Commission Regulation (EC) No 2870/2000Dangos y teitl llawn

[F1 IX. EGG YOLK. DETERMINATION OF EGG YOLK CONCENTRATION IN SPIRIT DRINKS — PHOTOMETRIC METHOD U.K.

1. Scope U.K.

This method is suitable for the determination of egg yolk concentration in the range of 40 to 250 g/l in egg liqueur and liqueur with egg.

2. Normative references U.K.

ISO 3696:1897 Water for analytical laboratory use — Specifications and test methods.

3. Principle U.K.

The ethanol-soluble phosphorus compounds found in egg yolk are extracted and assayed photometrically as a phosphorus molybdate complex.

4. Reagents and materials U.K.

4.1. Double-distilled water U.K.

4.2. Diatomaceous earth U.K.

4.3. Ethanol 96 % vol. (CAS 64-17-5) U.K.

4.4. 15 % magnesium acetate (CAS 16674-78-5) solution U.K.

4.5. 10 % sulphuric acid (CAS 7664-93-9) U.K.

4.6. 1 N sulphuric acid. U.K.

4.7. 0,16 g/l potassium dihydrogen phosphate (CAS 778-77-0), KH ₂ PO ₄ solution U.K.

4.8. Reagent for phosphate determination: U.K.

• dissolve 20 g of ammonium molybdate (CAS 12054-85-2), (NH ₄ ) ₆ Mo ₇ O ₂₄ .4H ₂ O in 400 ml water at 50 ^o C;

• dissolve, in another vessel, 1 g of ammonium vanadate (CAS 7803-55-6), NH ₄ VO ₃ , in 300 ml hot water, allow to cool, then add 140 ml of concentrated nitric acid (CAS 7697-37-2). Combine the cooled solutions in a 1 000 ml volumetric flask and make up to the 1 000 ml mark.

5. Apparatus and equipment U.K.

5.1. 100 ml conical flask U.K.

5.2. Ultrasonic bath (or magnetic stirrer) U.K.

5.3. 100 ml volumetric flask U.K.

5.4. 20 ^o C water bath U.K.

5.5. Filter (Whatman No 4 or equivalent) U.K.

5.6. Porcelain (or platinum) crucible U.K.

5.7. Boiling water bath U.K.

5.8. Hot plate U.K.

5.9. Muffle furnace U.K.

5.10. 50 ml volumetric flask U.K.

5.11. 20 ml volumetric flask U.K.

5.12. Spectrophotometer set at 420 nm U.K.

5.13. 1 cm cuvette. U.K.

6. Samples U.K.

Samples are stored at room temperature prior to analysis.

7. Procedure U.K.

7.1. Sample preparation U.K.

7.1.1. Weigh 10 g of the sample into a 100 ml conical flask (5.1). U.K.

7.1.2. Add gradually 70 ml of ethanol (4.3) in small portions, swirling with each addition, and place in an ultrasonic bath (5.2) for 15 minutes (or stir the mixture with a magnetic stirrer (5.2) for 10 minutes at room temperature). U.K.

7.1.3. Transfer the contents of the flask to a 100 ml volumetric flask (5.3) with washings of ethanol (4.3). Adjust to the calibration mark with ethanol (4.3) and place the flasks in a 20 ^o C water bath (5.4). Adjust to the calibration mark at 20 ^o C. U.K.

7.1.4. Add a small amount of diatomaceous earth (4.2) and filter (5.5), discarding the first 20 ml. U.K.

7.1.5. Transfer 25 ml of the filtrate to a porcelain (or platinum) crucible (5.6). The filtrate must then be concentrated by gentle evaporation in a boiling water bath (5.7), with the addition of 5 ml of 15 % magnesium acetate solution (4.4). U.K.

7.1.6. Place the crucibles on a hot plate (5.8) and heat until just dry. U.K.

7.1.7. Ash the residue by heating to incandescence at 600 ^o C in a muffle furnace (5.9) until the ash is white, minimum of one and a half hours but can be left overnight. U.K.

7.1.8. Take up the ash with 10 ml of 10 % sulphuric acid (4.5) and transfer it with washings of distilled water (4.1) to a 50 ml volumetric flask (5.10), and fill to the mark at room temperature with distilled water (4.1). A 5 ml aliquot of this ash solution is to be used to prepare the sample solution of the photometric phosphate assay. U.K.

7.2. Photometric phosphate assay U.K.

7.2.1. Comparative solution U.K.

7.2.1.1. Place 10 ml of 10 % sulphuric acid (4.5) in a 50 ml volumetric flask (5.10) and fill to the mark with distilled water (4.1). U.K.

