.2 Operating conditions for analysis using GC-MS/MS ......................................................................... 14

Bibliography ................................................................................................................................................................. 17

---------------------- Page: 4 ----------------------
kSIST-TS FprCEN/TS 17743:2021
FprCEN/TS 17743:2021 (E)
European foreword
This document (FprCEN/TS 17743:2021) 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 Vote on TS.
WARNING — The application of this Technical Specification can involve hazardous

materials, operations and equipment. This Technical Specification does not claim to

address all the safety problems associated with its use. It is the responsibility of the user of

this Technical Specification to establish appropriate safety and health practices and to

determine the applicability of regulatory limitations prior to use.
---------------------- Page: 5 ----------------------
kSIST-TS FprCEN/TS 17743:2021
FprCEN/TS 17743:2021 (E)
1 Scope

This document specifies a method for the analysis of pesticide residues in foods of plant and of

animal origin by ethyl acetate extraction using GC- and LC-MS/MS (SweEt).
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 17061:2020, Foodstuffs — Guideline for the calibration and quantitative determination of

chromatographic methods for the determination of pesticide residues and organic contaminants

3 Terms and Definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following

addresses:
• IEC Electropedia: available at https://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
4 Principle

The method is based on extraction with ethyl acetate followed by determination with liquid

chromatography (LC) and gas chromatography (GC). No clean-up is needed for low fat/oil fruit,

vegetables and cereals or honey. However, for commodities with high fat/oil content, ethyl

acetate/cyclohexane is used for extraction. Furthermore, a clean-up step using gel permeation

chromatography (GPC) is needed. The animal products with low fat/oil content are extracted with

ethyl acetate followed by clean-up using Primary Secondary Amine (PSA) and C18 as sorbents.

For extraction of cereals, acidified ethyl acetate is used, which makes it possible to analyze acidic

pesticides such as phenoxy acids.

Low fat/oil fruits, vegetables and honey: The homogenous sample is extracted with ethyl

acetate after addition of NaHCO . After extraction, Na SO is added to remove water. The sample

3 2 4
extract is centrifuged and filtered prior to injection to GC-MS/MS and LC-MS/MS.

Cereals: The grinded sample is extracted after water addition with acidified ethyl acetate (5.21).

After extraction, Na SO is added to remove water. The sample extract is centrifuged and filtered

2 4
prior to injection to GC-MS/MS and LC-MS/MS.
Animal origin: The homogenous sample is extracted using ethyl acetate or ethyl

acetate + cyclohexane (V + V , 1 + 1). After extraction, Na SO is added to remove water. The

1 2
2 4

choice of extraction solvent depends on the fat/oil content of the sample. Samples with a fat/oil

content of a mass fraction ≤ 10 % are extracted with ethyl acetate and purified with the sorbents

PSA and C18 whereas for samples with higher fat/oil content ethyl acetate + cyclohexane (V + V ,

1 2

1 + 1) followed by GPC is used. The sample extract is finally filtered prior to injection to GC-

MS/MS and LC-MS/MS.
Octadecylsilyl chemically bonded silica gel
---------------------- Page: 6 ----------------------
kSIST-TS FprCEN/TS 17743:2021
FprCEN/TS 17743:2021 (E)
5 Reagents

Unless otherwise specified, use reagents of recognized analytical grade. Take every precaution to

avoid possible contamination of water, solvents, sorbents, inorganic salts, etc.
5.1 Water, HPLC quality.
5.2 Methanol, HPLC quality.
5.3 Acetonitrile, HPLC quality.
5.4 Ethyl acetate, pesticide residue grade.
5.5 Cyclohexane, pesticide residue grade.
5.6 Toluene, pesticide residue grade.
5.7 Acetic acid, purity greater than mass fraction ≥ 98 %.
5.8 Ammonia, mass fraction of 25 % NH3 (approximately 13,4 mol/l).
5.9 Formic acid, mass fraction of 98 % to 100 %.
5.10 Sulphuric acid, mass fraction of 95 % to 97 %.
5.11 Sodium hydroxide.
5.12 Sodium sulphate, Na SO , water free, p.a.
2 4
5.13 Sodium hydrogen monocarbonate, NaHCO , water free, p.a.
® 2
5.14 BioBeads S-X3 resin, 38 µm to 75 µm (200 to 400 mesh) .
5.15 Primary secondary amino sorbent (PSA).

