S.L. 437.77 Art. 5C, point 1). with a 1-mm (4.6). Add 25 Stopper the flask, shake or filter paper 5.3. Insert into plug (3.9), fill sulphate Check that silica gel (3.8) bubbles. sulphate sodium Mix the 50 quantitatively above the add 100 ml of sodium per minute and Then elute whole of the Evaporate under a stream tra

Procedure

5.1. Preparation of the sample (see under ‘Observations’, Part Grind the sample so that the whole of it will pass through a sieve mesh (in accordance with recommendation ISO R 565). 5.2. Extraction Put 50 g of ground, homogenized sample into a 500-ml conical flask g of Kieselguhr (3.14), 25 ml of water and 250 ml of chloroform (3.2). stir for 30 minutes with the apparatus (4.2) and filter through a fluted (4.3). Discard the first 10 ml of the filtrate and then collect 50 ml.

Column clean-up the lower end of a chromatography tube (4.4) a cotton or glass wool two-thirds of the tube with chloroform (3.2) and add 5 g of sodium (3.10). the upper surface of the sodium sulphate layer is flat, thenadd 10 g of in small portions. Stir carefully after each addition to eliminate air Leave to stand for 15 minutes and then carefully add 15 g of sodium (3.10). Let the liquid fall until it is just above the upper surface of the sulphate layer. ml of extract collected in 5.2 with 100 ml of n-hexane (3.3) and transfer the mixture to the column. Let the liquid fall until it is just upper surface of the sodium sulphate layer. Discard this washing. Then diethylether (3.5) and again allow it to fall to the upper surface of the sulphate layer. During these operations see that the rate of flow is 8 to 12 ml that the column does not run dry. Discard the liquid that comes out. with 150 ml of the chloroform/methanol mixture (3.7) and collect the eluate. the latter almost to dryness at a temperature not exceeding 50ºC and of inert gas (3.11) with the rotary evaporator (4.5). Quantitatively residue, using chloroform (3.2) or the benzeneacetonitrile mixture (3.6), graduated tube (4.10). Concentrate the solution under a stream of inert gas then adjust the volume to 2 ml with chloroform (3.2) or the benzene/ mixture (3.6).

Thin-layer chromatography TLC plate (4.8), 2 cm from the lower edge and at intervals of 2 cm, the indicated below of the standard solution and the extract: 30 and 40 µl of the standard aflatoxin B solution (3.16); 1 extract obtained in 5.3 and, superimposed on the same point, 20 µl of solution (3.16);

µl of the extract obtained in 5.3. chromatogram in the dark with one of the developing solvents (3.18). the solvent must be made beforehand, by depositing 25 µl of the standard solution (3.17) on the plate and checking that, when developed, and B are completely separated. Let the solvents evaporate in the dark 2 irradiate with UV light, placing the plate 10 cm from the lamp (4.9). The aflatoxin B give a blue fluorescence. 1 Quantitative determinations either visually or by fluorodensitometry as indicated below. Visual measurements the quantity of aflatoxin B in the extract by matching the fluorescence 1 the extract spots with that of one of the standard solution spots. necessary. The fluorescence obtained by the superimposition of the standard solution must be more intense than that of the 10 µl of extract not be more than one visible spot. If the fluorescence intensity given extract is greater than that of the 40 µl of standard solution, dilute the extract 10 or 100 times with chloroform (3.2) or the benzene/acetonitrile (3.6) before subjecting it again to thin-layer chromatography. 5.5.2. Measurements by fluorodensitometry

Measure the fluorescence intensity of the aflatoxin B spots 1 fluorodensitometer (4.12) at an excitation wavelength of 365 nm and wavelength of 443 nm. Determine the quantity of aflatoxin B in the extract 1 comparison of their fluorescence intensities with that of the standard spots.

5.6. Confirmation of the identity of aflatoxin B 1 Confirm the identity of the aflatoxin B in the extract by the processes 1 below. 5.6.1. Treatment with sulphuric acid Spray sulphuric acid (3.13) chromatogram obtained in 5.4. The fluorescence of the aflatoxin B 1 from blue to yellow under UV irradiation.

