Commission Regulation (EC) No 2870/2000 of 19 December 2000 laying down Community reference methods for the analysis of spirits drinks

The method has been validated in an interlaboratory study for pastis, distilled anis, cherry liqueur, crème de (followed by the name of a fruit or the raw material used) and crème de cassis, at levels ranging from 10,86 g/l to 509,7 g/l. However, linearity of the instrument response was proven for the concentration range 2,5 g/l to 20,0 g/l.

This method is not intended for determining low levels of sugars.

ISO 3696:1987 Waters for analytical use — Specifications and test methods.

High-performance liquid chromatography assays of sugar solutions, in order to determine their glucose, fructose, sucrose, maltose and lactose concentrations.

This method uses an alkylamine stationary phase and differential refractometry detection and is given as an example. The use of anion exchange resins as stationary phase would also be possible.

4.1. Glucose (CAS 50-99-7), at least 99 % pure.

4.2. Fructose (CAS 57-48-7), at least 99 % pure.

4.3. Sucrose (CAS 57-50-1), at least 99 % pure.

4.4. Lactose (CAS 5965-66-2), at least 99 % pure.

4.5. Maltose monohydrate (CAS 6363-53-7), at least 99 % pure.

4.6. Pure acetonitrile (CAS 75-05-8) for HPLC analysis.

4.7. Distilled or demineralised water, preferably microfiltered.

4.8. The elution solvent is composed of: Pass helium through at a slow rate for 5-10 minutes prior to use to degas. If the water being used has not been microfiltered, the solvent should be filtered with a filter for organic solvents with a pore size less than or equal to 0,45 μm.

4.9. Ethanol absolute (CAS 64-17-5).

4.10. Ethanol solution (5 %, v/v).

4.11. Weigh 2 g each of the sugars to be analysed (4.1 to 4.5), transfer them without loss to a 100 ml volumetric flask. (NB 2,11 g of maltose monohydrate is equivalent to 2 g of maltose). Adjust to 100 ml with a 5 % vol. alcohol solution (4.10), shake and store at around + 4 °C. Prepare a new stock solution once a week.

4.12. Dilute the stock solution, 20 g/l (4.11) appropriately with a 5 % vol. alcohol solution (4.10) to give five working standards of 2,5, 5,0, 7,5, 10,0 and 20,0 g/l. Filter with a filter of a pore size less than or equal to 0,45 μm (5.3).

5.1.1. High-performance liquid chromatograph with a six-way injection valve fitted with a 10 μl loop or any other device, whether automatic or manual, for the reliable injection of microvolumes.

5.1.2. Pumping system enabling one to achieve and maintain a constant or programmed rate of flow with great precision.

5.1.3. Differential refractometer.

5.1.4. Computational integrator or recorder, the performance of which is compatible with the rest of the set-up.

5.1.5. Pre-column: It is recommended that a suitable pre-column is attached to the analytical column.

| 5.1.6. | Column (example): Material: stainless steel or glass. Internal diameter: 2-5 mm. Length: 100-250 mm (depending on the packing particle size), for example, 250 mm if the particles are 5 μm in diameter. Stationary phase: alkylamine functional groups bonded to silica, maximum particle size 5 μm. |

| --- | --- | | Material: | stainless steel or glass. | | Internal diameter: | 2-5 mm. | | Length: | 100-250 mm (depending on the packing particle size), for example, 250 mm if the particles are 5 μm in diameter. | | Stationary phase: | alkylamine functional groups bonded to silica, maximum particle size 5 μm. |

5.1.7. Chromatography conditions (example): To make certain that the detector is perfectly stable, it should be switched on a few hours before use. The reference cell must be filled with the elution solvent.

5.2. Analytical balance accurate to 0,1 mg.

5.3. Filtration set-up for small volumes using a 0,45 μm micromembrane.

On receipt, samples are to be stored at room temperature prior to analysis.

7.1.1. Shake the sample.

7.1.2. Filter the sample through a filter with a pore size less than or equal to 0,45 μm (5.3).

Inject 10 μl of the standard solutions (4.12) and samples (7.1.2). Perform the analysis under suitable chromatography conditions, for example those described above.

7.2.2. Should any peak of a sample have a greater area (or height) than the corresponding peak in the most concentrated standard, then the sample should be diluted with distilled water and reanalysed.

8. Calculation

Compare the two chromatograms obtained for the standard solution and spirit. Identify the peaks by their retention times. Measure their areas (or heights) to calculate the concentrations by the external standard method. Take into account any dilutions made to the sample.

The final result is the sum of sucrose, maltose, lactose, glucose and fructose, expressed as invert sugar in g/l.

Invert sugar is calculated as the sum of all monosaccharides and reducing disaccharides present, plus the stoichiometric amount of glucose and fructose calculated from the sucrose present.

The following data were obtained from an international method performance study carried out to internationally agreed procedures (¹) (²).

Year of interlaboratory test	2000
Number of laboratories	24
Number of samples	8

(1)‘Protocol for the design, conduct and interpretation of method-performance studies’, Horwitz, W. (1995) Pure and Applied Chemistry, 67, 332-343.

