Commission Regulation (EC) No 1459/98 of 8 July 1998 establishing a reference method for the determination of vanillin in concentrated butter, butter or cream
Date: 1998-07-08
Institutions: European Commission
Subjects: butter, cream, food inspection, food standard, foodstuffs legislation

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9. 7. 98

COMMISSION REGULATION (EC) No 1459/98

of 8 July 1998

establishing a reference method for

the determination of

vanillin in

concentrated butter, butter or cream

THE COMMISSION OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the 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 products (1), as last amended by Regula-
tion (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 for cream, butter and concentrated
butter for use in the manufacture of pastry products, ice-
cream and other foodstuffs (3), provides for the tracing of
cream, butter and concentrated butter in certain circum-
stances in order to ensure the correct end use of these
products;

Whereas,
in view of the importance of tracing to the
proper functioning of the scheme and in order to ensure
the equal treatment of operators who participate in it, it is
appropriate to establish common methods for the deter-
mination of the tracers referred to in Regulation (EC) No
2571/97;

Whereas it is difficult to establish such reference methods
for all
simultaneously; whereas establishing a
reference method for the determination of vanillin in

tracers

concentrated butter, butter or cream 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 Milk Products,

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 concentrated butter, 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 in accordance with the speci-
fications 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.

Done at Brussels, 8 July 1998.

For the Commission

Franz FISCHLER

Member of the Commission

(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

Determination of vanillin content in concentrated butter, butter or cream by 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 cooling between –15 HC and –20 HC, followed by centrifuga-
tion.

After dilution with water determination of the vanillin content by high-performance liquid chromato-
graphy (HPLC).

3.

Apparatus

3.1.

Usual laboratory apparatus and, in particular, the following:
freezer, operating in the temperature range –15 HC to –20HC;
syringes, disposable of 2 ml capacity;

3.2.
3.3. membrane microfilters of 0,45 lm pore size, resistant to a solution containing 5 % extraction solu-

tion (4.4);
liquid chromatography system consisting of a pump (flow of 1,0 ml/min), an injector (20 ll injection,
automatic or manual), an UV detector (operated at 306 nm, 0,01 AU full scale), a recorder or inte-
grator and a column thermostat operating at 25 HC;
analytical column (250 mm (cid:253) 4,6 mm ID) packed with LiChrospher RP 18 (Merck, 5 lm) or
equivalent;
guard column (ca. 20 mm (cid:253) 3 mm ID) dry-packed with Perisorb RP 18 (30 to 40 lm) or equivalent;

Reagents

All reagents used shall be of recognized analytical quality.

Isopropanol

Ethanol 96 % (v/v)

Acetonitrile

Extraction solution

3.4.

3.5.

3.6.

4.

4.1.

4.2.

4.3.

4.4.

Mix isopropanol (4.1), ethanol (4.2) and acetonitrile (4.3) in the ratio of 1:1:2 (V/V).

Vanillin (4-hydroxy-3-methoxybenzaldehyde)

4.5.
4.5.1. Vanillin stock solution (=500 lg/ml)

Weigh accurately to 0,1 mg about 50 mg (CM mg) vanillin (4.5) in a 100 ml volumetric flask, add 25
ml extraction solution (4.4) and make up with water.

4.5.2. Vanillin standard solution (=10 lg/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 quality

4.7.

Acetic acid, glacial

4.8. Water, HPLC quality

4.9. 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 lm filter.

5.

5.1.

Procedure

Preparation of

the test sample

5.1.1. Butter

Heat the sample until melting starts. Avoid local overheating above 40 HC. 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.

