Commission Regulation (EC) No 900/2008Show full title

ANNEX III

Detection of common wheat flour or meal in macaroni, spaghetti and similar products (pasta) (by the Young and Gilles method, modified by Bernaerts and Gruner)

I. PRINCIPLE

An extract of the sample of the pasta for analysis is prepared by using a non-polar solvent.

This extract is chromatographed on a thin layer of silica gel so as to separate the sterols present in various band form fractions.

According to the number of brightly coloured bands it is possible to determine whether the product under examination has been manufactured exclusively from durum wheat or common wheat, or from a mixture of the two. It is also possible to determine whether eggs have been added.

II. APPARATUS AND REAGENTS

1. Homogeniser or grinder to obtain a grist that will pass through a standard sieve with a 0,200 mm mesh;

2. Standard sieve with a 0,200 mm mesh;

3. Evaporator with a water bath for evaporation under reduced pressure;

4. Glass plate, aluminium sheet or other appropriate backing measuring 20 cm × 20 cm covered with a thin layer of silica gel. If the thin layer has to be prepared, silica gel mixed with about 13 % plaster should be used, and it should be applied in a 0,25 mm layer with suitable apparatus in accordance with the manufacturer's instructions;

5. Micropipette for measuring 20 microlitres;

6. Container with lid suitable for the development of chromatograms;

7. Atomiser;

8. Petroleum ether with a boiling point between 40 and 60 °C, redistilled before use;

9. Anhydrous ethyl ether for analysis;

10. Carbon tetrachloride for chromatography, redistilled before use;

11. Phosphomolybdic acid for analysis;

12. 94° ethyl alcohol.

III. METHOD

Grind about 20 g of the sample for analysis so that all of it passes through the sieve. Put the sample in an Erlenmeyer flask and cover with 150 ml petroleum ether. Leave at normal temperature until the following day. Shake from time to time.

Then filter on a Büchner funnel fitted with a filtering aid or on a sintered filter. Gradually transfer the clear solution thus obtained into a 100 ml calibrated flask. Evaporate the solvent under reduced pressure by heating the flask in a water bath at 40 °C to 50 °C. When the solvent has evaporated, heat under reduced pressure for a further ten minutes.

When the flask has cooled, determine the weight of the extract. Dilute the extract in ethyl ether on the basis of 1 ml ethyl ether per 60 mg of extract.

Activate the thin layers by bringing them to 130 °C for three hours. Leave to cool in a desiccator containing silica gel. Plates which are not used immediately can be preserved in the same desiccator.

Apply, drop by drop, 20 microlitres of the clear solution to form a band of constant width and 3 cm in length on a layer preferably newly activated. Let the solvent evaporate.

Develop the chromatogram under normal temperature with carbon tetrachloride using a chromatographic container the walls of which are covered with filter paper soaked in solvent. After about an hour the solvent will reach a height of 18 cm. Remove the plate and leave the solvent to evaporate in the open. For better separation of the bands, develop the chromatogram a second time. Again let the solvent evaporate in the open.

Spray the thin layer of silica gel with a solution of 20 % phosphomolybdic acid in ethyl alcohol. The colour of the layer must be a uniform yellow. Develop the bands by the heating the sprayed plate at 110 °C for five minutes.

IV. INTERPRETATION OF THE CHROMATOGRAM

If the chromatogram shows a single main brightly coloured band with an Rf of about 0,4 to 0,5, the wheat used for the manufacture of the pasta in question is durum wheat. If, on the other hand, two main bands of equal brightness appear, the raw material used is common wheat. Mixtures of durum wheat and common wheat can be assessed by an evaluation of the relative brightness of the two bands.

If there are three bands (two bands at the height where the main bands for common wheat are to be found, with a further band between them) eggs have been added to pasta. In this case, the raw material used is durum wheat if the middle band is brighter than the upper band. On the other hand, if the upper band is brighter than the middle band, the raw material used is common wheat.