SIST EN ISO 14501:2021

Milk and milk powder - Determination of aflatoxin M1 content - Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography (ISO 14501:2021)

This document specifies a method for the determination of aflatoxin M1 content in milk and milk powder. The lowest level of validation is 0,08 µg/kg for whole milk powder, i.e. 0,008 µg/l for reconstituted liquid milk. The limit of detection (LOD) is 0,05 μg/kg for milk powder and 0,005 μg/kg for liquid milk. The limit of quantification (LOQ) is 0,1 μg/kg for milk powder and 0,01 μg/kg for liquid milk.
The method is also applicable to low-fat milk, skimmed milk, low-fat milk powder and skimmed milk powder.

Milch und Milchpulver - Bestimmung des Gehalts an Aflatoxin M1 - Reinigung durch Immunaffinitäts-Chromatographie und Bestimmung mit Hochleistungs-Flüssigchromatographie (ISO 14501:2021)

Dieses Dokument legt ein Verfahren zur Bestimmung des Aflatoxin-M1-Gehalts von Milch und Milchpulver fest. Der kleinste Validierungswert beträgt 0,08 μg/kg für Vollmilchpulver, d. h. 0,008 μg/l für rekonstituierte flüssige Milch. Die Nachweisgrenze (LOD, en: limit of detection) beträgt 0,05 μg/kg für Milchpulver sowie 0,005 μg/kg für flüssige Milch. Die Bestimmungsgrenze (LOQ, en: limit of quantification) beträgt 0,1 μg/kg für Milchpulver sowie 0,01 μg/kg für flüssige Milch.
Das Verfahren ist auch auf fettarme Milch, Magermilch, fettarmes Milchpulver und Magermilchpulver anwendbar.

Lait et lait en poudre - Détermination de la teneur en aflatoxine M1 - Purification par chromatographie d'immunoaffinité et détermination par chromatographie en phase liquide à haute performance (ISO 14501:2021)

Le présent document spécifie une méthode pour le dosage de l’aflatoxine M1 dans le lait et le lait en poudre. Le niveau de validation le plus bas est de 0,08 µg/kg de poudre de lait entier, ce qui correspond à 0,008 µg/l de lait liquide reconstitué. La limite de détection (LOD) est de 0,05 μg/kg pour le lait en poudre et de 0,005 μg/kg pour le lait liquide. La limite de quantification (LOQ) est de 0,1 μg/kg pour le lait en poudre et de 0,01 μg/kg pour le lait liquide.
La méthode est également applicable au lait à faible teneur en matière grasse, au lait écrémé et au lait en poudre à faible teneur en matière grasse ou écrémé.

Mleko in mleko v prahu - Določevanje vsebnosti aflatoksina M1 - Čiščenje z imunoafinitetno kromatografijo in določevanje s tekočinsko kromatografijo visoke ločljivosti (ISO 14501:2021)

General Information

• Status: Published
• Public Enquiry End Date: 19-Feb-2020
• Publication Date: 16-Jun-2021
• ICS: 67.100.10 - Milk and processed milk products
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 03-Jun-2021
• Due Date: 08-Aug-2021
• Completion Date: 17-Jun-2021
• Ref Project: EN ISO 14501:2021 - Milk and milk powder - Determination of aflatoxin M1 content - Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography (ISO 14501:2021)

Relations

• Replaces: SIST EN ISO 14501:2007 - Milk and milk powder - Determination of aflatoxin M1 content - Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography (ISO 14501:2007)
• Effective Date: 02-Jun-2021

Standards Content (Sample)

