SIST EN ISO 14501:2007
(Main)Milk and milk powder - Determination of aflatoxin M1 content - Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography (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)
This International Standard specifies a method for the determination of aflatoxin M1 content in milk and milk powder. The limit of detection is 0,08 g/kg for whole milk powder, i.e. 0,008 g/l for reconstituted 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- Reiningung durch Immunaffinitäts-Chromatographie und Bestimmung mit Hochleistungs-Flüssigchromatographie (ISO 14501:2007)
Diese Internationale Norm legt ein Verfahren zur Bestimmung des Aflatoxin M1 Gehalts von Milch und Milchpulver fest. Der unterste Grenzwert der Validierung ist 0,08 µg/kg für Vollmilchpulver, d. h. 0,008 µg/l für rekonstituierte Milch.
Das Verfahren ist auch auf fettarme Milch, Magermilch, fettarmes Milchpulver und Magermilchpulver anwendbar.
WARNUNG
1 Bei dem in diesem Dokument beschriebenen Verfahren müssen Lösungen von Aflatoxin M1 verwendet werden. Aflatoxine sind für den Menschen karzinogen. Zu beachten sind die Feststellungen der International Agency for Research on Cancer [4], [5].
2 Das die Untersuchungen durchführende Laboratorium ist ausreichend vor Tageslicht zu schützen, und die Aflatoxin-Standardlösungen sind gegen Licht zu schützen, z. B. mit Hilfe von Aluminiumfolie.
3 Die Verwendung von Glasgeräten (z. B. Röhrchen, Flaschen, Kolben, Bechergläser, Spritzen), die nicht mit Säure gewaschen worden sind, kann Verluste an Aflatoxin verursachen.
Deshalb sollten neue Laborgeräte aus Glas, die mit wässrigen Aflatoxinlösungen in Berührung kommen, für einige Stunden in verdünnter Säure (z. B. Schwefelsäure, c = 2 mol/l) gelagert und danach zur Beseitigung aller Säurespuren gut mit destilliertem Wasser gespült werden. Es ist sicherzustellen, dass der pH-Wert zwischen 6 und 8 liegt.
4 Laboratoriumsabfälle wie feste chemische Stoffe, Lösungen in organischen Lösemitteln, wässrige Lösungen und vergossene Flüssigkeiten sowie Glasgeräte, die in Kontakt mit karzinogenen Materialien kommen, sind einer Dekontamination zu unterziehen. Geeignete Verfahren zur Dekontamination wurden von der International Agency for Research on Cancer entwickelt und validiert [4], [5].
Lait et lait en poudre - Détermination de la teneur en aflatoxine M1 - Purification par chromatographie d'immunoaffinité et détermination par chromatographie en phase liquide a haute performance (ISO 14501:2007)
L'ISO 14501| FIL 171:2007 spécifie une méthode pour le dosage de l'aflatoxine M1 dans le lait et le lait en poudre. La limite de détection se situe à 0,08 mg/kg de poudre de lait entier, ce qui correspond à 0,008 mg/l de lait liquide reconstitué.