7.2.1.2. Add to a 5 ml aliquot of this solution (7.2.1.1), contained in a 20 ml volumetric flask (5.11), 1 ml of 1 N sulphuric acid (4.6) and 2 ml of the phosphate reagent (4.8) and make up to 20 ml with distilled water (4.1). U.K.

7.2.1.3. Stopper with a loosely inserted stopper, shake, and heat in a boiling water bath (5.7) for 10 minutes, then cool in a 20 ^o C water bath (5.4) for 20 minutes. U.K.

7.2.1.4. Fill a 1 cm cuvette (5.13) with this comparative solution. U.K.

7.2.2. Sample solution U.K.

7.2.2.1. Add to a 5 ml aliquot of the ash solution (7.1.8), contained in a 20 ml volumetric flask (5.11), 1 ml of 1 N sulphuric acid (4.6) and 2 ml of the phosphate reagent (4.8) and make up to 20 ml with distilled water (4.1). U.K.

7.2.2.2. Stopper with a loosely inserted stopper, shake, and heat in a boiling water bath (5.7) for 10 minutes, then cool in a 20 ^o C water bath (5.4) for 20 minutes. U.K.

7.2.2.3. The yellow solution that develops is immediately analysed spectrophotometrically (5.12) in a 1 cm cuvette (5.13) at 420 nm against the comparative solution (7.2.1.4). U.K.

7.2.3. Calibration curve U.K.

7.2.3.1. To construct the calibration curve, add 2 ml aliquots of the phosphate reagent (4.8) to 20 ml volumetric flasks (5.11) each containing 1 ml of 1 N sulphuric acid (4.6) and 0, 2, 4, 6, 8, and 10 ml of the potassium dihydrogen phosphate solution (4.7) respectively, and make up to the 20 ml mark with distilled water (4.1). U.K.

7.2.3.2. Stopper with a loosely inserted stopper, shake, and heat in a boiling water bath (5.7) for 10 minutes, then cool in a 20 ^o C water bath (5.4) for 20 minutes and analyse spectrophotometrically (5.12) in a 1 cm cuvette (5.13) at 420 nm against the comparative solution (7.2.1.4). U.K.

7.2.3.3. Construction of the calibration curve: U.K.

dihydrogen phosphate solution (ml)	0	2	4	6	8	10
P 2 O 5 (mg)	0	0,167	0,334	0,501	0,668	0,835

8. Expression of results U.K.

The egg yolk content in g/l is calculated from the following formula:

where:

110

conversion factor for total P ₂ O ₅ in g in 100 g of egg yolk

mg P ₂ O ₅

value established from the calibration curve

density

mass per unit volume (g/ml) of the egg-based liqueur at 20 ^o C

E

weight of the egg-based liqueur in g

40

dilution factor for a 5 ml aliquot of ash solution.

9. Method performance characteristics (precision) U.K.

Statistical results of the interlaboratory test:

• the following table gives the values for egg yolk.

• The following data were obtained from an international method performance study carried out to internationally agreed procedures.

• Year of interlaboratory test:	1998
  Number of laboratories:	24
  Number of samples:	5
  Analyte:	Egg yolk

  Samples	A	B	C	D	E
  Number of laboratories retained after eliminating outliers	19	20	22	20	22
  Number of outliers (laboratories)	3	4	2	4	2
  Number of accepted results	38	40	44	40	44
  Mean value	147,3	241,1	227,4	51,9 (*) 72,8 (*)	191,1
  Repeatability standard deviation (S r ) g/l	2,44	4,24	3,93	1,83	3,25
  Repeatability relative standard deviation (RSD r ) (%)	1,7	1,8	1,8	2,9	1,7
  Repeatability limit (r) g/l	6,8	11,9	11,0	5,1	9,1
  Reproducibility standard deviation (S R ) g/l	5,01	6,06	6,66	3,42	6,87
  Reproducibility relative standard deviation (RSD R ) (%)	3,4	2,5	2,9	5,5	3,6
  Reproducibility limit (R) g/l	14,0	17,0	18,7	9,6	19,2

  Sample types

  A

  Advocaat, blind duplicates

  B

  Advocaat, blind duplicates

  C

  Advocaat, blind duplicates

  D

  Advocaat (diluted), split levels (*)

  E

  Advocaat, blind duplicates]

Textual Amendments

F1 Inserted by Commission Regulation (EC) No 2091/2002 of 26 November 2002 amending Regulation (EC) No 2870/2000 laying down Community reference methods for the analysis of spirits drinks.