Commission Regulation (EC) No 273/2008 (repealed)Show full title

5. PROCEDURE

5.1. Preparation of the test sample

5.1.1. Butter

Heat the sample until melting starts. Avoid local overheating at about 30 °C. The butter may not separate in two phases, in any case. When the sample becomes sufficiently plastic, homogenise it by shaking. Stir the butter for 15 s before taking a sample. Weigh, to the nearest 1 mg, about 5 g (SM g) of butter into a 100 ml volumetric flask.

5.1.2. Concentrated butter

Immediately before sampling place the container, with concentrated butter, into an oven at 40 to 50 °C until it is melted completely. Mix the sample by swirling or stirring, avoiding formation of air bubbles by too vigorous stirring. Weigh, to the nearest 1 mg, about 4 g (SM g) of concentrated butter into a 100 ml volumetric flask.

5.1.3. Cream

Heat the sample in a waterbath or incubator at a temperature of 35 to 40 °C. Distribute the fat homogeneously by swirling and, if necessary, by stirring. Cool the sample quickly to 20 ± 2 °C. The sample should look homogenous; otherwise the procedure should be repeated. Weigh, to the nearest 1 mg, about 10 g (SM g) of cream into a 100 ml volumetric flask.

5.2. Preparation of the test solution

Add about 75 ml extraction solution (4.4) to the test portion (5.1.1, 5.1.2 or 5.1.3), stir, or shake vigorously, for about 15 minutes and make up with extraction solution (4.4). Transfer about 10 ml of this extract to a test tube fitted with stopper. Place the test tube in the freezer (3.1) and allow it to stand for about 30 minutes. Centrifuge the cold extract for 5 minutes at approximately 2 000 rpm and decant immediately. Allow the decanted solution to adjust to room temperature. Pipette 5 ml of the decanted solution into a 100 ml volumetric flask and make up with water. Filter an aliquot through a membrane microfilter (3.3) using a syringue (3.2). The filtrate is ready for determination by HPLC.

5.3. Calibration

Pipette 5 ml of the vanillin standard solution (4.5.2) into a 100 ml volumetric flask. Add 5 ml extraction solution (4.4) and make up to the mark with water. This solution contains 0,5 μg/ml of vanillin.

5.4. Determination by HPLC

Allow the chromatographic system to stabilise for about 30 minutes. Inject the standard solution (5.3). Repeat this until the difference in peak area or peak height between two successive injections is less than 2 %. Under the conditions described the retention time of vanillin is about 9 minutes. Analyse the standard solution (5.3) in duplicate by injecting 20 μl. Inject 20 μl of the test solutions (5.2). Determine the area or height of the vanillin peak obtained. Repeat the duplicate injection of the standard solution (5.3) after 10 injections of test samples (5.2).