ISO 23851

DRAFT
International
Standard
ISO/DIS 23851
ISO/TC 34/SC 6
Determination of marker residues
Secretariat: SAC
of nicarbazin in chicken tissue and
Voting begins on:
eggs — Liquid chromatography
2025-07-21
tandem mass spectrometry method
Voting terminates on:
ICS: 67.050; 67.120.20; 11.120.10
2025-10-13
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
This document is circulated as received from the committee secretariat.
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS.
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.
Reference number
ISO/DIS 23851:2025(en)
DRAFT
ISO/DIS 23851:2025(en)
International
Standard
ISO/DIS 23851
ISO/TC 34/SC 6
Determination of marker residues
Secretariat: SAC
of nicarbazin in chicken tissue and
Voting begins on:
eggs — Liquid chromatography
tandem mass spectrometry method
Voting terminates on:
ICS: 67.050; 67.120.20; 11.120.10
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2025
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
STANDARDS MAY ON OCCASION HAVE TO
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
This document is circulated as received from the committee secretariat. BE CONSIDERED IN THE LIGHT OF THEIR
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
or ISO’s member body in the country of the requester.
NATIONAL REGULATIONS.
ISO copyright office
RECIPIENTS OF THIS DRAFT ARE INVITED
CP 401 • Ch. de Blandonnet 8
TO SUBMIT, WITH THEIR COMMENTS,
CH-1214 Vernier, Geneva
NOTIFICATION OF ANY RELEVANT PATENT
Phone: +41 22 749 01 11
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION.
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland Reference number
ISO/DIS 23851:2025(en)
ii
ISO/DIS 23851:2025(en)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Sampling . 1
6 Preparation of the test sample. 2
6.1 Preparation .2
6.1.1 Eggs .2
6.1.2 Chicken tissue .2
6.2 Storage .2
7 Reagents and materials . 2
8 Apparatus . 4
9 Procedure . 4
9.1 General .4
9.2 Extraction .4
9.3 Purification .4
9.3.1 Chicken and eggs .4
9.3.2 Chicken kidney and liver .4
9.4 LC-MS/MS analysis .5
9.4.1 HPLC conditions .5
9.4.2 MS parameters .5
9.5 Identification criteria .5
9.6 Determination .6
9.7 Blank test .6
9.8 Calculation .6
9.9 Limit of quantification .7
9.10 Precision . . .7
10 Test report . 7
Annex A (informative) Information of 4,4-dinitrocarbanilide and 4,4-dinitrocarbanilide-d . 8
Annex B (informative) LC-MS/MS chromatograms for 4,4-dinitrocarbanilide and
4,4-dinitrocarbanilide-d standard solutions . 9
Annex C (informative) Precision of the method . 10
Bibliography .11

iii
ISO/DIS 23851:2025(en)
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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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
DRAFT International Standard ISO/DIS 23851:2025(en)
Determination of marker residues of nicarbazin in chicken
tissue and eggs — Liquid chromatography tandem mass
spectrometry method
1 Scope
This document specifies a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the
determination of marker residues of nicarbazin (4,4-dinitrocarbanilide) in chicken tissue and eggs.
This document is applicable to the determination of marker residues of nicarbazin (4,4-dinitrocarbanilide)
in chicken tissue (including muscle, liver and kidney) and eggs.
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
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
The test portion is extracted with acetonitrile and then purified by acetonitrile-saturated n-hexane or solid
phase extraction column. The 4,4-dinitrocarbanilide is determined and confirmed by LC-MS/MS, and is
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 with a mass between 0,5 kg and 1 kg.
Store the sample in such a way that deterioration and change in composition are prevented.

ISO/DIS 23851:2025(en)
6 Preparation of the test sample
6.1 Preparation
6.1.1 Eggs
Take approximately 15 to 20 fresh eggs (with a total mass of about 1 kg) and remove the shells. Smash and
homogenize the eggs with the homogenizer (8.6). Place the egg sample in a polyethylene bottle.
6.1.2 Chicken tissue
Allow the sample to reach the room te
...

ISO/FDIS 23851:2026(en)
ISO/TC 34/SC 6/WG 28
Secretariat: SAC
Date: 2025-12-312026-02-11
Chicken tissue and eggs — Determination of marker residues of
nicarbazin — Liquid chromatography tandem mass spectrometry
method
FDIS stage
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: + 41 22 749 01 11
E-mail: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ISO/FDIS 23851:2026(en)
Contents
Foreword . iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents and materials . 1
6 Apparatus . 3
7 Preparation and preservation of test samples . 3
7.1 Sampling . 3
7.2 Preparation . 3
7.3 Preservation . 4
8 Procedure . 4
8.1 General . 4
8.2 Extraction . 4
8.3 Purification . 4
8.4 LC-MS/MS analysis . 4
8.5 Identification criteria . 5
8.6 Determination . 6
8.7 Blank test . 6
9 Expression of results . 6
9.1 Calculation . 6
9.2 Precision . 7
9.3 Limit of quantification. 7
10 Test report . 7
Annex A (informative) Information of 4,4-dinitrocarbanilide and 4,4-dinitrocarbanilide-d . 8
Annex B (informative) LC-MS/MS chromatograms for 4,4-dinitrocarbanilide and 4,4-
dinitrocarbanilide-d . 9
Annex C (informative) Precision of the method . 10
Bibliography . 11