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 14501:2007)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 14501:2007
01-december-2007
1DGRPHãþD
SIST EN ISO 14501:1999
0OHNRLQPOHNRYSUDKX'RORþHYDQMHYVHEQRVWLDIODWRNVLQD0ýLãþHQMH]
LPXQRDILQLWHWQRNURPDWRJUDILMRLQGRORþHYDQMHVWHNRþLQVNRNURPDWRJUDILMRYLVRNH
ORþOMLYRVWL ,62
Milk and milk powder - Determination of aflatoxin M1 content - Clean-up by
immunoaffinity chromatography and determination by high-performance liquid
chromatography (ISO 14501:2007)
Milch und Milchpulver - Bestimmung des Gehalts an Aflatoxin M1- Reiningung durch
Immunaffinitäts-Chromatographie und Bestimmung mit Hochleistungs-
Flüssigchromatographie (ISO 14501:2007)
Lait et lait en poudre - Détermination de la teneur en aflatoxine M1 - Purification par
chromatographie d'immunoaffinité et détermination par chromatographie en phase
liquide a haute performance (ISO 14501:2007)
Ta slovenski standard je istoveten z: EN ISO 14501:2007
ICS:
67.100.10 0OHNRLQSUHGHODQLPOHþQL Milk and processed milk
SURL]YRGL products
SIST EN ISO 14501:2007 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
EUROPEAN STANDARD
EN ISO 14501
NORME EUROPÉENNE
EUROPÄISCHE NORM
October 2007
ICS 67.100.10 Supersedes EN ISO 14501:1998
English Version
Milk and milk powder - Determination of aflatoxin M1 content -
Clean-up by immunoaffinity chromatography and determination
by high-performance liquid chromatography (ISO 14501:2007)
Lait et lait en poudre - Détermination de la teneur en Milch und Milchpulver - Bestimmung des Gehalts an
aflatoxine M1 - Purification par chromatographie Aflatoxin M1- Reiningung durch Immunaffinitäts-
d'immunoaffinité et détermination par chromatographie en Chromatographie und Bestimmung mit Hochleistungs-
phase liquide à haute performance (ISO 14501:2007) Flüssigchromatographie (ISO 14501:2007)
This European Standard was approved by CEN on 13 October 2007.
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 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 Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2007 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 14501:2007: E
worldwide for CEN national Members.
---------------------- Page: 2 ----------------------
EN ISO 14501:2007 (E)
Contents Page
Foreword.3
2
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EN ISO 14501:2007 (E)
Foreword
This document (EN ISO 14501:2007) has been prepared by Technical Committee ISO/TC 34 "Agricultural
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 April 2008, and conflicting national standards shall be withdrawn at the
latest by April 2008.
This document supersedes EN ISO 14501:1998.
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, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 14501:2007 has been approved by CEN as a EN ISO 14501:2007 without any modification.
3
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INTERNATIONAL ISO
STANDARD 14501
IDF
171
Second edition
2007-10-15
Milk and milk powder — Determination of
aflatoxin M content — Clean-up by
1
immunoaffinity chromatography and
determination by high-performance liquid
chromatography
Lait et lait en poudre — Détermination de la teneur en aflatoxine M —
1
Purification par chromatographie d'immunoaffinité et détermination par
chromatographie en phase liquide à haute performance
Reference numbers
ISO 14501:2007(E)
IDF 171:2007(E)
©
ISO and IDF 2007
---------------------- Page: 5 ----------------------
ISO 14501:2007(E)
IDF 171:2007(E)
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Published in Switzerland
ii © ISO and IDF 2007 – All rights reserved
---------------------- Page: 6 ----------------------
ISO 14501:2007(E)
IDF 171:2007(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee has
been established has the right to be represented on that committee. International organizations, governmental
and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 14501⎪IDF 171 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.
This second edition of ISO 14501⎪IDF 171 cancels and replaces the first edition (ISO 14501:1998), which has
been technically revised.
© ISO and IDF 2007 – All rights reserved iii
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ISO 14501:2007(E)
IDF 171:2007(E)
Foreword
IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National
Committee in every member country. Every National Committee has the right to be represented on the IDF
Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of
standard methods of analysis and sampling for milk and milk products.
Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the
National Committees for voting. Publication as an International Standard requires approval by at least 50 % of
the IDF National Committees casting a vote.
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.
ISO 14501⎪IDF 171 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.
All work was carried out by the Joint IDF-ISO Action Team on Organic contaminants of the Standing
Committee on Analytical methods for additives and contaminants under the aegis of its project leader,
Mr. L. Sørensen (DK).
