1996R1082 — EN — 03.02.1999 — 001.001 — 1
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►B COMMISSION REGULATION (EC) No 1082/96
of 14 June 1996
establishing a reference method for the determination of the ethyl ester of beta-apo-8 /prime1carotenic
acid in concentrated butter and butter
(OJ L 142, 15.6.1996, p. 26)
Amended by:
Official Journal
No page date
►M1Commission Regulation (EC) No 175/1999 of 26 January 1999 L 20 22 27.1.1999
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COMMISSION REGULATION (EC) No 1082/96
of 14 June 1996
establishing a reference method for the determination of the ethyl
esterofbeta-apo-8 /prime1carotenicacidinconcentratedbutterandbutter
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Havingreg ard to the Treaty establishingthe European Community,
Having regard to Council Regulation (EEC) No 804/68 of 27 June 1968
on the common organization of the market in milk and milk prod-ucts(
1), as last amended by Commission Regulation (EC) No 2931/
95 (2), and in particular Article 6 (7) and Article 12 (3) thereof,
Whereas Commission Regulation (EEC) No 570/88 of 16 February
1988 on the sale of butter at reduced prices and the granting of aid forbutter and for concentrated butter for use in the manufacture of pastryproducts, ice-cream and other foodstuffs(
3), as last amended by Regula-
tion (EC) No 531/96 (4), provides for the tracingof the subsidized butter
and concentrated butter in certain circumstances in order to ensure thecorrect end use of these products;
Whereas, in view of the importance of tracingto the proper functioning
of the scheme and in order to ensure the equal treatment of operatorswho participate in it, it is appropriate to establish common methods forthe determination of the tracers referred to in Regulation (EEC) No570/88;
Whereas it is difficult to establish such reference methods for all tracers
simultaneously; whereas establishinga reference method for the deter-mination of the ethyl ester of beta-apo-8 /prime1carotenic acid in concentrated
butter or butter constitutes a further step in this direction;
Whereas the measures provided for in this Regulation are in accordance
with the opinion of the Management Committee for Milk and MilkProducts,
HAS ADOPTED THIS REGULATION:
Article 1
The reference method of analysis specified in the Annex shall be
applied for the determination of the beta-apo-8 /prime1carotenic acid ethyl
ester content of concentrated butter or butter under ►M1Regulation
(EC) No 2571/97 ◄.
Concentrated butter or butter have been traced in conformity with
Article 6 of ►M1Regulation (EC) No 2571/97 ◄if the results
obtained are in accordance with the specifications of paragraph 8 of theAnnex.
Article 2
This Regulation shall enter into force on the third day following its
publication in the Official Journal of the European Communities .
It shall apply from 1 October 1996.
This Regulation shall be binding in its entirety and directly applicable in
all Member States.
(1) OJ No L 148, 28. 6. 1968, p. 13.
(2) OJ No L 307, 20. 12. 1995, p. 10.
(3) OJ No L 55, 1. 3. 1988, p. 31.
(4) OJ No L 78, 28. 3. 1996, p. 13.
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ANNEX
DETERMINATION OF THE ETHYL ESTER OF BETA-APO-8 /prime1-
CAROTENIC ACID IN CONCENTRATED BUTTER AND BUTTER BY
SPECTROMETRY
1.Scope and field of application
The method describes a procedure for the quantitative determination of the
ethyl ester of beta-apo-8 /prime1-carotenic acid (apo carotenic ester) in concen-
trated butter and butter. The apo carotenic ester is the sum of allsubstances present in an extract of samples obtained under the conditionsdescribed in the method which absorb light at 440 nm. It is applicable tosamples received under Regulation (EEC) No 570/88.
2.Principle
The butterfat is dissolved in light petroleum and the absorbance measured
at 440 nm. The apo carotenic ester content is determined by reference toan external standard.
3.Apparatus
3.1. Pipettes — graduated, of capacity 0,25, 0,50, 0,75 and 1,0 ml3.2. Spectrophotometer — suitable for use at 440 nm (and 447 — 449 nm) and
equipped with cells of optical path length 1 cm
3.3. Volumetric flasks e.g. 20 ml and 100 ml
4.Reagents
All reagents must be of recognized analytical grade.
4.1. Apo carotenic ester suspension (approximately 20 %)4.1.1. Establish the content of the suspension as follows:
weigh accurately approximately 400 mg into a 100 ml volumetric flask
and dissolve in petroleum spirit (4.2), dilute to volume with petroleumspirit. Dilute 5,0 ml of this solution to 100 ml with petroleum spirit.(Solution A.) Dilute 5,0 ml of Solution A to 100 ml with petroleum spirit.Measure the absorbance at 447 — 449 nm (measure the maximum againstpetroleum spirit as a blank using1 cm cells).
Apo carotenic ester content (%) =A
max·40 000
A·2 550
Amax= absorbance of the measuringsolution at the maximum
A = weight of sample (g)2 550 = reference A (1 %, 1 cm) value
The purity of the suspension is P (%).Note:Apo carotenic ester suspension is sensitive to air, heat and light. In
the unopened, original container (sealed under nitrogen) and in acool place it can be stored for about 12 months. After openingthecontents should be used within a short period.
