S.L. 437.77 ст. 3Analyses for the contentofaflatoxin B . 1

The competent authority shall require that analyses for official controls of feedingstuffs, as regards their content of aflatoxin B , be carried out in accordance with the methods 1 described in the Schedule.

*These rules have been repealed by Legal Notice 37 of 2025. SCHEDULE

DETERMINATION OF AFLATOXIN B 1 ONE-DIMENSIONAL THIN LAYER CHROMATOGRAPHIC METHOD Purpose and scope makes it possible to determine the level of aflatoxin B in raw 1 straight feedingstuffs. This method can not be applied in the presence The lower limit of determination is 0,01 mg/kg (10 ppb). presence of interfering substances it is necessary to repeat the analysis using (high performance liquid chromatography). Principle is subjected to extraction with chloroform. The extract is filtered, and portion taken and purified by column chromatography on silica gel. The evaporated and the residue redissolved in a specific volume of chloroform mixture of benzene and acetonitrile. An aliquot portion of this solution is thin-layer chromatography (TLC). The quantity of aflatoxin B is 1 under UV irradiation of the chromatogram, either visually or by fluorodensitometry, by comparison with known quantities of standard aflatoxin B . 1 of the aflatoxin B extracted from the feedingstuff must be confirmed 1 procedure indicated. Reagents reagents must be of ‘analytical reagent’ quality unless otherwise

Acetone.

Chloroform, stabilized with 0ꞏ5 to 1ꞏ0% of 96% ethanol (v/v). N-hexane. Methanol.

Anhydrous diethyl ether, free from peroxides. Mixture of benzene and acetonitrile: 98/2 (v/v). Mixture of chloroform (3.2) and methanol (3.4): 97/3 (v/v).

Silica gel, for column chromatography, particle size 0ꞏ05 to 0ꞏ20 mm. Absorbent cotton wool, previously defatted with chloroform, or glass

Sodium sulphate, anhydrous, granular. Inert gas, e.g. nitrogen.

N Hydrochloric acid. 50% (v/v) sulphuric acid. Kieselguhr (hyflosupercel), washed in acid.

Silica gel G-HR or equivalent, for TLC. Standard solution with about 0ꞏ1 µg of aflatoxin B per ml in chloroform 1 benzene/acetonitrile mixture (3.6), prepared and checked as indicated in

3.17. Standard solution for qualitative testing purposes containing µg of aflatoxin B and B per ml in chloroform (3.2) or the benzene/acetonitrile 1 2 mixture (3.6). These concentrations are given as a guide. They must as to obtain the same intensity of fluorescence for both aflatoxins.

3.18. Developing solvents: 3.18.1. Chloroform (3.2)/acetone (3.1): 9/1 (v/v), unsaturated tank; 3.18.2. Diethyl ether (3.5)/methanol (3.4)/water: 96/3/1 (v/v/v), tank; 3.18.3. Diethyl ether (3.5)/methanol (3.4)/water: 94/4ꞏ5/1ꞏ5 (v/v/v), tank; 3.18.4. Chloroform (3.2)/methanol (3.4): 94/6 (v/v), saturated tank;

3.18.5. Chloroform (3.2)/methanol (3.4): 97/3 (v/v), saturated tank.