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5.1.2. Concentrated butter

Immediately before sampling place the container, with concentrated butter, into an oven at 40 to
50 HC until it is melted completely. Mix the sample by swirling or stirring, avoiding formation of air
bubbles by too vigorous stirring. Weigh, to the nearest 1 mg, about 4 g (SM g) of concentrated 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 HC. Distribute the fat homo-
geneously by swirling and, if necessary, by stirring. Cool the sample quickly to 20 5 2 HC. The
sample should look homogenous, otherwise the procedure should be repeated. Weigh, to the nearest
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 vig-
orously, for about 15 minutes and make up with extraction solution (4.4). Transfer about 10 ml of this
extract to a test tube fitted with stopper. Place the test tube in the freezer (3.1) and allow it to stand
for about 30 minutes. Centrifuge the cold extract for 5 minutes at approximately 2 000 rpm and
decant immediately. Allow the decanted solution to cool to room temperature. Pipette 5 ml of the
decanted solution into a 100 ml volumetric flask and make up with water. Filter an aliquot through a
membrane microfilter (3.3). The filtrate is ready for determination 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 extrac-
tion solution (4.4) and make up to the mark with water. This solution contains 0,5 lg/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 or peak height between two successive injections is less
than 2 %. Under the conditions described the retention time of vanillin is about 9 minutes. Analyze
the standard solution (5.3) in duplicate by injecting 20 ll. Inject 20 ll of the test solutions (5.2).
Determine the area or height of the vanillin peak obtained. Repeat the duplicate of the standard solu-
tion (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 bracketing
duplicate injections for each batch of test solutions (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 (cid:253) 20 (cid:253) 0,96
SM (cid:253) R

where:

AS = peak area of the vanillin peak of the test sample

SM = mass of test sample in g (5.1.1, 5.1.2 or 5.1.3)

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 using the same apparatus 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 labor-
atories, using different apparatus on identical test material shall not exceed 27 mg/kg.

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8.

8.1.

8.2.

Tolerance limits

Three samples must be taken from the traced product in order to check homogeneity.

Tracer obtained either from vanilla or from synthetic vanillin:

8.2.1. The incorporation rate from 4-hydroxy-3-methoxybenzaldehyde is 250 grams per tonne.

8.2.2. Taking the critical difference for a 95 % probability level (CrD95) into consideration the mean of
single analyses undertaken on each of the three samples taken to check homogeneity shall not be less
than 236,0 mg/kg.

8.2.3.

In addition to the criterion given in 8.2.2 the lowest result obtained from analysis of the product is
used to check homogeneity of tracer distribution. This is done by a comparison with the following
limits:

— 221,5 mg/kg (95 % of the minimum incorporation rate, single sample CrD95 taken into consid-

eration),

— 159,0 mg/kg (70 % of the minimum incorporation rate, single sample CrD95 taken into consid-

eration).

The tracer concentration in the sample giving the lowest results is used in conjunction with inter-
polation between 221,5 mg/kg and 159,0 mg/kg.

8.3.

Tracer obtained exclusively from vanilla beans or integral extracts thereof:

8.3.1. The incorporation rate for 4-hydroxy-3-methoxybenzaldehyde is 100 grams per tonne.

8.3.2. Taking the critical difference for a 95 % probability level (CrD95) into consideration the mean of
single analyses undertaken on each of three samples taken to check homogeneity shall not be less
than 86,0 mg/kg.

8.3.3.

In addition to criterion given in 8.3.2, the lowest result obtained from analysis of the product is used
to check homogeneity of tracer distribution. This is done by comparison with the following limits:

— 79,0 mg/kg (95 % of the minimum incorporation rate, single sample CrD95 taken into considera-

tion),

— 54,0 mg/kg (70 % of the minimum incorporation rate, single sample CrD95 taken into considera-

tion).

The tracer concentration in the sample giving the lowest result is used in conjunction with interpola-
tion between 79,0 mg/kg and 54,0 mg/kg.

9.

9.1.

9.2.

9.3.

9.4.

9.5.

Notes

The repeatability r is the value below which the absolute difference between two single test results
obtained with the same method on identical test material, under the same conditions (same apparatus,
same laboratory, and in a short interval of time), may be expected to lie with a specified probability;
in the absence of other indications, the probability is 95 %.

The reproducibility R is the value below which the absolute difference between two single test results
obtained with the same method on identical test material, under different conditions (different opera-
tors, different apparatus, different laboratories and/or different time), may be expected to lie with a
specified probability; in the absence of other indications, the probability is 95 %.

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 a standard deviation of 2,7 %.

The standard solution contains 5 % extraction solution to compensate for peak broadening caused by
the presence of 5 % of the extraction solution of the test samples. This enables quantification by
peak height.

The analysis is based on a linear calibration line with a zero intercept. By using appropriate dilutions
of the standard solution (4.5.2), the linearity should be checked the first time the analysis is carried
out and furthermore at regular intervals and after changes in or repair of the HPLC equipment.