ISO 7124:2023

Eggs and egg products — Determination of fipronil and metabolites residues — Liquid chromatography tandem mass spectrometry method

This document specifies a liquid chromatography tandem mass spectrometry method (LC-MS/MS) for the determination of fipronil and metabolites (including fipronil-desulfinyl, fipronil-sulfide and fipronil-sulfone) residues in eggs and egg products.

Œufs et ovoproduits — Dosage des résidus de fipronil et de ses métabolites — Méthode de chromatographie en phase liquide couplée à la spectrométrie de masse en tandem

General Information

• Status: Published
• Publication Date: 12-Apr-2023
• ICS: 67.120.20 - Poultry and eggs
• Technical Committee: ISO/TC 34/SC 6 - Meat, poultry, fish, eggs and their products
• Drafting Committee: ISO/TC 34/SC 6 - Meat, poultry, fish, eggs and their products
• Current Stage: 6060 - International Standard published
• Start Date: 13-Apr-2023
• Due Date: 04-Jun-2024
• Completion Date: 13-Apr-2023

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 7124
First edition
2023-04
Eggs and egg products —
Determination of fipronil and
metabolites residues — Liquid
chromatography tandem mass
spectrometry method
Œufs et ovoproduits — Dosage des résidus de fipronil et de ses
métabolites — Méthode de chromatographie en phase liquide couplée
à la spectrométrie de masse en tandem
Reference number
ISO 7124:2023(E)
© ISO 2023

---------------------- Page: 1 ----------------------
ISO 7124:2023(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
  © ISO 2023 – All rights reserved

---------------------- Page: 2 ----------------------
ISO 7124:2023(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Sampling . 1
6 Preparing and storing the test sample . 2
6.1 Preparing . 2
6.1.1 Fresh eggs and egg products with shell. 2
6.1.2 Other egg products . 2
6.2 Storing . 2
7 Liquid chromatography tandem mass spectrometry method . 2
7.1 Reagents . 2
7.2 Preparation of solutions . 2
7.3 Standard materials . 3
7.4 Preparation of standard solutions . 3
7.5 Materials . 3
7.6 Apparatus . 3
7.7 Procedure . 4
7.7.1 General . 4
7.7.2 Extraction . 4
7.7.3 Purification . 4
7.7.4 LC-MS/MS analysis . 4
7.8 Identification criteria . 5
7.9 Determination . 6
7.10 Blank test . 6
7.11 Calculation . 6
7.12 Limit of quantification . 6
7.13 Precision . . . 7
8 Test report . 7
Annex A (informative) Information of fipronil and metabolites . 8
Annex B (informative) LC-MS/MS chromatograms for fipronil, fipronil-desulfinyl, fipronil-
13
sulfide, fipronil-sulfone and fipronil- C6 mixed standard solutions .10
Annex C (informative) Precision of the method .12
Bibliography .13
iii
© ISO 2023 – All rights reserved

---------------------- Page: 3 ----------------------
ISO 7124:2023(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 6,
Meat, poultry, fish, eggs and their products.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
  © ISO 2023 – All rights reserved

---------------------- Page: 4 ----------------------
INTERNATIONAL STANDARD ISO 7124:2023(E)
Eggs and egg products — Determination of fipronil and
metabolites residues — Liquid chromatography tandem
mass spectrometry method
1 Scope
This document specifies a liquid chromatography tandem mass spectrometry method (LC-MS/MS)
for the determination of fipronil and metabolites (including fipronil-desulfinyl, fipronil-sulfide and
fipronil-sulfone) residues in eggs and egg products.
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.
ISO 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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/
3.1
fipronil and metabolites content
ω
mass fraction of fipronil and metabolites residues in egg and egg products
Note 1 to entry: The fipronil and metabolites content is determined according to the procedure specified in this
document.
Note 2 to entry: The fipronil and metabolites content is expressed in micrograms per kilogram.
4 Principle
The test portion is extracted with acetonitrile. The extract is cleaned up with dispersive solid phase
extraction. The fipronil and metabolites are determined and confirmed by LC-MS/MS, and are
quantified with an internal standard method.
5 Sampling
Sampling is not part of the method specified in this document. A recommended sampling method is
given in CAC/GL 50-2004.
It is important that the laboratory receive a sample which is truly representative and has not been
damaged or changed during transport or storage.
Start from a representative sample of at least 1 kg.
1
© ISO 2023 – All rights reserved

