ISO 14501 pdf download

ISO 14501 pdf download Milk and milk powder — Determination of aflatoxin M 1 content — Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography
Aflatoxin Mh is extracted by passing the test portion through an immunoaffinity column that containsspecific antibodies bound onto a solid support material.
As the sample passes through the column, the antibodies are selectively bound with any aflatoxin M1(antigen] present and form an antibody-antigen complex. All other components of the sample matrixare washed off the column with water. Then aflatoxin Mn is eluted from the column and the eluate iscollected.The amount of aflatoxin Mi present in this eluate is determined by means of high-performanceliquid chromatography (HPLC) coupled with fluorimetric detection.
5Reagents
During the analysis，unless otherwise stated, use only reagents of recognized analytical grade anddistilled or demineralized water or water of equivalent purity.
5.1 Immunoaffinity column.
The immunoaffinity column shall contain antibodies against aflatoxin Mi. The column shall have amaximum capacity of not less than 100 ng of aflatoxin Mi (which corresponds to 2 ug/l when a volumeof 50 ml of a test portion is applied). lt shall give a recovery of not less than 80 % for aflatoxin Mi whena standard solution containing 4 ng of toxin is applied (which corresponds to 80 ng/l when a volumeof 50 ml of sample is applied).Any immunoaffinity column meeting the performance specificationsmentioned above can be used.1
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 Mi 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 Mi 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 M1 standard working solution of 0,005 ug/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 Mi eluted from the column by HPLC after preparing a
suitable dilution of the final eluate;
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 ug/1.Recovery checks can also be conducted with commercially available referencematerials.
5.2Acetonitrile, pure, HPLC grade.
5.2.1Acetonitrile 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 maybe used. Degas the solution (eluent) before using it.
5.2.2Acetonitrile 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 maybe used. Degas the solution (eluent) before using it.
5.3 Nitrogen gas.
5.4Aflatoxin M1 standard solutions.
5.4.1 Aflatoxin Mi standard calibration solution, (mass concentration p = 10 ug/ml aflatoxin M1 inacetonitrile).
Prepare an aflatoxin Mi standard calibration solution by dissolving aflatoxin M1 (C1Hrz07) inacetonitrile (5.2) to give a nominal concentration of 10 ug/ml. Determine the actual aflatoxin M1concentration by measurement of the absorbance at the maximum absorption wavelength of thesolution as follows.
Use the spectrophotometer (6.13) to record the absorbance of the aflatoxin Mi standard calibrationsolution against acetonitrile(5.2 ) as blank at wavelengths between 330 nm and 370 nm.Measure theabsorbance,A, at its maximum absorption wavelength,?max, which is close to 350 nm.
Calculate the concentration, p1, expressed in micrograms per millilitre, by using Formula(1);:
x where
is the numerical value of the absorbance at 2max;
is the molar mass of aflatoxin M1, in grams per mole (M = 328 g/mol);is the optical path length, in centimetres (d = 1 cm);
is the numerical value of the absorption coefficient of the toxin in acetonitrile, in squaremetres per mole (e = 1 985 m2:mol-1).
Alternatively , certified reference materials are available commercially (for example BCR-423 10 ug/mlaflatoxin M1 in chloroform).