Commission Regulation (EEC) No 2568/91Show full title

ANNEX VI U.K. DETERMINATION OF ERYTHRODIOL AND UVAOL

INTRODUCTION U.K.

Erythrodiol (commonly understood as the glycols erythrodiol and uvaol together) is a constituent of the unsaponifiable fraction, characteristic of some types of fatty substances. It is found at considerably higher concentrations in solvent-extracted olive oil than in other oils, such as pressed olive oil and grape pip oil, which also contain it, and so its presence may demonstrate the presence of solvent-extract olive oil.

1. SCOPE U.K.

The method describes a procedure for detecting erythrodiol in fatty substances.

2. PRINCIPLE OF THE METHOD U.K.

The fatty substance is saponified with potassium [X1 hydroxide in ethanolic solution. The unsaponifiable fraction is then extracted with diethyl ether] and purified by passage over a column of alumina.

Editorial Information

X1 Substituted by Corrigendum to Commission Regulation (EEC) No 2568/91 of 11 July 1991 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis (Official Journal of the European Communities No L 248 of 5 September 1991).

The unsaponifiables are subjected to thin-layer chromatography on a silica gel plate until the bands corresponding to the sterol and erythrodiol fractions are separated. The sterols and the erythrodiol recovered from the plate are transformed into trimethylsilyl ethers and the mixture is analysed by gas chromatography.

The result is expressed as the percentage of erythrodiol in the mixture of erythrodiol and sterols.

3. APPARATUS U.K.

3.1. The apparatus described in Annex V (determination of the content of sterols). U.K.

4. REAGENTS U.K.

4.1. The reagents described in Annex V (determination of the content of sterols). U.K.

4.2. Reference solution of erythrodiol, 0,5 % solution in chloroform. U.K.

5. PROCEDURE U.K.

5.1. Preparation of the unsaponifiables. U.K.

As described at paragraph 5.1.2 of Annex V.

5.2. Separation of erythrodiol and the sterols. U.K.

5.2.1. See paragraph 5.2.1 of Annex V. U.K.

5.2.2. See paragraph 5.2.2 of Annex V. U.K.

5.2.3. Prepare a 5 % solution of the unsaponifiables in chloroform. U.K.

Using the 0,1 ml microsyringe, streak a chromatographic plate with 0,3 ml of solution approximately 1,5 cm from the lower edge in a streak which is as thin and uniform as possible.

At one end of the plate place a few microlitres of the solutions of cholesterol and erythrodiol to serve as a reference.

5.2.4. Place the plate in the developing chamber prepared as specified in 5.2.1. The ambient temperature should be about 20 °C. Immediately close the chamber with the cover and allow to elute until the solvent front reaches approximately 1 cm from the upper edge of the plate. Remove the plate from the developing chamber and evaporate the solvent in a flow of hot air. U.K.

5.2.5. Spray the plate lightly and uniformly with the alcoholic 2,7-dichlorofluoroscein solution. When the plate is observed under ultralviolet light the sterol and erythrodiol bands can be identified through being aligned with the references. Mark with a spot just outside the edges of the fluorescence. U.K.

5.2.6. Using a metal spatula scrape off the silica gel in the marked areas. Place the material from the plate in a 50 ml flask. Add 15 ml of hot chloroform, shake well and filter through a funnel with a sintered glass disc so that the silica gel is transferred to the filter. Wash three times with hot chloroform (10 ml each time) collecting the filtrate in a 100 ml flask. Evaporate the filtrate to a volume of 4 to 5 ml, transfer to a calibrated 10 ml conical-bottomed centrifuge tube, dry by gently heating in a current of nitrogen and weigh. U.K.

5.3. Preparation of the trimethylsily esters U.K.

As described in paragraph 5.3 of Annex V.

5.4. Gas chromatographic analysis U.K.

As described in paragraph 5.4 of the above method. The operating conditions of the gas chromatograph in analysis must be such as to perform the sterol analysis and separate the TMSE from the erythrodiol and uvaol.

Once the sample has been injected, continue recording until the sterols present, the erythrodiol and the uvaol have been eluted. Then identify the peaks (the retention times for erythrodiol and uvaol relative to β-sitosterol are about 1,45 and 1,55 respectively) and calculate the areas as for the sterols.

6. EXPRESSION OF THE RESULTS U.K.

where:

A₁

=

peak area for erythrodiol[F1 in square millimetres;

A₂

=

peak area for uvaol in square millimetres;

A_sterols

=

total peak area for sterols in square millimetres].

Textual Amendments

F1 Deleted by Commission Regulation (EEC) No 183/93 of 29 January 1993 amending Regulation (EEC) No 2568/91 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis.

The result is expressed to one decimal place.