---------------------- Page: 5 ----------------------
ISO 7124:2023(E)
Store the sample in such a way that deterioration and change in composition are prevented.
6 Preparing and storing the test sample
6.1 Preparing
6.1.1 Fresh eggs and egg products with shell
Wash approximately 15 to 20 fresh eggs (with a mass of about 1 kg) and remove the shells. Smash and
homogenize the egg samples with the appropriate equipment (7.6.6). Place the sample in a polyethylene
bottle.
Weigh about 1 kg of egg products (with shells) and then remove the shells. Smash and homogenize the
egg samples with the appropriate equipment (7.6.6). Place the sample in a polyethylene bottle.
6.1.2 Other egg products
Homogenize the egg product samples. Place each sample in a polyethylene bottle.
6.2 Storing
Store the test sample and a standby sample at a temperature of −20 °C to −16 °C, respectively.
7 Liquid chromatography tandem mass spectrometry method
7.1 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
7.1.1 Water, conforming to grade 1 of ISO 3696.
7.1.2 Acetonitrile, HPLC grade.
7.1.3 Methanol, HPLC grade.
7.1.4 Formic acid, HPLC grade.
7.1.5 Ammonium acetate, HPLC grade.
7.1.6 Anhydrous magnesium sulfate.
7.1.7 Sodium chloride.
7.1.8 Anhydrous sodium sulfate.
7.2 Preparation of solutions
7.2.1 Formic ac
...

© ISO 2023 – All rights reserved
ISO/DIS PRF 7124:20222023(E)
Date: 2022-11-042023-02-15
ISO/TC 34/SC 6/WG 24
Secretariat: SAC
Eggs and egg products—Determination of fipronil and metabolites
residues — Liquid chromatography tandem mass spectrometry
method

---------------------- Page: 1 ----------------------
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of
this publication may be reproduced or utilized otherwise in any form or by any means, electronic or
mechanical, including photocopying, or posting on the internet or an intranet, without prior written
permission. Permission can be requested from either ISO at the address below or ISO’s member body in the
country of the requester.
ISO Copyright Office
CP 401 • CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
Email: [e-mail]
Website: www.iso.org
Published in Switzerland.

---------------------- Page: 2 ----------------------
ISO/PRF 7124:2023(E)
Contents
Foreword . iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Sampling . 1
6 Preparing and storing the test sample . 2
6.1 Preparing . 2
6.1.1 Fresh eggs and egg products with shell . 2
6.1.2 Other egg products . 2
6.2 Storing. 2
7 Liquid chromatography tandem mass spectrometry method . 2
7.1 Reagents . 2
7.2 Preparation of solutions . 2
7.3 Standard materials . 3
7.4 Preparation of standard solutions . 3
7.5 Materials . 3
7.6 Apparatus . 3
7.7 Procedure . 4
7.7.1 General . 4
7.7.2 Extraction . 4
7.7.3 Purification . 4
7.7.4 LC-MS/MS analysis . 4
7.8 Identification criteria . 5
7.9 Determination . 6
7.10 Blank test . 6
7.11 Calculation . 6
7.12 Limit of quantification . 7
7.13 Precision . 7
8 Test report . 7
Annex A (informative) Information of fipronil and metabolites . 8
Annex B (informative) LC-MS/MS chromatograms for fipronil, fipronil-desulfinyl, fipronil-
13
sulfide, fipronil-sulfone and fipronil- C6 mixed standard solutions . 10
Annex C (informative) Precision of the method . 13
Bibliography . 14
© ISO 2023 – All rights reserved iii

---------------------- Page: 3 ----------------------
ISO/PRF 7124:2023(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO
collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any
patent rights identified during the development of the document will be in the Introduction and/or on
the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the World
Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 6,
Meat, poultry, fish, eggs and their products.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv © ISO 2023 – All rights reserved

---------------------- Page: 4 ----------------------
INTERNATIONAL STANDARD ISO/PRF 7124:2023(E)

