oSIST prEN ISO 14501:2020

Milk and milk powder - Determination of aflatoxin M1 content - Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography (ISO/DIS 14501:2019)

Milch und Milchpulver - Bestimmung des Gehalts an Aflatoxin M1 - Reinigung durch Immunaffinitäts-Chromatographie und Bestimmung mit Hochleistungs-Flüssigchromatographie (ISO/DIS 14501:2019)

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/DIS 14501:2019)

Mleko in mleko v prahu - Določevanje vsebnosti aflatoksina M1 - Čiščenje z imunoafinitetno kromatografijo in določevanje s tekočinsko kromatografijo visoke ločljivosti (ISO/DIS 14501:2019)

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN ISO 14501:2020
01-februar-2020
Mleko in mleko v prahu - Določevanje vsebnosti aflatoksina M1 - Čiščenje z
imunoafinitetno kromatografijo in določevanje s tekočinsko kromatografijo visoke
ločljivosti (ISO/DIS 14501:2019)
Milk and milk powder - Determination of aflatoxin M1 content - Clean-up by
immunoaffinity chromatography and determination by high-performance liquid
chromatography (ISO/DIS 14501:2019)
Milch und Milchpulver - Bestimmung des Gehalts an Aflatoxin M1 - Reinigung durch
Immunaffinitäts-Chromatographie und Bestimmung mit Hochleistungs-
Flüssigchromatographie (ISO/DIS 14501:2019)

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/DIS 14501:2019)
Ta slovenski standard je istoveten z: prEN ISO 14501
ICS:
67.100.10 Mleko in predelani mlečni Milk and processed milk
proizvodi products
oSIST prEN ISO 14501:2020 en,fr,de

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

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oSIST prEN ISO 14501:2020
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oSIST prEN ISO 14501:2020
DRAFT INTERNATIONAL STANDARD
ISO/DIS 14501
IDF 171
ISO/TC 34/SC 5 Secretariat: NEN
Voting begins on: Voting terminates on:
2019-12-12 2020-03-05
Milk and milk powder — Determination of aflatoxin M1
content — Clean-up by immunoaffinity chromatography
and determination by high-performance liquid
chromatography

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

ICS: 67.100.10
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT 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
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
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STANDARDS MAY ON OCCASION HAVE TO
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WHICH REFERENCE MAY BE MADE IN
Reference numbers
NATIONAL REGULATIONS.
ISO/DIS 14501:2019(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
IDF 171:2019(E)
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO and IDF 2019
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oSIST prEN ISO 14501:2020
ISO/DIS 14501:2019(E)
IDF 171:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO and IDF 2019

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.
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ii © ISO and IDF 2019 – All rights reserved
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oSIST prEN ISO 14501:2020
ISO/DIS 14501:2019(E)
IDF 171:2019(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 Preparation of test samples ......................................................................................................................................................... 5

8.1.1 Milk .............................................................................................................................................................................................. 5

8.1.2 Milk powder ......................................................................................................................................................................... 5

8.2 Immunoaffinity column preparation ................................................................................................................................... 5

8.3 Test sample purification ................................................................................................................................................................. 5

8.4 High performance liquid chromatography ..................................................................................................................... 6

8.4.1 Pump setting........................................................................................................................................................................ 6

8.4.2 Chromatographic performance ........................................................................................................................... 6

8.4.3 Calibration curve of aflatoxin M ........................................................................................................................

1 6

8.4.4 Analysis of the purified extracts and injection scheme .................................................................. 6

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 ..................................................................................................................................................................... 7

9.2.1 Calculation ............................................................................................................................................................................. 7

9.2.2 Expression of results .................................................................................................................................................... 7

10 Precision ....................................................................................................................................................................................................................... 8

10.1 Interlaboratory test............................................................................................................................................................................. 8

10.2 Repeatability ............................................................................................................................................................................................. 8

10.3 Reproducibility ....................................................................................................................................................................................... 8

11 Test report ................................................................................................................................................................................................................... 8

Annex A (normative) Results of an interlaboratory trial ................................................................................................................ 9

Annex B (informative) Example of an emission spectrum of aflatoxin M .................................................................10

Bibliography .............................................................................................................................................................................................................................11

© ISO and IDF 2019 – All rights reserved iii
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oSIST prEN ISO 14501:2020
ISO/DIS 14501:2019(E)
IDF 171:2019(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 on 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 the following

URL: 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). It is being published jointly by ISO

and IDF.
iv © ISO and IDF 2019 – All rights reserved
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oSIST prEN ISO 14501:2020
ISO/DIS 14501:2019(E)
IDF 171:2019(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 and endorsement.

