Commission Regulation (EC) No 273/2008 (repealed)Show full title

DETERMINING THE ETHYL ESTER OF BETA-APO-8'-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'-carotenic acid (apo-carotenic ester) in concentrated butter and butter. The apo-carotenic ester is the sum of all substances present in an extract of samples obtained under the conditions described in the method which absorb light at 440 nm.

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 to an external standard.

3. APPARATUS

3.1. Pipettes — graduated, of capacity 0,25, 0,50, 0,75 and 1,0 ml

3.2. 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, 20 ml and 100 ml

3.4. Analytical balance, sensitivity 0,1 mg capable of weighing to the nearest 1 mg, with a readability of 0,1 mg

3.5. Oven, 45 °C ± 1 °C

3.6. Fast-filtering ashless filters.

4. REAGENTS

All reagents must be of recognised analytical grade.

4.1.

Apo-carotenic ester suspension (approximately 20 %)

4.1.1. Establish the content of the suspension as follows:

Warm the suspension between 45 °C and 50 °C and homogenize in the unopened original container. Weigh about 400 mg in a volumetric flask (100 ml), dissolve in 20 ml chloroform (4.4) and make up the volume with cyclohexane (4.5). Dilute 5 ml of this solution to 100 ml with cyclohexane (solution A). Dilute 5 ml of solution A to 100 ml with cyclohexane. Measure the absorbance at 447-449 nm (measure the maximum against cyclohexane as a blank using cells with 1 cm optical path length).

Apo-carotenic ester content P (%) = (Abs_max × 40 000) / (M_susp × 2 550) or develop: (Abs_max / 2 550) × (100 / 5) × (100 / 5) × (100 / M_susp)

Abs_max

=

absorbance of the measuring solution at the maximum

M_susp

=

mass of suspension (g)

2 550

=

reference Abs (1 %, 1 cm) value

P

=

Purity (content) of the suspension (%)

Note: Apo-carotenic ester suspension is sensitive to air, heat and light. In the unopened, original container (sealed under nitrogen) and in a cool place it can be stored for about 12 months. After opening the contents should be used within a short period.

4.1.2. Apo-carotenic ester standard solution, approx. 0,2 mg/ml

Weigh to the nearest 1 mg about 0,100 g of apo-carotenic ester suspension (4.1.1) (W), dissolve in petroleum spirit (4.2), transfer quantitatively into a volumetric flask of capacity 100 ml, and make up to the mark with petroleum spirit.

This solution contains (W × P) / 10 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

4.4. Chloroform

4.5. Cyclohexane

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 representative portion through a filter containing about 10 g of anhydrous sodium sulphate (4.3) in an environment shielded from strong natural and artificial light and maintained at 45 °C. Collect a suitable amount of butterfat.

5.2. Determination

Weigh, to the nearest 1 mg approximately 1 g of concentrated butter (or extracted butterfat (5.1.2)), (M). Transfer quantitatively to a 20 ml (V) volumetric flask using petroleum spirit (4.2), make up to the mark and mix thoroughly.

Transfer an aliquot to a 1 cm cell and measure the absorbance at 440 nm, against a petroleum spirit blank. Obtain the concentration of apo-carotenic ester in the solution by referring to the obtained standard curve (C μ/ml).

5.3. Calibration

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 with petroleum 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 the standard solution (4.1.2) (W × P) / 10 mg/ml. Measure the absorbances at 440 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. Calculate the equation of the standard curve.

6. CALCULATION OF THE RESULTS

6.1. Apo-carotenic ester content, expressed as mg/kg product, is given by:

Concentrated butter: (C × V)/M

Butter: 0,82 (C × V)M

where:

C

=

apo-carotenic ester content, μg/ml, read from the calibration graph (5.3)

V

=

volume (ml) of the test solution (5.2)

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 using the same apparatus on identical test material, may 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 using the same apparatus on identical test material, may 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, using different apparatus on identical test material, may not exceed 4,7 mg/kg.

7.3. Concentrated butter analysis

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 5,3 mg/kg.

7.4. Source of precision data

The precision data were determined from an experiment conducted in 1995 involving 11 laboratories and 12 traced samples (six blind duplicates) for butter and 12 traced (six blind duplicates) for concentrated butter.

8. TOLERANCE LIMITS

8.1. Three samples must be taken from the traced product in order to check on the correct tracing of the product.

8.2. Butter

8.2.1. The incorporation rate for butter, taking into account background absorbance, is 22 mg/kg.

8.2.2. The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits:

• 17,7 mg/kg (95 % of the minimum incorporation rate),

• 12,2 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 17,7 mg/kg and 12,2 mg/kg.

8.3. Concentrated butter

8.3.1. The incorporation rate for concentrated butter, taking into account background absorbance, is 24 mg/kg

The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits:

• 19,2 mg/kg (95 % of the minimum incorporation rate),

• 13,2 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 19,2 mg/kg and 13,2 mg/kg.