Eggs and egg products — Determination of fipronil and
metabolites residues — Liquid chromatography tandem mass
spectrometry method
1 Scope
This document specifies a liquid chromatography tandem mass spectrometry method (LC-MS/MS) for
the determination of fipronil and metabolites (including fipronil-desulfinyl, fipronil-sulfide and fipronil-
sulfone) residues in eggs and egg products.
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.
ISO 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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/
3.1
fipronil and metabolites content
ω
mass fraction of fipronil and metabolites residues in egg and egg products
Note 1 to entry: FipronilThe fipronil and metabolites content areis determined according to the procedure specified
in this document.
Note 2 to entry: FipronilThe fipronil and metabolites content areis expressed in micrograms per kilogram.
4 Principle
The test portion is extracted with acetonitrile. The extract is cleaned up with dispersive solid phase
extraction. The fipronil and metabolites are determined and confirmed by LC-MS/MS, and are quantified
with an internal standard method.
5 Sampling
Sampling is not part of the method specified in this document. A recommended sampling method is given
in CAC/GL 50-2004.
© ISO 2023 – All rights reserved 1

---------------------- Page: 5 ----------------------
ISO/PRF 7124:2023(E)
It is important that the laboratory receive a sample which is truly representative and has not been
damaged or changed during transport or storage.
Start from a representative sample of at least 1 kg.
Store the sample in such a way that deterioration and change in composition are prevented.
6 Preparing and storing the test sample
6.1 Preparing
6.1.1 Fresh eggs and egg products with shell
Wash approximately 15 to 20 fresh eggs (with a mass of about 1 kg) and remove the shells. Smash and
homogenize the egg samples with the appropriate equipment (7.6.6). Place the sample in a polyethylene
bottle.
Weigh about 1 kg of egg products (with shells) and then remove the shells. Smash and homogenize the
egg samples with the appropriate equipment (7.6.6). Place the sample in a polyethylene bottle.
6.1.2 Other egg products
Homogenize the egg product samples. Place each sample in a polyethylene bottle.
6.2 Storing
Store the test sample and a standby sample at a temperature of −20 °C to −16 °C, respectively.
7 Liquid chromatography tandem mass spectrometry method
7.1 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
7.1.1 Water, conforming to grade 1 of ISO 3696.
7.1.2 Acetonitrile, HPLC grade.
7.1.3 Methanol, HPLC grade.
7.1.4 Formic acid, HPLC grade.
7.1.5 Ammonium acetate, HPLC grade.
7.1.6 Anhydrous magnesium sulfate.
7.1.7 Sodium chloride.
7.1.8 Anhydrous sodium sulfate.
7.2 Preparation of solutions
7.2.1 Formic acid solution, 1 ml/l.
Pipette 1 ml formic acid (7.1.4) to a 1 000 ml one-mark volumetric flask. Dilute to the mark with water
and mix.
7.2.2 Ammonium acetate-formic acid solution, c(CH COONH ) = 5 mmol/l.
3 4
2 © ISO 2023 – All rights reserved

---------------------- Page: 6 ----------------------
ISO/PRF 7124:2023(E)
Dissolve 0,385 4 g ammonium acetate in 1 ml/l formic acid solution (7.2.1), dilute to 1 000 ml and mix.
7.3 Standard materials
7.3.1 Fipronil and its metabolites, purity ≥ 95 %, see Annex A.
13
7.3.2 Fipronil isotope internal: standard stock solution of fipronil- C6, 100 mg/l.
7.4 Preparation of standard solutions
7.4.1 Standard stock solution, 100 mg/l.
Weigh, to the nearest 0,1 mg, 10 mg of fipronil, fipronil-desulfinyl, fipronil-sulfide and fipr
...

INTERNATIONAL ISO
STANDARD 7124
First edition
Eggs and egg products —
Determination of fipronil and
metabolites residues — Liquid
chromatography tandem mass
spectrometry method
Œufs et ovoproduits — Dosage des résidus de fipronil et de ses
métabolites — Méthode de chromatographie en phase liquide couplée
à la spectrométrie de masse en tandem
PROOF/ÉPREUVE
Reference number
ISO 7124:2023(E)
© ISO 2023

---------------------- Page: 1 ----------------------
ISO 7124:2023(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
PROOF/ÉPREUVE © ISO 2023 – All rights reserved