EXAMPLE Bondesil-PSA 40 µm Agilent No 12213023 . Other amino sorbents may be used, but

investigations may be necessary to prove equivalency especially regarding analyte losses and pH of the final

extracts.
5.16 C18 sorbent
® 3
EXAMPLE Bondesil-C18 40 µm, Agilent .

BioBeads -S-X3 resin, 38 µm to 75 µm (200 to 400 mesh) is a product of Bio-Rad. This information is

given for the convenience of users of this document and does not constitute an endorsement by CEN of the

product named. Equivalent products may be used if they can be shown to lead to the same results.

® ®

Bondesil-PSA 40 µm Agilent No 12213023 and Bondesil-C18 40 µm are products of Agilent. This

information is given for the convenience of users of this document and does not constitute an endorsement

by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same

results.
---------------------- Page: 7 ----------------------
kSIST-TS FprCEN/TS 17743:2021
FprCEN/TS 17743:2021 (E)
5.17 Ammonium formate solution (50 mM), pH 4,1.

Add 1,92 ml ± 0,02 ml 98 % formic acid (26 mol/l; 5.9) in 900 ml ± 5 ml water. Adjust the pH

between 4,1 and 4,15 with ammonia (5.8). Dilute 9 parts buffer solution to 1 part water.

The buffer solution is stable up to 3 months at +4 °C.
5.18 Mobile phase A, 10 mmol/l ammonium formate, pH 4,0 to 4,2.

200 ml ± 2 ml (volumetric glas) of 50 mmol/l buffer (5.17) in 800 ml ± 5 ml of MilliQ water (5.1)

The buffer solution is stable up to 3 months at +4 °C.
5.19 Mobile phase B, Methanol (5.2).
5.20 Wash solution for HPLC, acetonitrile + water, V1 + V2, 80 + 20.
Add 200 ml ± 2 ml water (5.1) to 800 ml ± 5 ml acetonitrile (5.3)
5.21 Acidified ethyl acetate for extraction, volume fraction of 1 % acetic acid.
Mix 495 ml of ethyl acetate (5.4) with 5 ml acetic acid (5.7).
5.22 Sodium hydroxide, 5 mol/l.

Dissolve 20 g of NaOH (5.11) in approximately 80 ml of water (5.1) and dilute to 100 ml.

5.23 Sodium chloride, NaCl.
5.24 Sulphuric acid, H2SO4, 2,5 mol/l.
Mix 7 ml of H SO (5.10) with 50 ml water (5.1)
2 4
5.25 Primary pesticide standards.
Primary standards are purchased with purity > 95 %.
5.26 Pesticide stock solutions.

Stock solutions are prepared in the most suitable solvent with regards to their solubility and

stability.
5.27 Intermediate pesticide working solutions.

Intermediate standard solutions of pesticide mixtures (40 µg/ml) are prepared in methanol,

acetonitrile or toluene.
5.28 Pesticide calibration solutions.

Working solutions used for calibration on LC-MS/MS in fruit and vegetables are prepared in blank

matrix (generic matrix) with low suppression, e.g. banana or carrot. By doing so the matrix effect

can be compensated and the stability of pesticides improved as well.

Working solutions for the calibration on GC-MS/MS are prepared in generic matrix, e.g. cucumber

with no or little enhancement of the signal. True matrix matched working solutions are always

used for the calibration on both types of analytical techniques for cereals and products of animal

origin.

