SIST EN ISO 14501:1999
(Main)Milk and milk powder - Determination of aflatoxin M1 content - Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography (ISO 14501:1998)
Milk and milk powder - Determination of aflatoxin M1 content - Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography (ISO 14501:1998)
Milch und Milchpulver - Bestimmung des Gehalts an Aflatoxin M1 - Reinigung durch Immunaffinitäts-Chromatographie und Bestimmung mit Hochleistungs-Flüssigchromatographie (ISO 14501:1998)
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 ug/kg für Vollmilchpulver, d.h. 0,008 ug/l für rekonstituierte 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 a haute performance (ISO 14501:1998)
Mleko in mleko v prahu – Določevanje vsebnosti aflatoksina M1 - Čiščenje z imunoafinitetno kromatografijo in določevanje s tekočinsko kromatografijo visoke ločljivosti (ISO 14501:1998)
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 14501:1999
01-maj-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 14501:1998)
Milch und Milchpulver - Bestimmung des Gehalts an Aflatoxin M1 - Reinigung durch
Immunaffinitäts-Chromatographie und Bestimmung mit Hochleistungs-
Flüssigchromatographie (ISO 14501:1998)
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 14501:1998)
Ta slovenski standard je istoveten z: EN ISO 14501:1998
ICS:
67.100.10 0OHNRLQSUHGHODQLPOHþQL Milk and processed milk
SURL]YRGL products
SIST EN ISO 14501:1999 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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INTERNATIONAL ISO
STANDARD 14501
First edition
1998-11-15
Milk and milk powder — Determination of
aflatoxin M content — Clean-up
1
by 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
A
Reference number
ISO 14501:1998(E)
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ISO 14501:1998(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 3.
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.
International Standard ISO 14501 was prepared by Technical Committee
ISO/TC 34, Agricultural food products, Subcommittee SC 5, Milk and milk
products, in collaboration with International Dairy Federation (IDF) and the
Association of Official Analytical Chemists (AOAC International), and will
also be published by these organizations.
Annex A of this International Standard is for information only.
© ISO 1998
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced
or utilized in any form or by any means, electronic or mechanical, including photocopying and
microfilm, without permission in writing from the publisher.
International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet iso@iso.ch
Printed in Switzerland
ii
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INTERNATIONAL STANDARD © ISO ISO 14501:1998(E)
Milk and milk powder — Determination of aflatoxin M content —
1
Clean-up by immunoaffinity chromatography and determination by
high-performance liquid chromatography
WARNINGS
1 The method described in this International Standard requires the use of chloroform and aflatoxin M
1
solutions. Chloroform is an ozone-depleting substance. Aflatoxins are carcinogenic to human subjects.
Attention is drawn to the statement made by the International Agency for Research on Cancer (WHO) [4, 5].
2 Adequately protect from daylight the laboratory where the analyses are performed, and keep
aflatoxin standard solutions protected from light, for example 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 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 a decontamination procedure for laboratory wastes such as solid compounds, solutions in
organic solvents, glassware, aqueous solutions and spills. The procedure for decontamination was
developed and validated in a programme of the International Agency for Research on Cancer (WHO) [4, 5]
1 Scope
This International Standard specifies a method for the determination of aflatoxin M
content of milk and milk
1
powder. The lowest level of validation is 0,08 mg/kg for whole milk powder i.e. 0,008 mg/l for reconstituted liquid milk.
The method is also applicable to low fat milk, skimmed milk, low fat milk powder and skimmed milk powder.
2 Term and definition
For the purposes of this International Standard, the following term and definition apply.
2.1
aflatoxin M content
1
mass fraction of substances determined by the procedure specified in this International Standard
NOTE The aflatoxin M content is expressed as micrograms per litre or micrograms per kilogram.
1
1
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© ISO
ISO 14501:1998(E)
3 Principle
Aflatoxin M is extracted by passing the test portion through an immunoaffinity column. The column contains
1
specific antibodies bound onto a solid support material. As the sample passes through the column, the antibodies
selectively bind with any aflatoxin M (antigen) present and form an antibody-antigen complex. All other
1
components of the sample matrix are washed off the column with water. Then aflatoxin M is eluted from the
1
column and the eluate is collected. The amount of aflatoxin M present in this eluate is determined by means of
1
high-performance liquid chromatography (HPLC) coupled with fluorimetric detection.
4 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and 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 test portion
1
ed), and shall give a recovery of not less than 80 % for aflatoxin M when a standard solution containing 4 ng
is appli
1
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 4.1.1 and 4.1.2).
4.1.1 Capacity check
By means of a pipette (5.4) transfer 1,0 ml of the stock aflatoxin M solution (4.5.2) to a 20 ml conical tube (5.9).
1
Evaporate the solution slowly to dryness using a constant stream of nitrogen (4.3) and dissolve the residue obtained
in 10 ml of the 10 % acetonitrile solution (4.2.2). Shake vigorously.
Add this solution to 40 ml of water. Mix well and apply the whole volume to the immunoaffinity column. Be careful to
follow the recommendations given by the manufacturer for the use of the columns. Wash the column, elute the toxin
and determine the amount bound to the column by HPLC after suitable dilution of the final eluate.
