Commission Regulation (EC) No 273/2008 (repealed)Show full title

8. PROCEDURE

8.1. Preparation of the test sample

Transfer the milk powder into a container with a capacity of about twice the volume of the powder, fitted with an airtight lid. Close the container immediately. Mix the milk powder well by means of repeated inversion of the container.

8.2. Test portion

Weight 2,000 ± 0,001 g of test sample into a centrifuge tube (6.2) or a suitable stoppered flask (50 ml).

8.3. Removal of fat and proteins

8.3.1. Add 20,0 ml of warm water (50 °C) to the test portion. Dissolve the powder by shaking for five minutes using a mechanical shaker (6.3). Place the tube into the water bath (6.10) and allow to equilibrate to 25 °C

8.3.2. Add 10,0 ml of the trichloroacetic acid solution (5.1) of ca. 25 °C in two minutes, while stirring vigorously with the aid of the magnetic stirrer (6.4). Place the tube in a water bath (6.10) and leave for 60 minutes

8.3.3. Centrifuge (6.2) for 10 minutes at 2 200 g, or filter through paper (6.6), discarding the first 5 ml of filtrate

8.4. Chromatographic determination

8.4.1. Inject 15 to 30 μl of accurately measured supernatant or filtrate (8.3.3) into the HPLC apparatus (6.11) operating at a flow rate of 1,0 ml of eluent solution (5.2) per minute

Note 1. Another flow rate may be used, dependent of the internal diameter of the columns used or the instructions of the manufacturer of the column.

Note 2. Keep the eluent solution (5.2) at 85 °C throughout the chromatographic analysis in order to keep the eluent degassed and to prevent bacterial growth. Any precaution with a similar effect may be used.

Note 3. Rinse the columns with water during each interruption. Never leave the eluent solution in them (5.2).

Prior to any interruption of more than 24 hours, rinse the columns with water then wash them with solution (5.3) for at least three hours at a flow rate of 0,2 ml per minute.

8.4.2. The results of chromatographic analysis of the test sample [E] are obtained in the form of chromatogram in which each peak is identified by its retention time RT as follows:

Peak II:	The second peak of the chromatogram having an RT of about 12,5 minutes.
Peak III:	The third peak of the chromatogram, corresponding to the CMP, having an RT of 15,5 minutes.

The choice of the column(s) can effect the retention times of the individual peaks considerably.

The integrator (6.11.6) automatically calculates the area A of each peak:

AII:	area of peak II,
AIII:	area of peak III,

It is essential to examine the appearance of each chromatogram prior to quantitative interpretation, in order to detect any abnormalities due either to malfunctioning of the apparatus or the columns, or to the origin and nature of the sample analysed.

If in doubt, repeat the analysis.

8.5. Calibration

8.5.1. Apply exactly the procedure described from point 8.2 to point 8.4.2 to the standard samples (5.4)

Use freshly prepared solutions, because CMP degrade in an 8 % trichloroacetic environment. The loss is estimated at 0,2 % per hour at 30 °C.

8.5.2. Prior to chromatographic determination of the samples, condition the columns by repeatedly injecting the standard sample (5.4.2) in solution (8.5.1) until the area and retention time of the peak corresponding to the CMP are constant

8.5.3. Determine the response factors R by injecting the same volume of filtrates (8.5.1) as used for the samples