SLOVENSKI STANDARD
01-julij-2021
Nadomešča:
SIST EN ISO 14501:2007
Mleko in mleko v prahu - Določevanje vsebnosti aflatoksina M1 - Čiščenje z
imunoafinitetno kromatografijo in določevanje s tekočinsko kromatografijo visoke
ločljivosti (ISO 14501:2021)
Milk and milk powder - Determination of aflatoxin M1 content - Clean-up by
immunoaffinity chromatography and determination by high-performance liquid
chromatography (ISO 14501:2021)
Milch und Milchpulver - Bestimmung des Gehalts an Aflatoxin M1 - Reinigung durch
Immunaffinitäts-Chromatographie und Bestimmung mit Hochleistungs-
Flüssigchromatographie (ISO 14501:2021)
Lait et lait en poudre - Détermination de la teneur en aflatoxine M1 - Purification par
chromatographie d'immunoaffinité et détermination par chromatographie en phase
liquide à haute performance (ISO 14501:2021)
Ta slovenski standard je istoveten z: EN ISO 14501:2021
ICS:
67.100.10 Mleko in predelani mlečni Milk and processed milk
proizvodi products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 14501
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2021
EUROPÄISCHE NORM
ICS 67.100.10 Supersedes EN ISO 14501:2007
English Version
Milk and milk powder - Determination of aflatoxin M1
content - Clean-up by immunoaffinity chromatography and
determination by high-performance liquid
chromatography (ISO 14501:2021)
Lait et lait en poudre - Détermination de la teneur en Milch und Milchpulver - Bestimmung des Gehalts an
aflatoxine M1 - Purification par chromatographie Aflatoxin M1 - Reinigung durch Immunaffinitäts-
d'immunoaffinité et détermination par Chromatographie und Bestimmung mit Hochleistungs-
chromatographie en phase liquide à haute Flüssigchromatographie (ISO 14501:2021)
performance (ISO 14501:2021)
This European Standard was approved by CEN on 12 April 2021.

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. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists 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, 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
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 14501:2021 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 14501:2021) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 302 “Milk and milk products - Methods of
sampling and analysis” the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by November 2021, and conflicting national standards
shall be withdrawn at the latest by November 2021.
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.
This document supersedes EN ISO 14501:2007.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: 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.
Endorsement notice
The text of ISO 14501:2021 has been approved by CEN as EN ISO 14501:2021 without any modification.

INTERNATIONAL ISO
STANDARD 14501
IDF 171
Third edition
2021-05
Milk and milk powder —
Determination of aflatoxin M content
— Clean-up by immunoaffinity
chromatography and determination
by high-performance liquid
chromatography
Lait et lait en poudre — Détermination de la teneur en aflatoxine
M — Purification par chromatographie d'immunoaffinité et
détermination par chromatographie en phase liquide à haute
performance
Reference numbers
ISO 14501:2021(E)
IDF 171:2021(E)
©
ISO and IDF 2021
ISO 14501:2021(E)
IDF 171:2021(E)
© ISO and IDF 2021
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 International Dairy Federation
CP 401 • Ch. de Blandonnet 8 Silver Building • Bd Auguste Reyers 70/B
CH-1214 Vernier, Geneva B-1030 Brussels
Phone: [phone] Phone: [phone]
Fax: [phone]
Email: [e-mail] Email: [e-mail]
Website: www.iso.org Website: www.fil-idf.org
Published in Switzerland
ii © ISO and IDF 2021 – All rights reserved

ISO 14501:2021(E)
IDF 171:2021(E)
Contents Page
Forewords .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 2
6 Apparatus . 4
7 Sampling . 5
8 Procedure. 5
8.1 General . 5
8.2 Preparation of test samples . 5
8.2.1 Milk . 5
8.2.2 Milk powder . 5
8.3 Immunoaffinity column preparation . 6
8.4 Test sample purification . 6
8.5 High performance liquid chromatography (HPLC) . 6
8.5.1 Pump setting. 6
8.5.2 Chromatographic performance . 6
8.5.3 Calibration curve of aflatoxin M . 7
8.5.4 Analysis of the purified extracts and injection scheme . 7
9 Calculation and expression of results . 7
9.1 Skimmed milk . 7
9.1.1 Calculation . 7
9.1.2 Expression of results . 7
9.2 Skimmed milk powder . 8
9.2.1 Calculation . 8
9.2.2 Expression of results . 8
10 Precision . 8
10.1 Interlaboratory test. 8
10.2 Repeatability . 8
10.3 Reproducibility . 8
11 Test report . 9
Annex A (informative) Results of an interlaboratory trial .10
Bibliography .11
ISO 14501:2021(E)
IDF 171:2021(E)
Forewords
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 5,
Milk and milk products, and the International Dairy Federation (IDF), in collaboration with the European
Committee for Standardization (CEN) Technical Committee CEN/TC 302, Milk and milk products -
Methods of sampling and analysis, in accordance with the Agreement on technical cooperation between
ISO and CEN (Vienna Agreement). It is being published jointly by ISO and IDF.
This third edition cancels and replaces the second edition (ISO 14501 | IDF 171:2007), which has been
technically revised. The main changes compared with the previous edition are as follows:
— the lack of detailed explanation in some clauses was leading to variability in the way the method
was executed from one laboratory to another. Practical information from skilled end users familiar
with routine analysis using this protocol was taken into account in this revision to clarify those
ambiguous clauses.
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 and IDF 2021 – All rights reserved