iii
ISO/FDIS 23851:2026(en)
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights
in respect thereof. As of the date of publication of this document, ISO had not received notice of (a) patent(s)
which may be required to implement this document. However, implementers are cautioned that this may not
represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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 [or Project Committee] ISO/TC [or ISO/PC] ###, [name
of committee], Subcommittee SC ##, [name of subcommittee], in collaboration with the European Committee
for Standardization (CEN) Technical Committee CEN/TC ###, [name of committee], in accordance with the
Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
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
Chicken tissue and eggs — Determination of marker residues of
nicarbazin — Liquid chromatography tandem mass spectrometry
method
1 Scope
This document specifies a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the
determination of marker residues of nicarbazin (4,4-dinitrocarbanilide) in chicken tissue and eggs.
This document is applicable to the determination of marker residues of nicarbazin (4,4-dinitrocarbanilide) in
chicken tissue (including muscle, liver and kidney) and eggs.
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
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
The test portion is extracted with acetonitrile and then purified by acetonitrile-saturated n-hexane or solid
phase extraction column. The 4,4-dinitrocarbanilide is determined and confirmed by LC-MS/MS, and is
quantified with an internal standard method.
5 Reagents and materials
Use only reagents of recognized analytical grade, unless otherwise specified.
5.1 Water, at least grade 1 conforming to ISO 3696.
5.2 Acetonitrile, HPLC grade.
5.3 Formic acid, HPLC grade.
5.4 Ammonium acetate, HPLC grade.
5.5 N,N-Dimethylformamide.
5.6 n-hexane.
5.7 Anhydrous sodium sulfate.
ISO/FDIS 23851:2026(en)
5.8 Formic acid solution, 1 ml/l.
Pipette 1,0 ml formic acid (5.3) into a 1 000 ml one-mark volumetric flask. Dilute to the mark with water (5.1)
and mix well.
5.9 Ammonium acetate-formic acid solution, c(CH COONH ) = 5 mmol/l.
3 4
Weigh 0,38 g of ammonium acetate (5.4), dissolve and dilute to 1 000 ml with 1 ml/l formic acid solution (5.8),
and mix well.
5.10 Acetonitrile-saturated n-hexane.
Add 20 ml of acetonitrile (5.2) to 100 ml of n-hexane (5.6) and mix well. Allow the mixture to stand for a
sufficient period to enable phase separation, and then collect the supernatant.
5.11 4,4-dinitrocarbanilide, purity ≥ 98 %, see Annex A.
5.12 4,4-dinitrocarbanilide-d , purity ≥ 98 %, see Annex A.
5.13 4,4-dinitrocarbanilide stock standard solution, 100 mg/l.
Weigh, to the nearest 0,1 mg, 10 mg of 4,4-dinitrocarbanilide (5.11) and transfer it to a 100 ml one-mark
volumetric flask. Dilute to the mark with N,N-Dimethylformamide (5.5), and mix well.
This stock solution is stable for three months when stored in the dark at −18 °C.
5.14 4,4-dinitrocarbanilide intermediate standard solution, 1 mg/l.
Pipette 1,0 ml of the 4,4-dinitrocarbanilide stock standard solution (5.13) into a 100 ml one-mark volumetric
flask. Dilute to the mark with acetonitrile (5.2) and mix well.
This solution is stable for one month when stored in the dark at a temperature of 0 °C to 4 °C.
5.15 4,4-dinitrocarbanilide-d stock standard solution, 100 mg/l.
Weigh, to the nearest 0,1 mg, 10 mg of 4,4-dinitrocarbanilide-d (5.12) and transfer it to a 100 ml one-mark
volumetric flask. Dilute to the mark with N,N-Dimethylformamide (5.5), and mix well.
This stock solution is stable for three months when stored in the dark at −18 °C.
5.16 4,4-dinitrocarbanilide-d intermediate standard solution, 1 mg/l.
Pipette 1,0 ml of the 4,4-dinitrocarbanilide-d stock standard solution (5.15) into a 100 ml one-mark
volumetric flask. Dilute to the mark with acetonitrile (5.2) and mix well.
This solution is stable for one month when stored in the dark at a temperature of 0 °C to 4 °C.
5.17 Calibration curve standard working solutions
Pipette 0,08 ml, 0,2 ml, 0,5 ml, 2,0 ml and 5,0 ml of 4,4-dinitrocarbanilide intermediate standard solution
(5.14) and 0,5 ml of 4,4-dinitrocarbanilide-d intermediate standard solution (5.16) to five 100 ml one-mark
volumetric flasks. Dilute with acetonitrile (5.2) to obtain standard solutions with a 4,4-dinitrocarbanilide
concentration of 0,8 μg/l, 2,0 μg/l, 5,0 μg/l, 20,0 μg/l and 50,0 μg/l. The concentration of 4,4-
dinitrocarbanilide-d is 5,0
...