ISO 14501:2021 INTERNATIONAL STANDARDIDF 171:2021(E)Milk and milk powder — Determination of aflatoxin M 1content — Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography

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INTERNATIONAL STANDARDIDF 171:2021(E)Milk and milk powder — Determination of aflatoxin M 1content — Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography是国际标准化组织于2021-05发布的ISO标准，适用于国际范围。

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INTERNATIONAL STANDARD

IDF 171:2021(E)

Milk and milk powder — Determination of aflatoxin M 1

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

[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 1

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 1

glassware, before coming into contact with aqueous solutions of aflatoxin M , should be soaked 1

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. 1

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://w ww.e lectropedia. org/ — ISO Online browsing platform: available at https://w ww. iso .org/ obp

3.1

aflatoxin M content

1

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.

IDF 171:2021(E)

4 Principle

Aflatoxin M is extracted by passing the test portion through an immunoaffinity column that contains 1

specific antibodies bound onto a solid support material.

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 1

collected. The amount of aflatoxin M present in this eluate is determined by means of high-performance 1

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 1

maximum capacity of not less than 100 ng of aflatoxin M (which corresponds to 2 µg/l when a volume 1

of 50 ml of a test portion is applied). It shall give a recovery of not less than 80 % for aflatoxin M when 1

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 1)

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;

1

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 1

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 1

(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 1

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.

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