This edition of ISO 14501⎪IDF 171 cancels and replaces IDF 171:1995, which has been technically revised.
iv © ISO and IDF 2007 – All rights reserved
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ISO 14501:2007(E)
INTERNATIONAL STANDARD
IDF 171:2007(E)
Milk and milk powder — Determination of aflatoxin M
1
content — Clean-up by immunoaffinity chromatography and
determination by high-performance liquid chromatography
1 Scope
This International Standard specifies a method for the determination of aflatoxin M content in milk and milk
1
powder. The limit of detection is 0,08 µg/kg for whole milk powder, i.e. 0,008 µg/l for reconstituted 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
1
carcinogenic to humans. Attention is drawn to the statement made by the International Agency for
[4], [5]
Research on Cancer .
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.
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).
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 validated
[4], [5]
by the International Agency for Research on Cancer .
2 Terms and definitions
For the purposes of this document the following terms and definitions apply.
2.1
aflatoxin M content
1
concentration or mass fraction of substances determined by the procedure specified in this International
Standard.
NOTE The aflatoxin M concentration is expressed in micrograms per litre and the mass fraction in micrograms per
1
kilogram.
3 Principle
Aflatoxin M is extracted by passing the test portion through an immunoaffinity column that contains specific
1
antibodies bound onto a solid support material.
© ISO and IDF 2007 – All rights reserved 1
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ISO 14501:2007(E)
IDF 171:2007(E)
As the sample passes through the column, the antibodies are selectively bound with any aflatoxin M
1
(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
1
amount of aflatoxin M present in this eluate is determined by means of high-performance liquid
1
chromatography (HPLC) coupled with fluorimetric detection.
4 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and only distilled or
demineralized water or water of equivalent purity.
4.1 Immunoaffinity column.
The immunoaffinity column shall contain antibodies against aflatoxin M . The column shall have a maximum
1
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
1
portion is applied). It shall give a recovery of not less than 80 % for aflatoxin M when a standard solution
1
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 4.1.1 and 4.1.2).
4.1.1 Capacity check.
Dilute 1,0 ml of aflatoxin M standard stock solution (4.4.2) to 50 ml with water. Mix well and apply the whole
1
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
1
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 4.1.
4.1.2 Recovery check.
Use a pipette (5.4) to dilute 0,8 ml of aflatoxin M standard working solution of 0,005 µg/ml (4.4.3) to 10 ml
1
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
1
the final eluate.
Calculate the recovery for the aflatoxin. Compare the result with the requirements given in 4.1.
4.2 Acetonitrile, pure, HPLC grade.
4.2.1 Acetonitrile solution, 25 %.
Add 250 ml of acetonitrile (4.2) to 750 ml of water and mix. Other volumes in the same proportion may be
used. Degas the solution (eluent) before using it.
4.2.2 Acetonitrile solution, 10 %.
Add 100 ml of acetonitrile (4.2) to 900 ml of water and mix. Other volumes in the same proportion may be
used. Degas the solution before using it.
4.3 Nitrogen gas.
4.4 Aflatoxin M standard solutions.
1
4.4.1 Aflatoxin M standard calibration solution.
1
2 © ISO and IDF 2007 – All rights reserved
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ISO 14501:2007(E)
IDF 171:2007(E)
Prepare an aflatoxin M standard calibration solution by dissolving aflatoxin M (C H O ) in acetonitrile (4.2)
1 1 17 12 7
to give a nominal concentration of 10 µg/ml. Determine the actual aflatoxin M concentration by measurement
1
of the absorbance at the maximum absorption wavelength of the solution as follows.
Use the spectrophotometer (5.14) to record the absorbance of the aflatoxin M standard calibration solution
1
against acetonitrile (4.2) 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, c , expressed in micrograms per millilitre, by using Equation (1):
1
100
cA=×M× (1)
1
d×ε
where:
A is the numerical value of the absorbance at λ ;
max
M is the molar mass, in grams per mole, of aflatoxin M (M = 328 g/mol);
1
d is the optical pathlength, in centimetres (d = 1 cm);
ε is the numerical value of t
...
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