---------------------- Page: 2 ----------------------
ISO 7124:2023(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Sampling . 1
6 Preparing and storing the test sample . 2
6.1 Preparing . 2
6.1.1 Fresh eggs and egg products with shell. 2
6.1.2 Other egg products . 2
6.2 Storing . 2
7 Liquid chromatography tandem mass spectrometry method . 2
7.1 Reagents . 2
7.2 Preparation of solutions . 2
7.3 Standard materials . 3
7.4 Preparation of standard solutions . 3
7.5 Materials . 3
7.6 Apparatus . 3
7.7 Procedure . 4
7.7.1 General . 4
7.7.2 Extraction . 4
7.7.3 Purification . 4
7.7.4 LC-MS/MS analysis . 4
7.8 Identification criteria . 5
7.9 Determination . 6
7.10 Blank test . 6
7.11 Calculation . 6
7.12 Limit of quantification . 6
7.13 Precision . . . 7
8 Test report . 7
Annex A (informative) Information of fipronil and metabolites . 8
Annex B (informative) LC-MS/MS chromatograms for fipronil, fipronil-desulfinyl, fipronil-
13
sulfide, fipronil-sulfone and fipronil- C6 mixed standard solutions .10
Annex C (informative) Precision of the method .12
Bibliography .13
iii
© ISO 2023 – All rights reserved PROOF/ÉPREUVE

---------------------- Page: 3 ----------------------
ISO 7124:2023(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 6,
Meat, poultry, fish, eggs and their products.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
PROOF/ÉPREUVE © ISO 2023 – All rights reserved

---------------------- Page: 4 ----------------------
INTERNATIONAL STANDARD ISO 7124:2023(E)
Eggs and egg products — Determination of fipronil and
metabolites residues — Liquid chromatography tandem
mass spectrometry method
1 Scope
This document specifies a liquid chromatography tandem mass spectrometry method (LC-MS/MS)
for the determination of fipronil and metabolites (including fipronil-desulfinyl, fipronil-sulfide and
fipronil-sulfone) residues in eggs and egg products.
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.
ISO 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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/
3.1
fipronil and metabolites content
ω
mass fraction of fipronil and metabolites residues in egg and egg products
Note 1 to entry: The fipronil and metabolites content is determined according to the procedure specified in this
document.
Note 2 to entry: The fipronil and metabolites content is expressed in micrograms per kilogram.
4 Principle
The test portion is extracted with acetonitrile. The extract is cleaned up with dispersive solid phase
extraction. The fipronil and metabolites are determined and confirmed by LC-MS/MS, and are
quantified with an internal standard method.
5 Sampling
Sampling is not part of the method specified in this document. A recommended sampling method is
given in CAC/GL 50-2004.
It is important that the laboratory receive a sample which is truly representative and has not been
damaged or changed during transport or storage.
Start from a representative sample of at least 1 kg.
1
© ISO 2023 – All rights reserved PROOF/ÉPREUVE

---------------------- Page: 5 ----------------------
ISO 7124:2023(E)
Store the sample in such a way that deterioration and change in composition are prevented.
6 Preparing and storing the test sample
6.1 Preparing
6.1.1 Fresh eggs and egg products with shell
Wash approximately 15 to 20 fresh eggs (with a mass of about 1 kg) and remove the shells. Smash and
homogenize the egg samples with the appropriate equipment (7.6.6). Place the sample in a polyethylene
bottle.
Weigh about 1 kg of egg products (with shells) and then remove the shells. Smash and homogenize the
egg samples with the appropriate equipment (7.6.6). Place the sample in a polyethylene bottle.
6.1.2 Other egg products
Homogenize the egg product samples. Place each sample in a polyethylene bottle.
6.2 Storing
Store the test sample and a standby sample at a temperature of −20 °C to −16 °C, respectively.
7 Liquid chromatography tandem mass spectrometry method
7.1 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
7.1.1 Water, conforming to grade 1 of ISO 3696.
7.1.2 Acetonitrile, HPLC grade.
7.1.3 Methanol, HPLC grade.
7.1.4 Formic acid, HPLC grade.
7.1.5 Ammonium acetate, HPLC grade.
7.1.6 Anhydrous magnesium sulfate.
7.1.7 Sodium chloride.
7.1.8 Anhydrous sodium sulfate.
7.2 Preparation of solutions
7.2.1 Formic acid solution, 1 ml/l.
Pipette 1 ml formic acid (7.1.4) to a 1 000
...