4.1.2. Apo carotenic ester standard solution, approx 0,2 mg/ml
Weig h to the nearest 0,1 mgabout 0,100 gof apo carotenic ester suspen-
sion (4.1.1) (Wg), dissolve in petroleum spirit (4.2), transfer quantitativelyinto a volumetric flask of capacity 100 ml, and make up to the mark withpetroleum spirit.
This solution contains (W.P)/100 mg/ml of apo carotenic ester.Note:The solution must be stored in a cool place in the dark. Discard
unused solution after one month.
4.2. Petroleum spirit (40 — 60 °C).
4.3. Sodium sulphate, anhydrous, granular previously dried at 102 °C for two
hours.
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5.Procedure
5.1. Preparation of the test sample
5.1.1. Concentrated butter
Melt the sample in an oven at approximately 45 °C.
5.1.2. Butter
Melt the sample in an oven at approximately 45 °C and filter a repres-
entative portion through a filter containing about 10 g of anhydroussodium sulphate (4.3) in an environment shielded from strongnatural andartificial light and maintained at 45 °C. Collect a suitable amount of
butterfat.
5.2. Determination
Weig h, to the nearest 1 mgapproximately 1 gof concentrated butter, (or
extracted butterfat (5.1.2)), (mg). Transfer quantitatively to a 20 ml (Vml)volumetric flask usingpetroleum spirit (4.2), make up to the mark andmix thoroughly.
Transfer an aliquot t oa1c m cell and measure the absorbance at 440 nm,
against a petroleum spirit blank. Obtain the concentration of apo carotenicester in the solution by reference to the calibration graph (C µg/ml).
5.3. Calibration graph
Pipette 0, 0,25, 0,5, 0,75 and 1,0 ml of apo carotenic ester standard
solution (4.1.2) into five 100 ml volumetric flasks. Dilute to volume withpetroleum spirit (4.2) and mix.
The approximate concentrations of the solutions range from 0 to 2 µg/ml
and are calculated accurately by reference to the concentration of thestandard solution (4.1.2) (W.P)/100 mg/ml. Measure the absorbances at440 nm against a petroleum spirit (4.2) blank.
Plot the values of absorbance on the y axis against apo carotenic ester
concentration on the x axis.
6.Calculation of results
6.1. Apo carotenic ester content, expressed as mg/kg product is given by:
Concentrated butter: (C.V)/mButter: 0,82 (C.V)/mwhereC = apo carotenic ester content, µg/ml, read from the calibration
graph (5.3),
m = mass (g) of the test portion (5.2),0,82 = a correction factor for the butterfat content of butter.
7.Accuracy of the method
7.1. Repeatability
7.1.1. Butter analysis
The difference between the results of two determinations carried out
within the shortest feasible time interval, by one operator usingthe sameapparatus on identical test material shall not exceed 1,4 mg/kg.
7.1.2. Concentrated butter analysis
The difference between the results of two determinations carried out
within the shortest feasible time interval, by one operator usingthe sameapparatus on identical test material shall not exceed 1,6 mg/kg.
7.2. Reproducibility
7.2.1. Butter analysis
The difference between the results of two determinations carried out by
operators in different laboratories, usingdifferent apparatus on identicaltest material shall not exceed 4,7 mg/kg.
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7.2.2. Concentrated butter analysis
The difference between the results of two determinations carried out by
operators in different laboratories, usingdifferent apparatus on identicaltest material shall not exceed 5,3 mg/kg.
7.3. Source of precision data
The precision data were determined from an experiment conducted in
1995 involving11 laboratories and 12 traced samples (six blind dupli-cates) for butter; 12 traced (six blind duplicates) for concentrated butter.
8.Tolerance limits
▼
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8.1. Three samples must be taken from the traced product in order to check on
the correct tracingof the product.
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8.2. Butter
8.2.1. The incorporation rate for butter, takinginto account backg round absor-
bance, is 22 mg/kg.
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8.2.2. The results of the three samples obtained from the analysis of the product
is used to check the rate and the homogeneity of tracer incorporation andthe lowest of these results is compared with the followinglimits (CriticalDifference for a 95 % probability level (CrD95) taken into consideration):
— 18,0 mg/kg (95 % of the minimum incorporation rate),
— 13,0 mg/kg (70 % of the minimum incorporation rate).
The tracer concentration of the sample giving the lowest result is used in
conjunction with interpolation between 18,0 mg/kg and 13,0 mg/kg.
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8.3. Concentrated butter
8.3.1. The incorporation rate for concentrated butter, takinginto account back-
ground absorbance, is 24 mg/kg.
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8.3.2. The results of the three samples obtained from the analysis of the product
is used to check the rate and the homogeneity of tracer incorporation andthe lowest of these results is compared with the followinglimits (CriticalDifference for a 95 % probability level (CrD95) taken into consideration):
— 20,0 mg/kg (95 % of the minimum incorporation rate),
— 14,0 mg/kg (70 % of the minimum incorporation rate).
The tracer concentration of the sample giving the lowest result is used in
conjunction with interpolation between 20,0 mg/kg and 14,0 mg/kg.