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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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There are currently no known outstanding effects for the Commission Implementing Regulation (EU) 2016/1240,
Appendix II
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This method allows detection of rennet whey in skimmed milk powder intended for public storage by determination of the caseinomacropeptides.
International Standard ISO 707 - Milk and Milk Products - Guidance on sampling.
The content of rennet whey solids is defined as the percentage by mass as determined by the caseinomacropeptide content by the procedure described.
Reconstitution of the skimmed milk powder, removal of fat and proteins with trichloroacetic acid, followed by centrifugation or filtration;
Determination of the quantity of caseinomacropeptides (CMP) in the supernatant by high-performance liquid chromatography (HPLC);
Evaluation of the result obtained for the samples by reference to standard samples consisting of skimmed milk powder with or without the addition of a known percentage of whey powder.
All reagents shall be of recognised analytical grade. The water used shall be distilled water or water of at least equivalent purity.
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.
Dissolve 1,74 g of dipotassium hydrogen phosphate (K 2 HPO 4 ), 12,37 g of potassium dihydrogen phosphate (KH 2 PO 4 ) and 21,41 g of sodium sulphate (Na 2 SO 4 ) in about 700 ml of water. Adjust, if necessary, to pH 6,0, using a solution of phosphoric acid or potassium hydroxide.
Make up to 1 000 ml with water and homogenise.
Note: The composition of the eluent can be updated to comply with the certificate of the standards or the recommendations of the manufacturer of the column packing material. U.K.
Filter the eluent solution, prior to use, through a membrane filter with a 0,45 μm pore diameter.
Mix one volume acetonitrile (CH 3 CN) with nine volumes water. Filter the mixture prior to use through a membrane filter with a 0,45 μm pore diameter.
Note: Any other flushing solvent with a bactericidal effect which does not impair the columns' resolution efficiency may be used. U.K.
Pump
Injector, hand or automatic, with a 15 to 30 μl capacity
Two TSK 2 000 -SW columns in series (length 30 cm, internal diameter 0,75 cm) or equivalent columns (e.g. single TSK 2 000 -SWxl, single Agilent Technologies Zorbax GF 250) and a precolumn (3 cm × 0,3 cm) packed with I 125 or material of equivalent effectiveness
Thermostatic column oven, set at 35 ± 1 °C
Variable wavelength UV detector, permitting measurements at 205 nm with a sensitivity of 0,008 Å
Integrator capable of valley-to-valley integration
Note: Working with columns kept at room temperature is possible, but their power of resolution is slightly lower. In that case, the temperature should vary by less than ± 5 °C in any one range of analyses. 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.
Weight 2,000 ± 0,001 g of test sample into a centrifuge tube (6.2) or a suitable stoppered flask (50 ml).
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. U.K.
Note 2. Rinse the columns with water during each interruption. Never leave the eluent solution in them (5.2). U.K.
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.
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 affect the retention times of the individual peaks considerably.
The integrator (6.11.6) automatically calculates the area A of each peak:
A II : | area of peak II, |
---|---|
A III : | 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.
Use freshly prepared solutions, because CMP degrade in an 8 % trichloroacetic environment. The loss is estimated at 0,2 % per hour at 30 °C.
Peak II: | R II = 100/(A II [0]) |
where:
=
the response factors of peaks II,
=
the areas of peaks II of the standard sample [0] obtained in 8.5.3.
Peak III: | R III = W/(A III [5] – A III [0]) |
where:
=
the response factor of peak III,
=
the areas of peak III in standard samples [0] and [5] respectively obtained in 8.5.3,
=
the quantity of whey in standard sample [5], i.e. 5.
S II [E] = R II × A II [E]
S III [E] = R III × A III [E]
S IV [E] = R IV × A IV [E]
where:
=
the relative areas of peaks II, III and IV respectively in the sample [E],
=
the areas of peaks II and III respectively in the sample [E] obtained in 8.4.2,
=
the response factors calculated in 9.1.1.
RRT III [E] = (RT III [E])/(RT III [5])
where:
=
the relative retention time of peak III in sample [E],
=
the retention time of peak III in sample [E] obtained in 8.4.2,
=
the retention time of peak III in control sample [5] obtained in 8.5.3.
The RRT III [E] is < 1,000 when the whey content is > 5 %;
The RRT III [E] is ≥ 1,000 when the whey content is ≤ 5 %.
The uncertainty allowed for the values of RRT III is ± 0,002.
Normally the value of RRT III [0] deviates little from 1,034. Depending on the condition of the columns, the value may approach 1,000, but it shall always be greater.
W = S III [E] – [1, 3 + (S III [0] – 0,9)]
where:
=
the percentage m/m of rennet whey in the sample [E];
=
the relative area of peak III of test sample [E] obtained as in 9.1.2;
=
represents the relative average area of peak III expressed in grams of rennet whey per 100 g determined in non-adulterated skimmed milk powder of various origins. This figure was obtained experimentally;
=
represents the relative area of peak III which is equal to R III × A III [0]. These values are obtained in 9.1.1 and 8.5.3 respectively;
=
represents the correction to be made to the relative average area 1,3 when S III [0] is not equal to 0,9. Experimentally the relative average area of peak III of the control sample [0] is 0,9.
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.
The difference between two single and independent results, obtained in two different laboratories on identical test material shall not exceed 0,4 % m/m.
where
2,0 | is the maximum value allowed for the relative area of peak III taking into account the relative average area of peak III, i.e. 1,3, the uncertainty due to variations in the composition of skimmed milk powder and the reproducibility of the method (9.3.2), |
(S III [0] – 0,9) | is the correction to be made when the area S III [0] is different from 0,9 (see point 9.2) |
The continuous line represents the linear regression, the coefficients of which are calculated by the least squares method.
The dashed straight line fixes the upper limit of the relative area of peak III with a probability of not being exceeded in 90 % of cases.
The equations for the dashed straight lines of graphs 1 and 2 are:
S III = 0,376 P % – 10,7 | (graph 1), |
S III = 0,0123 S II [E] + 0,93 | (graph 2), |
respectively where:
is the relative area of peak III calculated either according to total protein content or according to the relative area of peak S II [E],
is the total protein content expressed as a percentage, by weight,
is the relative area of sample calculated in point 9.1.2.
These equations are equivalent to the figure of 1,3 mentioned in point 9.2.
The discrepancy (T 1 and T 2 ) between the relative area S III [E] found and the relative area S III is given by means of the following: T 1 = S III [E] – [(0,376 P% – 10,7) + (S III [0] – 0,9)]T 2 = S III [E] – [(0,0123 S II [E] + 0,93) + (S III [0] – 0,9)]
If T 1 and/or T 2 | are zero or less, the presence of rennet whey cannot be determined. |
If T 1 and T 2 | exceed zero, rennet whey is present. |
The rennet whey content is calculated according to the following formula: W = T 2 + 0,91
where:
0,91 is the distance on the vertical axis between the continuous and dotted straight lines.]
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