This document was prepared by the IDF Standing Committee on Analytical Methods for Composition and

the 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 Composition under the aegis of its project leader Mr. Paul Jamieson.
© ISO and IDF 2019 – All rights reserved v
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oSIST prEN ISO 14501:2020
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oSIST prEN ISO 14501:2020
ISO/DIS 14501:2019(E)
DRAFT INTERNATIONAL STANDARD
IDF 171:2019(E)
Milk and milk powder — Determination of aflatoxin M1
content — Clean-up by immunoaffinity chromatography
and determination by high-performance liquid
chromatography
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 LOD is 0,005 μg/kg for liquid milk.

The limit of quantification(LOQ) is 0,1 μg/kg for milk powder and LOQ is 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.

CAUTION 1 The method described in this protocol requires the use of solutions of aflatoxin M . Aflatoxins

are carcinogenic to humans. Attention is drawn to the statement made by the International Agency for Research

[1,2]
on Cancer (WHO) .

CAUTION 2 Protect the laboratory in which the analyses are performed adequately from daylight and keep

aflatoxin 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 may cause loss of aflatoxin.

Moreover, brand new laboratory glassware, before coming into contact with aqueous solutions of aflatoxin,

should be soaked in dilute acid (e.g. sulfuric acid, 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, aequeous solutions and spills, and for glassware coming into contact with carcinogenic

materials. Suitable decontamination procedures have been developed and validated by the International Agency

[1,2]
for Research on Cancer (WHO) .
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 (in μg/l) or mass fraction (in μg/kg) of substances determined by the procedure

specified in this document.
© ISO and IDF 2019 – All rights reserved 1
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oSIST prEN ISO 14501:2020
ISO/DIS 14501:2019(E)
IDF 171:2019(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 detection.
5 Reagents

During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and

distilled or demineralized water or water of equivalent purity.
5.1 Immunoaffinity column

The immunoaffinity column shall contain antibodies against aflatoxin M . The column shall have a

maximum capacity of not less than 100 ng of aflatoxin M (which corresponds to 2 µg/l when a volume

of 50 ml of a test portion is applied). It shall give a recovery of not less than 80 % for aflatoxin M when

a standard solution containing 4 ng of toxin is applied (which corresponds to 80 ng/l when a volume

of 50 ml of sample is applied). Any immunoaffinity column meeting the performance specifications

mentioned above can be used.

The performance of the columns shall be checked regularly and at least once for every batch of columns

(see the procedure in 5.1.1 and 5.1.2).
5.1.1 Capacity check

Dilute 2,0 ml of aflatoxin M standard stock solution (5.4.2) to 50 ml with water. Mix well and apply

the whole volume to the immunoaffinity column carefully following the recommendations given by the

manufacturer for the use of columns. Wash the column and elute the toxin. Determine the amount of

aflatoxin M eluted from the column by HPLC after preparing a suitable dilution of the final eluate.

Calculate the capacity for the aflatoxin. Compare the result with the requirements given in 5.1.

5.1.2 Recovery check

Use a pipette (6.4) to dilute 0,8 ml of aflatoxin M standard working solution of 0,005 µg/ml (5.4.3) to 50

ml with water. Mix well and apply the whole volume to the immunoaffinity column carefully following

the recommendations given by the manufacturer for the use of columns. Wash the column and elute the

toxin. Determine the amount of aflatoxin M eluted from the column by HPLC after preparing a suitable

dilution of the final eluate.

Calculate the recovery for the aflatoxin. Compare the result with the requirements given in 5.1.

The concentration shall
...