5.6.2. Two-dimensional chromatography involving the formation B - hemiacetal (aflatoxin B ) 1 2a NB: The operations described below must be carried out following diagram in figure 3.

5.6.2.1. Application of the solutions Score two straight lines on the plate (4.8) parallel to two contiguous from each side) to limit migration of the solvent fronts. Spot the following on the plate using capillary pipettes or microsyringes:

- on point A: a volume of purified extract of the sample, obtained containing about 2ꞏ5 nm of aflatoxin B ; 1 - 25 µl of the standard solution (3.16). on points B and C: 5.6.2.2. Development

Develop the chromatogram indirection I, in the dark, using the developing (3.18.1) (1 cm layer in an unsaturated tank) until the solvent front solvent limit line. Remove the plate from the tank and allow to dry in the dark temperature for five minutes. Then spray hydrochloric acid (3.12) along cm high, covering points A and B (indicated by the hatched area in figure darkens, protecting the rest of the plate with a glass sheet. Allow minutes in the dark and dry with a stream of air at ambient temperature. Next, develop the chromatogram indirection II, in the dark, using solvent (3.18.1) (1 cm layer in an unsaturated tank) until the solvent the solvent limit line. Remove the plate from the tank and allow to temperature.

5.6.2.3. Interpretation of the chromatogram Examine the chromatogram UV light (4.9) and check for the following features.

(a) Appearance of a blue fluorescent spot of aflatoxin B originating 1 the standard solution applied at C (migration in the direction

(b) Appearance of a blue fluorescent spot of unreacted hydrochloric acid) aflatoxin B and a more intense blue fluorescent 1 of aflatoxin B - hemiacetal, both originating from the standard 1 applied at B (migration in direction II). Appearance of spots matching those described in (b), originating from the sample extract applied at A. The position of these spots is defined first by the migration distance of the aflatoxin B from point A in 1 direction I (the same as that traveled by the standard applied at C), and then by the migration distances from there in direction II of the aflatoxin B - hemiacetal (same as those travelled by the standard 1 applied at B). The fluorescence intensities of hemiacetal spots originating from the extract and from the standard applied at B should match. Calculation of the results

From the visual measurements in micrograms of aflatoxin B per kg of sample (ppb) is given by the 1

S • Y • V W • X

respectively the volumes in microlitres of the standard solution of (3.16) and of the extract having a similar intensity of fluorescence;

concentration in micrograms of aflatoxin B per ml in the standard solution 1

volume of the extract in microlitres, allowing for any dilution that was in grammes of the sample corresponding to the volume of extract column clean-up.

From the fluorodensitometric measurements in micrograms of aflatoxin B per kg of sample is given by the 1

S • V W • Y

in microlitres of the extract spotted on the plate (10 or 20 µl); in nanograms of aflatoxin B in the extract spot (proportional to the 1 taken), deduced from the measurements; volume of the extract in microlitres, allowing for any dilution that was in grammes of the sample corresponding to the volume of extract column clean-up. Preparation and testing of the standard solution (3.16)

Determination of the concentration of aflatoxin B 1 standard solution of aflatoxin B in the chloroform (3.2) or the benzene/ 1 mixture (3.6) with a concentration of 8 to 10 µg/ml. Determine the spectrum between 330 and 370 nm with the aid of the spectrophotometer

Measure the optical density (A) at 363 nm in the case of the chloroform or at 348 nm in the case of the solution in the benzene/acetonitrile mixture. Calculate the concentration in micrograms of aflatoxin B per ml of 1 the formulae below:

312 • A • 1000 for the chloroform solution; 20600

312 • A • 1000 for the solution in the benzene/acetonitrile mixture. 19800

Dilute as appropriate, away from day light, to obtain a working standard with a concentration of aflatoxin B of about 0ꞏ1 µg/ml. If kept in a 1 4ºC, this solution is stable for two weeks. 7.2. Testing of chromatographic purity Spot on a plate (4.8) 5 µl of the standard solution of aflatoxin B 1 10 µg/ml (7.1). Develop the chromatogram as indicated in 5.4. In chromatogram should show only one spot and no fluorescence must be the original deposit zone.