Commission Regulation (EEC) No 2568/91Dangos y teitl llawn

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.