ISO 14501:2021(E)
IDF 171:2021(E)
IDF (the International Dairy Federation) is a non-profit private sector organization representing the
interests of various stakeholders in dairying at the global level. IDF members are organized in National
Committees, which are national associations composed of representatives of dairy-related national
interest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and
governments/food control authorities.
ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis
and sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International
Standards using the logos and reference numbers of both organizations.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. IDF 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.
This document was prepared by the IDF Standing Committee on Analytical Methods for Additives and
Contaminants and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk
products. It is being published jointly by ISO and IDF.
The work was carried out by the IDF-ISO Action Team on A12 of the Standing Committee on Analytical
Methods for Additives and Contaminants under the aegis of its project leader Mr Paul Jamieson.
ISO 14501:2021(E)
INTERNATIONAL STANDARD
IDF 171:2021(E)
Milk and milk powder — Determination of aflatoxin M
content — Clean-up by immunoaffinity chromatography
and determination by high-performance liquid
chromatography
CAUTION 1 — The method described in this document requires the use of solutions of
aflatoxin M . Aflatoxins are carcinogenic to humans. Attention is drawn to the statement made
[1][2]
by the International Agency for Research on Cancer (WHO) .
CAUTION 2 — Adequately protect the laboratory in which the analyses are performed from
daylight and keep aflatoxin M standard solutions protected from light, e.g. by using aluminium
foil.
CAUTION 3 — The use of non-acid-washed glassware (e.g. tubes, vials, flasks, beakers, syringes)
for aqueous aflatoxin solutions can cause loss of aflatoxin M . Moreover, brand new laboratory
glassware, before coming into contact with aqueous solutions of aflatoxin M , should be soaked
in dilute acid (e.g. sulfuric acid, c = 2 mol/l) for several hours, then rinsed well with distilled
water to remove all traces of acid (check to ensure pH is in the range 6 to 8).
CAUTION 4 — Use decontamination procedures for laboratory wastes such as solid compounds,
solutions in organic solvents, aqueous solutions and spills, and for glassware coming into contact
with carcinogenic materials. Suitable decontamination procedures have been developed and
[1][2]
validated by the International Agency for Research on Cancer (WHO) .
1 Scope
This document specifies a method for the determination of aflatoxin M content in milk and milk powder.
The lowest level of validation is 0,08 µg/kg for whole milk powder, i.e. 0,008 µg/l for reconstituted
liquid milk. The limit of detection (LOD) is 0,05 μg/kg for milk powder and 0,005 μg/kg for liquid milk.
The limit of quantification (LOQ) is 0,1 μg/kg for milk powder and 0,01 μg/kg for liquid milk.
The method is also applicable to low-fat milk, skimmed milk, low-fat milk powder and skimmed milk
powder.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at https:// www .iso .org/ obp
3.1
aflatoxin M content
concentration or mass fraction of substances determined by the procedure specified in this document
Note 1 to entry: Concentration is expressed in μg/l and mass fraction is expressed in in μg/kg.
ISO 14501:2021(E)
IDF 171:2021(E)
4 Principle
Aflatoxin M is extracted by passing the test portion through an immunoaffinity column that contains
specific antibodies bound onto a solid support material.
As the sample passes through the column, the antibodies are selectively bound with any aflatoxin M
(antigen) present and form an antibody-antigen complex. All other components of the sample matrix
are washed off the column with water. Then aflatoxin M is eluted from the column and the eluate is
collected. The amount of aflatoxin M present in this eluate is determined by means of high-performance
liquid chromatography (HPLC) coupled with fluorimetric d
...