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)

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
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
26-May-2021
Completion Date
26-May-2021

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SLOVENSKI STANDARD
SIST EN ISO 14501:2021
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
SIST EN ISO 14501:2021 en,fr,de

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

---------------------- Page: 1 ----------------------
SIST EN ISO 14501:2021
---------------------- Page: 2 ----------------------
SIST EN ISO 14501:2021
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.
---------------------- Page: 3 ----------------------
SIST EN ISO 14501:2021
EN ISO 14501:2021 (E)
Contents Page

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

---------------------- Page: 4 ----------------------
SIST EN ISO 14501:2021
EN ISO 14501:2021 (E)
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.

---------------------- Page: 5 ----------------------
SIST EN ISO 14501:2021
---------------------- Page: 6 ----------------------
SIST EN ISO 14501:2021
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
---------------------- Page: 7 ----------------------
SIST EN ISO 14501:2021
ISO 14501:2021(E)
IDF 171:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© 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: +41 22 749 01 11 Phone: +32 2 325 67 40
Fax: +32 2 325 67 41
Email: copyright@iso.org Email: info@fil-idf.org
Website: www.iso.org Website: www.fil-idf.org
Published in Switzerland
ii © ISO and IDF 2021 – All rights reserved
---------------------- Page: 8 ----------------------
SIST EN ISO 14501:2021
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 and IDF 2021 – All rights reserved iii
---------------------- Page: 9 ----------------------
SIST EN ISO 14501:2021
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
---------------------- Page: 10 ----------------------
SIST EN ISO 14501:2021
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 and IDF 2021 – All rights reserved v
---------------------- Page: 11 ----------------------
SIST EN ISO 14501:2021
---------------------- Page: 12 ----------------------
SIST EN ISO 14501:2021
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 and IDF 2021 – All rights reserved 1
---------------------- Page: 13 ----------------------
SIST EN ISO 14501:2021
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 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.

The procedure is as follows.
a) Capacity check:

1) dilute 2,0 ml of aflatoxin M standard stock solution (5.4.2) to 50 ml with water;

2) mix well and apply the whole volume to the immunoaffinity column, carefully following the

recommendations given by the manufacturer for the use of columns;
3) wash the column and elute the toxin;

4) determine the amount of aflatoxin M eluted from the column by HPLC after preparing a

suitable dilution of the final eluate;
5) calculate the capacity for the aflatoxin;
6) compare the result with the requirements given above.
b) Recovery check:

1) 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;

2) mix well and apply the whole volume to the immunoaffinity column, carefully following the

recommendations given by the manufacturer for the use of columns;
3) wash the column and elute the toxin;

4) determine the amount of aflatoxin M eluted from the column by HPLC after preparing a

suitable dilution of the final eluate;
® ®

1) Examples of immuno affinity columns: Afla Test P Vicam , Aflaprep M R-Biopharm. Similar products are

® ® ®

also available from Romer Labs , Bioo Scientific and Neogen . These products are examples of suitable products

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

constitute an endorsement by ISO and/or IDF of these products.
2 © ISO and IDF 2021 – All rights reserved
---------------------- Page: 14 ----------------------
SIST EN ISO 14501:2021
ISO 14501:2021(E)
IDF 171:2021(E)
5) calculate the recovery for the aflatoxin;

6) compare the result with the requirements given above. The concentration shall not be less

than 0,064 μg/l. Recovery checks can also be conducted with commercially available reference

materials.
5.2 Acetonitrile, pure, HPLC grade.
5.2.1 Acetonitrile solution, volume fraction of 25 % in water.

Add 250 ml of acetonitrile (5.2) to 750 ml of water and mix. Other volumes in the same proportion may

be used. Degas the solution (eluent) before using it.
5.2.2 Acetonitrile solution, volume fraction of 10 % in water.

Add 100 ml of acetonitrile (5.2) to 900 ml of water and mix. Other volumes in the same proportion may

be used. Degas the solution (eluent) before using it.
5.3 Nitrogen gas.
5.4 Aflatoxin M standard solutions.

5.4.1 Aflatoxin M standard calibration solution, (mass concentration ρ = 10 μg/ml aflatoxin M in

1 1
acetonitrile).

Prepare an aflatoxin M standard calibration solution by dissolving aflatoxin M (C H O ) in

1 1 17 12 7

acetonitrile (5.2) to give a nominal concentration of 10 μg/ml. Determine the actual aflatoxin M

concentration by measurement of the absorbance at the maximum absorption wavelength of the

solution as follows.

