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Commission Implementing Regulation (EU) 2016/1240 of 18 May 2016 laying down rules for the application of Regulation (EU) No 1308/2013 of the European Parliament and of the Council with regard to public intervention and aid for private storage (Text with EEA relevance)
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Version Superseded: 31/12/2020
Point in time view as at 07/02/2018.
There are currently no known outstanding effects for the Commission Implementing Regulation (EU) 2016/1240,
Appendix III
.
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The content of rennet whey solids is defined as the percentage by mass as determined by caseinomacropeptide content by the procedure described.
Samples are analysed for caseinomacropeptide A by a reversed-phase high-performance liquid chromatography procedure (HPLC procedure). Evaluation of the result is obtained by reference to standard samples consisting of skimmed milk powder with and without a known percentage of whey powder. Results higher than 1 % (m/m) show that rennet whey solids are present.
All reagents shall be of recognised analytical grade. The water used shall be distilled water or water of at least equivalent purity. Acetonitrile should be of spectroscopic or HPLC quality.
Dissolve 240 g of trichloroacetic acid (CCl 3 COOH) in water and make up to 1 000 ml. The solution should be clear and colourless.
Eluent A: 150 ml of acetonitrile (CH 3 CN), 20 ml of isopropanol (CH 3 CHOHCH 3 ), and 1,00 ml of trifluoroacetic acid (TFA, CF 3 COOH) are placed in a 1 000 ml volumetric flask. Make up to 1 000 ml with water.
Eluent B: 550 ml of acetonitrile, 20 ml of isopropanol and 1,00 ml of TFA are placed in a 1 000 ml volumetric flask. Make up to 1 000 ml with water. Filter the eluent solution, prior to use, through a membrane filter with a 0,45 μm pore diameter.
After the analyses the column is flushed with eluent B (via a gradient) and subsequently flushed with acetonitrile (via a gradient for 30 minutes). The column is stored in acetonitrile.
Binary gradient pumping system
Injector, hand or automatic, with a 100 μl capacity
Agilent Technologies Zorbax 300 SB-C3 column (length 25 cm, 0,46 cm internal diameter) or an equivalent wide-pore silica based reversed-phase column
Thermostatic column oven, set at 35 ± 1 °C
Variable wavelength UV detector, permitting measurements at 210 nm (if necessary, a higher wavelength up to 220 nm may be used) with a sensitivity of 0,02 Å
Integrator capable of setting the integration to common baseline or valley-to-valley
Note : Operation of the column at room temperature is possible, provided that the room temperature does not fluctuate more than 1 °C, otherwise too much variation in the retention time of CMP A takes place. U.K.
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.
Weigh 2,00 ± 0,001 g of test sample into a centrifuge tube (6.2) or suitable stoppered flask (50 ml).
Note: In the case of mixtures, weigh such an amount of the test sample that the defatted sample portion corresponds to 2,00 g. U.K.
Take solutions of the standard samples (5.4) without and with 50 % rennet whey.
Inject 100 μl of supernatant or filtrate (8.3.3) into the HPLC apparatus operating at the scouting gradient conditions given in Table 1.
Scouting gradient conditions for optimisation of the chromatography
Time (min) | Flow (ml/min) | % A | % B | Curve |
---|---|---|---|---|
Initial | 1,0 | 90 | 10 | * |
27 | 1,0 | 60 | 40 | linear |
32 | 1,0 | 10 | 90 | linear |
37 | 1,0 | 10 | 90 | linear |
42 | 1,0 | 90 | 10 | linear |
Comparison of the two chromatograms should reveal the location of the peak of CMP Α .
Using the formula given below, the initial solvent composition to be used for the normal gradient (see 8.4.3) can be calculated % B = 10 – 2,5 + (13,5 + (RT cmpA – 26) / 6) * 30 / 27 % B = 7,5 + (13,5 + (RT cmpA – 26) / 6) * 1,11
Where:
:
retention time of CMP Α in the scouting gradient
:
the initial % B of the scouting gradient
:
% B at midpoint minus % B at initial in the normal gradient
:
midpoint time of the scouting gradient
:
required retention time of CMP Α
:
ratio of slopes of the scouting and normal gradient
:
% B at initial minus % B at 27 minutes in the scouting gradient
:
run-time of the scouting gradient.
Inject 100 μl of accurately measured supernatant or filtrate (8.3.3) into the HPLC apparatus operating at a flow rate of 1,0 ml of eluent solution (5.2) per minute.
The composition of the eluent of the start of the analysis is obtained from 8.4.2. It is normally close to A:B = 76:24 (5.2). Immediately after the injection a linear gradient is started, which results in a 5 % higher percentage of B after 27 minutes. Subsequently a linear gradient is started, which brings the eluent composition to 90 % B in five minutes. This composition is maintained for five minutes, after which the composition is changed, via a linear gradient in five minutes to the initial composition. Depending on the internal volume of the pumping system, the next injection can be made 15 minutes after reaching the initial conditions.
Note 1. The retention time of the CMP A should be 26 ± 2 minutes. This can be achieved by varying the initial and end conditions of the first gradient. However, the difference in the % B for the initial and end conditions of the first gradient shall remain 5 % B. U.K.
Note 2. The eluents should be degassed sufficiently and should also remain degassed. This is essential for proper functioning of the gradient pumping system. The standard deviation for the retention time of the CMP A peak should be smaller than 0,1 minutes (n = 10). U.K.
Note 3. Every five samples the reference sample [5] should be injected and used to calculate a new response factor R. (9.1.1). U.K.
The integrator (6.11.6) automatically calculates the peak height H of the CMP A peak. The baseline location should be checked in every chromatogram. The analysis or the integration should be repeated if the baseline was incorrectly located.
Note: If the CMP A peak is sufficiently separated from other peaks valley-to-valley baseline allocation should be used, otherwise use dropping perpendiculars to a common baseline, which should have starting point close to the CMP A peak (thus not at t = 0 min!).Use for the standard and the samples the same type integration type and check in case of common baseline its consistency for the samples and the standard. U.K.
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 column, or to the origin and nature of the sample analysed. If in doubt, repeat the analysis.
Note: 8.4.2. may be omitted if the % B at initial conditions is known from previous analyses. U.K.
The chromatogram of the reference sample [5] should be analogous to Figure. 1. In this figure the CMP A peak is preceded by two small peaks. It is essential to obtain a similar separation.
The chromatogram should not show a peak at the retention time of the CMP A peak.
CMP A peak: R = W/H
Where:
=
the response factor of the CMP A peak
=
the height of the CMP A peak
=
the quantity of whey in the standard sample [5].
W(E) = R × H(E)
Where:
=
the percentage (m/m) of rennet whey in the sample (E).
=
the response factor of the CMP A peak (9.1.1)
=
the height of the CMP A peak of the sample (E)
If W(E) is greater than 1 % and the difference between the retention time and that of the standard sample [5] is smaller than 0,2 minutes then rennet whey solids are present.
The difference between the results of two determinations carried out simultaneously or in rapid succession by the same analyst using the same apparatus on identical test material shall not exceed 0,2 % m/m.
Not determined.
From 0 to 16 % of rennet whey a linear relationship should be obtained with a coefficient of correlation > 0,99.
The 1 % limit includes the uncertainty due to reproducibility.
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