Calculate the recovery for the aflatoxin. Compare the result with the specification under 4.1.
4.1.2 Recovery check
By means of a pipette (5.4) dilute 0,8 ml of the 0,005 mg/ml aflatoxin M working solution (4.5.3) to 10 ml with water.
1
Mix well and apply the whole volume to the immunoaffinity column. Be careful to follow the recommendations given
by the manufacturer for the use of the columns. Wash the column, elute the toxin and determine the amount bound
to the column by HPLC after suitable dilution of the final eluate. Calculate the recovery for the aflatoxin. Compare
the result with the specification under 4.1.
4.2 Acetonitrile, pure, HPLC grade.
4.2.1 Acetonitrile, in water, 25 % solution by volume.
Add 250 ml of acetonitrile (4.2) to 750 ml of water (degas before use).
4.2.2 Acetonitrile, in water, 10 % solution by volume.
Add 100 ml of acetonitrile (4.2) to 900 ml of water (degas before use).
2
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© ISO
ISO 14501:1998(E)
4.3 Nitrogen gas
4.4 Chloroform, stabilized with 0,5 % to 1,0 % ethanol (by mass).
4.5 Aflatoxin M standard solutions
1
4.5.1 Calibrant solution
Standard solution of aflatoxin M in chloroform with a nominal concentration of 10 μg/ml.
1
Determine the concentration by measurement of its absorbance at the wavelength for maximum absorption as
follows.
By using the spectrometer (5.11), record the absorbance of the calibrant solution against chloroform as blank
between 340 nm and 370 nm. Measure the absorbance, A, at the wavelength of maximum absorption, λ , close
max
to 360 nm. Calculate the concentration, c in micrograms per millilitre, using the following equation:
i
c = A · M · 100/ε
i
where
A is the numerical value of the absorbance at λ
max;
M is the numerical value of the molar mass of the aflatoxin M in grams per mole (M = 328 g/mol);
1
ε is the numerical value of the absorption coefficient of the toxin in chloroform, in square metres per mole
2
(ε = 1 995 m /mol).
4.5.2 Stock solution
After checking the concentration of the calibrant solution (4.5.1), dilute the calibrant solution in chloroform to give an
aflatoxin M stock solution of 0,1 μg/ml. The stock solution shall be well-stoppered and wrapped in aluminium foil to
1
exclude light.
Store the stock solution in a refrigerator at a temperature below 5 °C in the dark. Under these conditions the stock
solution is stable for about 2 months. After 2 months, the stability should be checked.
4.5.3 Working solutions of aflatoxin M
1
Before preparing working dilutions of the aflatoxin M standard solution, allow the stock solution (4.5.2) to attain
1
ambient temperature before removing aliquots of the solution for subsequent dilution. Prepare working solutions on
the day of use.
Prepare a solution with a concentration of 0,005 μg/ml as follows. By means of a pipette (5.4) transfer 1,0 ml of the
stock solution (4.5.2) to a 20 ml conical tube (5.9). Evaporate the solution to dryness using a gentle stream of
nitrogen (4.3) and dissolve the residue obtained in 20,0 ml of the diluted acetonitrile (4.2.2). Shake occasionally
over a period of 30 min.
Care should be taken when evaporating the solution to dryness to ensure the temperature does not drop so low that
condensation occurs.
Use this diluted solution for the preparation of a series of appropriate dilutions of aflatoxin M standard solution to
1
provide, depending on the injection loop volume, for injection of 0,05 ng, 0,1 ng, 0,2 ng and 0,4 ng of aflatoxin M .
1
Dilute by using diluted acetonitrile solution (4.2.2).
5 Apparatus
Usual laboratory equipment and, in particular, the following.
3
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© ISO
ISO 14501:1998(E)
5.1 Disposable syringes, of capacities 10 ml and 50 ml.
1)
5.2 Vacuum system (e.g. Büchner flask, Vac-Elute system or peristaltic pump).
5.3 Centrifuge, capable of producing a radial acceleration of 4 000 · g.
5.4 Pipettes, of capacities 1,0 ml, 2,0 ml and 50,0 ml.
, of capacity 250 ml.
5.5 Glass beakers
5.6 Volumetric flask, of capacity 100 ml.
, capable of operating at 30 C 2 C, 50 C 2 C, and between 35 C and 37 C.
5.7 Water baths ° – ° ° – ° ° °
1)
5.8 Filter paper (Whatman No. 4 , or equivalent).
5.9 Graduated conical glass tubes, with ground glass neck and stopper, of capacities of 5 ml, 10 ml and 20 ml.
5.10 HPLC-equipment
5.10.1 Pulse-free pump, suitable for constant volume flow rate of about 1 ml/min.
5.10.2 Injector system, with fixed or variable injection volume loop, suitable for injection of 50 μl to 500 μl.
5.10.3 Reversed-phase HPLC analytical column, with 3 μm or 5 μm packing of octadecyl silicagel plus guard
column filled with reverse-phase material.
5.10.4 Fluorescence detector, capable of providing about 365 nm excitation and 435 nm emission waveleng
...
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