1998R1459 — EN — 03.02.1999 — 001.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B COMMISSION REGULATION (EC) No 1459/98
of 8 July 1998
establishingareferencemethodforthedeterminationofvanillininconcentratedbutter,butteror
cream
(OJ L 193, 9.7.1998, p. 16)
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 1459/98
of 8 July 1998
establishingareferencemethodforthedeterminationofvanillinin
concentrated butter, butter or cream
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 organisation of the market in milk and milk prod-ucts(
1), as last amended by Regulation (EC) No 1587/96 (2), and in
particular Articles 6(6) and 12(3) thereof,
Whereas Commission Regulation (EC) No 2571/97 of 15 December
1997 on the sale of butter at reduced prices and the granting of aid forcream, butter and concentrated butter for use in the manufacture ofpastry products, ice-cream and other foodstuffs(
3), provides for the
tracingof cream, butter and concentrated butter in certain circumstancesin order to ensure the correct 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 (EC) No2571/97;
Whereas it is difficult to establish such reference methods for all tracers
simultaneously; whereas establishinga reference method for the deter-mination of vanillin in concentrated butter, butter or cream constitutes afurther 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 method of analysis specified in the Annex shall be applied as a
reference method for the determination of vanillin in concentratedbutter, butter or cream pursuant to Regulation (EC) No 2571/97.
Concentrated butter, butter or cream has been traced in conformity with
Article 6 of Regulation (EC) No 2571/97 if the results obtained are inaccordance with the specifications of paragraph 8 of the Annex.
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 September 1998.
This Regulation shall be binding in its entirety and directly applicable in
all Member States.
(1) OJ L 148, 28. 6. 1968, p. 13.
(2) OJ L 206, 16. 8. 1996, p. 21.
(3) OJ L 350, 20. 12. 1997, p. 3.
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ANNEX
Determinationofvanillincontentinconcentratedbutter,butterorcreamby
high-performance liquid chromatography
1.Scope and field of application
The method describes a procedure for the quantitative determination of
vanillin in concentrated butter, butter or cream.
It is applicable to samples received pursuant to Regulation (EC) No
2571/97.
2.Principle
Extraction of a known quantity of sample with a mixture of isopropanol/
ethanol/acetonitrile (1:1:2). Precipitation of the majority of fat by coolingbetween −15 °C and −20 °C, followed by centrifugation.
After dilution with water determination of the vanillin content by high-
performance liquid chromatography (HPLC).
3.Apparatus
Usual laboratory apparatus and, in particular, the following:
3.1. freezer, operatingin the temperature rang e −15 °Ct o− 2 0 °C;
3.2. syringes, disposable of 2 ml capacity;3.3. membrane microfilters of 0,45 µm pore size, resistant to a solution
containing5 % extraction solution (4.4);
3.4. liquid chromatography system consisting of a pump (flow of 1,0 ml/min),
an injector (20 µl injection, automatic or manual), an UV detector (oper-
ated at 306 nm, 0,01 AU full scale), a recorder or integrator and a columnthermostat operatingat 25 °C;
3.5. analytical column (250 mm × 4,6 mm ID) packed with LiChrospher RP
18 (Merck, 5 µm) or equivalent;
3.6. guard column (ca. 20 m m×3m mI D ) dry-packed with Perisorb RP 18
(30 to 40 µm) or equivalent;
4.Reagents
All reagents used shall be of recognized analytical quality.
4.1. Isopropanol4.2. Ethanol 96 % (v/v)4.3. Acetonitrile4.4. Extraction solution
Mix isopropanol (4.1), ethanol (4.2) and acetonitrile (4.3) in the ratio of
1:1:2 (V/V).
4.5. Vanillin (4-hydroxy-3-methoxybenzaldehyde)4.5.1. Vanillin stock solution (=500 µg/ml)
Weig h accurately to 0,1 mgabout 50 mg(CM mg ) vanillin (4.5) in a 100
ml volumetric flask, add 25 ml extraction solution (4.4) and make up withwater.
4.5.2. Vanillin standard solution (=10 µg/ml).
Pipet 5 ml of the vanillin stock solution (4.5.1) into a volumetric flask of
250 ml and make up with water.
4.6. Methanol, HPLC quality4.7. Acetic acid, glacial4.8. Water, HPLC quality4.9. HPLC mobile phase
Mix 300 ml methanol (4.6) about 500 ml water (4.8) and 20 ml acetic acid
(4.7) in a volumetric flask of 1 000 ml and make up with water (4.8).Filter through 0,45 µm filter.
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5.Procedure
5.1.Preparation of the test sample
5.1.1. Butter
Heat the sample until meltingstarts. Avoid local overheatingabove 40 °C.
When the sample becomes sufficiently plastic, homogenise it by shaking.Stir the butter for 15 s before taking a sample. Weigh, to the nearest 1 mg,about 5 g(SM g ) of butter into a 100 ml volumetric flask.
5.1.2. Concentrated butter
Immediately before samplingplace the container, with concentrated
butter, into an oven at 40 to 50 °C until it is melted completely. Mix the
sample by swirlingor stirring , avoidingformation of air bubbles by toovigorous stirring. Weigh, to the nearest 1 mg, about 4 g (SM g) ofconcentrated butter into a 100 ml volumetric flask.
