Textual Amendments
F1Annex 5 para. A1 inserted (E.) (28.2.2023 at 9.10 a.m.) by The Market Measures Payment Schemes (Amendments, Revocation and Transitional Provision) (England) Regulations 2023 (S.I. 2023/124), regs. 1(2)(b), 9(15) (with reg. 10)
Textual Amendments
lots containing up to 800 25-kg bags: at least eight;
lots containing more than 800 25-kg bags: at least eight, plus one for each additional 800 bags or fraction thereof.
where a composite sample shows a defect with regard to one parameter, the quantity from which the sample came is rejected;
where a composite sample shows a defect with regard to more than one parameter, the quantity from which the sample came is rejected and samples are taken from the remaining quantities from the same plant; the analysis of those samples shall be decisive. In that case:
the number of samples laid down in point 2 is doubled,
where a composite sample shows a defect with regard to one or more parameters, the quantity from which the sample came is rejected.
a Scorched particles' analyses may be conducted systematically. However, such analyses shall always be conducted if no sensory checks are performed. | |
b The method to be applied shall be approved by the paying agency (one or both methods). | |
c The method to be applied shall be approved by the paying agency. | |
d Sensory checks shall be performed where deemed necessary after risk based analysis approved by the paying agency. | |
Parameter | Method |
---|---|
Protein | ISO 8968 part 1 |
Fat | ISO 1736 |
Water | ISO 5537 |
Acidity | ISO 6091 |
Lactates | ISO 8069 |
Phosphatase test | ISO 11816 part 1 |
Insolubility index | ISO 8156 |
Scorched particles a | ADPI |
Micro-organisms | ISO 4833-part 1 |
Buttermilk | Appendix I |
Rennet whey b | Appendix II and III |
Acid whey c | ISO 8069 or On-the-spot inspections |
Sensory checks d | ISO 22935 part 2 and 3 |
Textual Amendments
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.
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.
Textual Amendments
F4Words in Annex 5 Pt. 1A Appendix 2 para. 7.1 substituted (31.12.2020) by The Market Measures Payment Schemes (Amendment) (EU Exit) Regulations 2019 (S.I. 2019/823), regs. 1, 6(47)(a)(i); 2020 c. 1, Sch. 5 para. 1(1)
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.
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.
Textual Amendments
F5Words in Annex 5 Pt. 1A Appendix 3 para. 7.1 substituted (31.12.2020) by The Market Measures Payment Schemes (Amendment) (EU Exit) Regulations 2019 (S.I. 2019/823), regs. 1, 6(47)(a)(ii); 2020 c. 1, Sch. 5 para. 1(1)
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.
the bags shall have at least three layers, which together correspond to at least 420 J/m2 TEA average;
the second layer shall be covered with a layer of polyethylene of at least 15 g/m2;
inside the paper layers, a polyethylene bag at least 0,08 mm thick shall be fused to the bottom;
bags shall conform to standard EN 770;
when filling, the powder should be well pressed down. Loose powder must on no account be allowed to penetrate between the various layers.
the approval number identifying the factory and [F6indicating that the skimmed milk powder was produced in the United Kingdom];
the date or, where appropriate, the week of production;
the number of the production batch;
the description ‘spray skimmed-milk powder’.
Textual Amendments
F6Words in Annex 5 Pt. 2 para. 2(a) substituted (31.12.2020) by The Market Measures Payment Schemes (Amendment) (EU Exit) Regulations 2019 (S.I. 2019/823), regs. 1, 6(47)(b); 2020 c. 1, Sch. 5 para. 1(1)