Use the spectrophotometer (6.13) to record the absorbance of the aflatoxin M standard calibration

solution against acetonitrile (5.2) as blank at wavelengths between 330 nm and 370 nm. Measure the

absorbance, A, at its maximum absorption wavelength, λ , which is close to 350 nm.

max

Calculate the concentration, ρ , expressed in micrograms per millilitre, by using Formula (1):

100
ρ =×AM× (1)
d×ε
where
A is the numerical value of the absorbance at λ ;
max
M is the molar mass of aflatoxin M , in grams per mole (M = 328 g/mol);
d is the optical path length, in centimetres (d = 1 cm);

ε is the numerical value of the absorption coefficient of the toxin in acetonitrile, in square

2 . −1
metres per mole (ε = 1 985 m mol ).

Alternatively, certified reference materials are available commercially (for example BCR-423 10 μg/ml

aflatoxin M in chloroform).

5.4.2 Aflatoxin M standard stock solution, (ρ = 0,1 μg/ml aflatoxin M in acetonitrile).

1 1

After checking its concentration, dilute the aflatoxin M standard calibration solution (5.4.1) with 25 %

acetonitrile (5.2.1) to an aflatoxin M standard stock solution of 0,1 µg/ml. The standard stock solution

shall be well-stoppered and wrapped in aluminium foil to protect it from light.
© ISO and IDF 2021 – All rights reserved 3
---------------------- Page: 15 ----------------------
SIST EN ISO 14501:2021
ISO 14501:2021(E)
IDF 171:2021(E)

Store the aflatoxin M standard stock solution in a refrigerator at a temperature between 1 °C and 5 °C

in the dark. Under these conditions the stock solution is stable for at least two months. If the standard

stock solution is more than two months old, determine the aflatoxin M concentration before use. If

there is any change, discard the solution and prepare a fresh standard stock solution.

5.4.3 Aflatoxin M standard working solutions, (ρ = 0,005 μg/ml aflatoxin M in a mixture of

1 1
nine parts per volume of water and one part per volume of acetonitrile).

Before preparing the aflatoxin M standard working solutions, allow the standard stock solution (5.4.2)

to attain ambient temperature. Prepare the standard working solutions on the day of use.

Dilute the aflatoxin M standard stock solution (5.4.2) with the 10 % acetonitrile solution (5.2.2) to an

aflatoxin M concentration of 0,005 µg/ml.

Remove aliquots of the diluted standard stock solution to prepare a series of five standard working

solutions containing, for example, 0,05 ng/ml, 0,10 ng/ml, 0,20 ng/ml, 0,40 ng/ml, and 0,80 ng/ml of

aflatoxin M by diluting with the 10 % acetonitrile solution (5.2.2). Other final dilutions may be chosen,

depending on the injection loop volume.

In some cases, better peak shape may be obtained with diluting the aflatoxin M standard stock

solution (5.4.2) with a mixture of water and acetonitrile in the same acetonitrile/water ratio as the

eluent (5.2.1).
6 Apparatus
Usual laboratory equipment and, in particular, the following:
6.1 Disposable syringes, of capacities 10 ml and 50 ml.
6.2 Vacuum system, e.g. Büchner flask, Vac-Elut system or peristaltic pump.
6.3 Centrifuge, capable of producing a radial acceleration of at least 2 000 g.
6.4 Pipettes, of capacities 1,0 ml, 2,0 ml and 50,0 ml, or suitable autopipette.
6.5 Glass beakers, of capacity 250 ml.
6.6 One-mark volumetric flask, of capacity 100 ml.

6.7 Water baths, capable of operating at 30 °C ± 2 °C, at between 35 °C and 37 °C and 50 °C ± 5 °C.

6.8 Filter paper, Whatman No. 4 or equivalent.

6.9 Graduated conical glass tubes, with ground glass neck and stopper of capacities 5 ml, 10 ml and

20 ml.
6.10 HPLC ap
...

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.

---------------------- Page: 1 ----------------------
oSIST prEN ISO 14501:2020
---------------------- Page: 2 ----------------------
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
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oSIST prEN ISO 14501:2020
ISO/DIS 14501:2019(E)
IDF 171:2019(E)
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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

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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).

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For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and

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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
...

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