The findings in fruit and vegetables above the maximum residue level (MRL) are always

confirmed against true matrix matched standards.
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kSIST-TS FprCEN/TS 17743:2021
FprCEN/TS 17743:2021 (E)

5.29 GPC eluent and extraction solvent for commodities with high fat/oil content,

ethyl acetate + cyclohexane (V1 + V2, 1 + 1).
6 Apparatus
Usual laboratory apparatus and, in particular, the following:

6.1 Automatic pipettes, suitable for handling volumes of 10 µL to 100 µL, 200 µL to 1 000 µL

and 2 ml to 10 ml.
NOTE Instead of the latter, 10 ml graduated glass pipettes can be used.
6.2 Centrifuge tubes with screw caps, 114 mm × 28 mm, polypropylene (50 ml).
6.3 Volumetric cylinder for ethyl acetate (25 ml).
6.4 Syringes, e.g. 10 ml disposable syringes.

6.5 Syringe filters, 0,20 μm pore size, polytetrafluoroethylene (PTFE) for filtration of final

extracts.
6.6 Injection vials, 1,5 ml suitable for LC and GC auto-samplers.
6.7 Analytical balance, accuracy 0,1 mg.
6.8 Laboratory balance, accuracy 0,01 g.
6.9 Centrifuge.
6.10 Ultrasonic bath.

6.11 Instrument for GPC, equipped with a column, 25 mm internal diameter, 50 cm length, and

5 ml sample loop.

6.12 Mechanical shaker (300 r/min) or a high-speed mechanical shaker, for example from

Spex SamplePrep shaking ≥ 1 500 r/min.
6.13 Rotavapor or similar for evaporation.
6.14 Nitrogen evaporator with thermostated water bath.
6.15 Vibration device, e.g. Vortex.
6.16 6.16LC-MS/MS system, see Annex A.
6.17 GC-MS/MS system, see Annex A.

A high-speed mechanical shaker from Spex SamplePrep is an example of a suitable product available

commercially. This information is given for the convenience of users of this document and does not

constitute an endorsement by CEN of those products.
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kSIST-TS FprCEN/TS 17743:2021
FprCEN/TS 17743:2021 (E)
7 Procedure
7.1 Sample preparation
7.1.1 Fruit and vegetables

Weigh a test portion of 10,0 g ± 0,1 g (m ) of the homogenous laboratory sample into a 50 ml

sample

centrifuge tube (6.2). For a recovery test (QC sample), add 50 µl to 250 µl of standard solution

(5.27) at this stage. Wait for 5 min before adding 3,0 g ± 0,1 g of sodium hydrogen monocarbonate

(NaHCO , 5.13) and add 20,0 ml ± 0,1 ml ethyl acetate (V , 5.4). Shake vigorously by hand for

3 ex

approximately 30 s and thereafter place the tubes on a mechanical shaker at 300 r/min for

15 min, alternatively at 1 500 r/min for 3 min (6.12 ). Add 10,0 g ± 0,5 g of sodium sulphate

(Na SO , 5.12) and shake the sample for 1 min. Centrifuge the tubes for 3 min at 3 200 g (6.9).

2 4

Filter the crude extract using a 0,2 µm PTFE syringe filter (6.5). The final extract has a

concentration of 0,50 g/ml sample in ethyl acetate and is analyzed directly by GC- and LC-MS/MS

(6.16, 6.17).

For dried fruit (water content about 20 % or less) and honey, water should be added prior to the

extraction. Weigh e.g. 10 g of sample, add 15 ml ± 0,1 ml cold water, shake for 5 min and extract as

above.

NOTE 1: To improve phase separation for extracted honey samples, 1,00 g ± 0,05 g of NaCl (5.23) should be

added prior to centrifugation, i.e. after extraction in combination with the addition of Na2SO4 (5.12).

7.1.2 Cereals

Weigh 5,0 g ± 0,1 g grinded, dry sample (m ) in a 50 ml centrifuge tube (6.2), add 10 ml ± 1 ml

sample

water (5.1) and shake to wet the sample. Add 10,0 ml ± 0,1 ml acidified ethyl acetate (V , 5.21),

and shake the mixture vigorously by hand or with a vibration device (6.15) for 30 s in order to get

the sample homogeneously mixed with the solvent and thereafter for 15 min on a mechanical

...

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