5.1.3. Cream
Heat the sample in a waterbath or incubator at a temperature of 35 to
40°C. Distribute the fat homogeneously by swirling and, if necessary, by
stirring. Cool the sample quickly to 20 ± 2 °C. The sample should look
homogenous, otherwise the procedure should be repeated. Weigh, to thenearest 1 mg, about 10 g (SM g) of cream into a 100 ml volumetric flask.
5.2.Preparation of the test solution
Add about 75 ml extraction solution (4.4) to the test portion (5.1.1, 5.1.2
or 5.1.3), stir, or shake vigorously, for about 15 minutes and make up withextraction solution (4.4). Transfer about 10 ml of this extract to a test tubefitted with stopper. Place the test tube in the freezer (3.1) and allow it tostand for about 30 minutes. Centrifuge the cold extract for 5 minutes atapproximately 2 000 rpm and decant immediately. Allow the decantedsolution to cool to room temperature. Pipette 5 ml of the decanted solutioninto a 100 ml volumetric flask and make up with water. Filter an aliquotthrough a membrane microfilter (3.3). The filtrate is ready for deter-mination by HPLC.
5.3.Calibration
Pipette 5 ml of the vanillin standard solution (4.5.2) into a 100 ml
volumetric flask. Add 5 ml extraction solution (4.4) and make up to themark with water. This solution contains 0,5 µg/ml of vanillin.
5.4.Determination of HPLC
Allow the chromatographic system to stabilise for about 30 minutes. Inject
the standard solution (5.3). Repeat this until the difference in peak area orpeak height between two successive injections is less than 2 %. Under theconditions described the retention time of vanillin is about 9 minutes.Analyze the standard solution (5.3) in duplicate by injecting20 µl. Inject
20µl of the test solutions (5.2). Determine the area or height of the
vanillin peak obtained. Repeat the duplicate of the standard solution (5.3)after 10 injections of test samples (5.2).
6.Calculation of results
Calculate the average peak area (or height) (AC), of the vanillin peaks
associated with the bracketingduplicate injections for each batch of testsolutions (four areas in total).
Calculate the response factor (R):
R = AC/CM
where CM is the mass of vanillin in mg(4.5.1).
The content (mg/kg) of vanillin (C) in the test sample is given by:
C=AS × 20 × 0,96
S M×R
where:AS = peak area of the vanillin peak of the test sample
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SM = mass of test sample in g(5.1.1, 5.1.2 or 5.1.3).
Where the cream is analysed for vanillin, the tracer concentration
has to be expressed as mgtracer/kgmilk fat. This is done bymultiplyingC with 100/f. f is the fat content of the cream inpercent (m/m).
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20 = factor which takes into account the dilutions of the standard and
the test sample
0,96 = correction factor for the fat content in first dilution of the test
sample
Note:
Instead of peak area, peak heights can be used (see 8.3).
7.Accuracy of the method
7.1. Repeatability (r)
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 16 mg/kg.
7.2. Reproducibility (R)
The difference between the results of two determinations carried out by
operators in different laboratories, usingdifferent apparatus on identicaltest material shall not exceed 27 mg/kg.
8.Tolerance limits
8.1. Three samples must be taken from the traced product in order to check
homogeneity.
8.2. Tracer obtained either from vanilla or from synthetic vanillin:
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8.2.1. The incorporation rate for 4-hydroxy-3-methoxybenzaldehyde is 250
grams per tonne of concentrated butter or butter. Where cream is traced,the incorporation rate is 250 grams per tonne of milk fat.
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):
— 221,0 mg/kg (95 % of the minimum incorporation rate),
— 159,0 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 221,0 mg/kg and 159,0 mg/kg.
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8.3. Tracer obtained exclusively from vanilla beans or integral extracts thereof:
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8.3.1. The incorporation rate for 4-hydroxy-3-methoxyzaldehyde is 100 grams
per tonne of concentrated butter or butter. Where cream is traced, theincorporation rate is 100 grams per tonne of milk fat.
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):
— 79,0 mg/kg (95 % of the minimum incorporation rate),
— 54,0 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 79,0 mg/kg and 54,0 mg/kg.
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9.Notes
9.1. The repeatability r is the value below which the absolute difference
between two single test results obtained with the same method on identicaltest material, under the same conditions (same apparatus, same laboratory,and in a short interval of time), may be expected to lie with a specifiedprobability; in the absence of other indications, the probability is 95 %.
9.2. The reproducibility R is the value below which the absolute difference
between two single test results obtained with the same method on identicaltest material, under different conditions (different operators, differentapparatus, different laboratories and/or different time), may be expected tolie with a specified probability; in the absence of other indications, theprobability is 95 %.
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9.3. Recovery of added vanillin at a level of 250 mg/kg butteroil varies from
97,0 to 103,8. The average content which where found was 99,9 % with astandard deviation of 2,7 %.
9.4. The standard solution contains 5 % extraction solution to compensate for
peak broadeningcaused by the presence of 5 % of the extraction solutionof the test samples. This enables quantification by peak height.
9.5. The analysis is based on a linear calibration line with a zero intercept. By
usingappropriate dilutions of the standard solution (4.5.2), the linearityshould be checked the first time the analysis is carried out and furthermoreat regular intervals and after changes in or repair of the HPLC equipment.