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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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The method describes a procedure for the quantitative determination of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in skimmed milk powder (SMP) and is suitable for detecting buttermilk solids in SMP.
:
the mass fraction of substance determined using the procedure here specified. The result is expressed as milligrams of phosphatidylethanolamine dipalmitoyl (PEDP) per 100 g powder.
Extraction of aminophospholipids by methanol from reconstituted milk powder. Determination of PS and PE as o-phthaldialdehyde (OPA) derivatives by reversed-phase (RP) HPLC and fluorescence detection. Quantification of PS and PE content in the test sample by reference to a standard sample containing a known amount of PEDP.
All reagents shall be of recognised analytical grade. Water shall be distilled or water of at least equivalent purity, unless otherwise specified.
Note: Standard material shall be stored at – 18 °C.U.K.
Sampling shall be carried out in accordance with ISO Standard 707.
Note: Test sample solution should be stored at 4 °C until the HPLC analysis is performed.U.K.
Note: Reference sample solution should be stored at 4 °C until the HPLC analysis is performed.U.K.
Weigh 25,0 ± 0,1 mg of OPA (4.3.4) into a 10 ml volumetric flask (5.6), add 0,5 ml (5.5) of methanol (4.2.1) and mix carefully to dissolve the OPA. Make up to the mark with boric acid solution (4.3.2) and add 20 μl of 2-mercaptoethanol (4.3.3) by syringe (5.7).
Note: The derivatising reagent should be stored at 4 °C in a brown glass vial and is stable for one week.U.K.
Solvent A: Solution of 0,3 mM sodium dihydrogen phosphate and 3 mM sodium acetate solution (adjusted to pH 6,5 ± 0,1 with acetic acid): methanol: tetrahydrofuran = 558:440:2 (v/v/v)
Solvent B: methanol
Time (min) | Solvent A (%) | Solvent B (%) | Flow rate (ml/min) |
---|---|---|---|
Initial | 40 | 60 | 0 |
0,1 | 40 | 60 | 0,1 |
5,0 | 40 | 60 | 0,1 |
6,0 | 40 | 60 | 1,0 |
6,5 | 40 | 60 | 1,0 |
9,0 | 36 | 64 | 1,0 |
10,0 | 20 | 80 | 1,0 |
11,5 | 16 | 84 | 1,0 |
12,0 | 16 | 84 | 1,0 |
16,0 | 10 | 90 | 1,0 |
19,0 | 0 | 100 | 1,0 |
20,0 | 0 | 100 | 1,0 |
21,0 | 40 | 60 | 1,0 |
29,0 | 40 | 60 | 1,0 |
30,0 | 40 | 60 | 0 |
Note: The eluting gradient may require slight modification in order to achieve the resolution shown in figure 1.U.K.
Column temperature: 30 °C.
Starting up the system on a daily basis, flush the column with 100 % solvent B for 15 minutes, then set at A:B = 40:60 and equilibrate at 1 ml/min for 15 minutes. Perform a blank run by injecting methanol (4.2.1).
Note: Before long-term storage flush the column with methanol: chloroform = 80:20 (v/v) for 30 minutes.U.K.
Note: The column shall be cleaned by flushing with 100 % solvent B (7.5.1) for at least 30 minutes every 20-25 runs.U.K.
PEDP is eluted as a single peak. Determine the peak area by valley-to- valley integration.
Tryptamine is eluted as a single peak (Figure 1). Determine the peak area by valley-to-valley integration.
Under the described conditions (Figure 1), PS elutes as two main partially unresolved peaks preceded by a minor peak. PE elutes as three main partially unresolved peaks. Determine the whole area of each peak cluster setting the baseline as reported in Figure 1.
PS and PE content in the test sample shall be calculated as follows:
C = 55,36 × ((A 2 )/(A 1 )) × ((T 1 )/(T 2 ))
where:
=
PS or PE content (mg/100 g powder) in the test sample
=
PEDP peak area of the standard sample solution (7.3)
=
PS or PE peak area of the test sample solution (7.2)
=
Tryptamine peak area of the standard sample solution (7.3)
=
Tryptamine peak area of the test sample solution (7.2).
Note: The values for repeatability were calculated according to the IDF International Standard (*).U.K.
The relative standard deviation of the repeatability, which expresses the variability of independent analytical results obtained by the same operator using the same apparatus under the same conditions on the same test sample and in a short interval of time, should not exceed 2 % relative. If two determinations are obtained under these conditions, the relative difference between the two results should not be greater than 6 % of the arithmetic mean of the results.
If two determinations are obtained by operators in different laboratories using different apparatus under different conditions for the analysis on the same test sample, the relative difference between the two results should not be greater than 11 % of the arithmetic mean of the results.
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