(2)Horwitz, W. (1982) Analytical Chemistry, 54, 67A-76A.

| Analyte | Fructose | Glucose | Maltose | | | | |

| --- | --- | --- | --- | --- | --- | --- | --- | | Samples (× 2) | Crème de Cassis | Standard (50 g/l) | Aniseed-flavoured spirit drink | Crème de Cassis | Standard (50 g/l) | Aniseed-flavoured spirit drink | Standard (10 g/l) | | Mean value (g/l) | 92,78 | 50,61 | 15,62 | 93,16 | 50,06 | 15,81 | 9,32 | | No of labs without outliers | 21 | 22 | 21 | 23 | 19 | 21 | 22 | | Repeatability standard deviation, sr, (g/l) | 2,34 | 2,12 | 0,43 | 3,47 | 1,01 | 0,48 | 0,54 | | Repeatability relative standard deviation, RSDr (%) | 2,53 | 4,2 | 2,76 | 3,72 | 2,03 | 3,02 | 5,77 | | Repeatability limit, r (g/l) (r = 2,8 × sr) | 6,56 | 5,95 | 1,21 | 9,71 | 2,84 | 1,34 | 1,51 | | Reproducibility standard deviation, sR (g/l) | 7,72 | 3,13 | 0,84 | 9,99 | 2,7 | 0,88 | 1,4 | | Reproducibility relative standard deviation, RSDR (%) | 8,32 | 6,18 | 5,37 | 10,72 | 5,4 | 5,54 | 15,06 | | Reproducibility limit, R (g/l) (R = 2,8 × sR) | 21,62 | 8,76 | 2,35 | 27,97 | 7,57 | 2,45 | 3,93 | | Analyte | Sucrose | | | | | | | --- | --- | --- | --- | --- | --- | --- | | Samples | Pastis | Ouzo | Cherry liqueur | Crème de Menthe | Crème de Cassis | Standard (100 g/l) | | Mean value (g/l) | 10,83 | 29,2 19,7 (¹) | 103,33 | 349,96 | 319,84 | 99,83 | | No of labs without outliers | 19 | 19 | 20 | 18 | 18 | 18 | | Repeatability standard deviation, sr (g/l) | 0,09 | 0,75 | 2,17 | 5,99 | 4,31 | 1,25 | | Repeatability relative standard deviation, RSDr (%) | 0,81 | 3,07 | 2,1 | 1,71 | 1,35 | 1,25 | | Repeatability limit, r (g/l) (r = 2,8 × sr) | 0,25 | 2,1 | 6,07 | 16,76 | 12,06 | 3,49 | | Reproducibility standard deviation, sR (g/l) | 0,79 | 0,92 | 4,18 | 9,94 | 16,11 | 4,63 | | Reproducibility relative standard deviation, RSDR (%) | 7,31 | 3,76 | 4,05 | 2,84 | 5,04 | 4,64 | | Reproducibility limit, R (g/l) (R = 2,8 × sR) | 2,22 | 2,57 | 11,7 | 27,84 | 45,12 | 12,97 | | (1) split level. | | | | | | |

| Samples | Pastis | Ouzo | Aniseed-flavoured spirit drink | Cherry liqueur | Crème de Menthe | Crème de Cassis | Standard (220 g/l) |

| --- | --- | --- | --- | --- | --- | --- | --- | | Mean value (g/l) | 10,86 | 29,2 19,7 (¹) | 31,59 | 103,33 | 349,73 | 509,69 | 218,78 | | No of Labs without outliers | 20 | 19 | 20 | 20 | 18 | 18 | 19 | | Repeatability standard deviation, sr (g/l) | 0,13 | 0,75 | 0,77 | 2,17 | 5,89 | 5,59 | 2,71 | | Repeatability relative standard deviation, RSDr (%) | 1,16 | 3,07 | 2,45 | 2,1 | 1,69 | 1,1 | 1,24 | | Repeatability limit, r (g/l) (r = 2,8 × sr) | 0,35 | 2,1 | 2,17 | 6,07 | 16,5 | 15,65 | 7,59 | | Reproducibility standard deviation sR (g/l) | 0,79 | 0,92 | 1,51 | 4,18 | 9,98 | 14,81 | 8,53 | | Reproducibility relative standard deviation, RSDR (%) | 7,25 | 3,76 | 4,79 | 4,04 | 2,85 | 2,91 | 3,9 | | Reproducibility limit R (g/l) (R = 2,8 × sR) | 2,21 | 2,57 | 4,24 | 11,7 | 27,94 | 41,48 | 23,89 | | (1) split level. | | | | | | | |

IX.   EGG YOLK. DETERMINATION OF EGG YOLK CONCENTRATION IN SPIRIT DRINKS — PHOTOMETRIC METHOD

1. Scope

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

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

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

4.1.Double-distilled water

4.2.Diatomaceous earth

4.3.Ethanol 96 % vol. (CAS 64-17-5)

4.4.15 % magnesium acetate (CAS 16674-78-5) solution

4.5.10 % sulphuric acid (CAS 7664-93-9)

4.6.1 N sulphuric acid.

4.7.0,16 g/l potassium dihydrogen phosphate (CAS 778-77-0), KH2PO4 solution

4.8.Reagent for phosphate determination:

5.1.100 ml conical flask

5.2.Ultrasonic bath (or magnetic stirrer)

5.3.100 ml volumetric flask

5.4.20 ^oC water bath

5.5.Filter (Whatman No 4 or equivalent)

5.6.Porcelain (or platinum) crucible

5.7.Boiling water bath

5.8.Hot plate

5.9.Muffle furnace

5.10.50 ml volumetric flask

5.11.20 ml volumetric flask

5.12.Spectrophotometer set at 420 nm

5.13.1 cm cuvette.

Samples are stored at room temperature prior to analysis.

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

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).

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 ^oC water bath (5.4). Adjust to the calibration mark at 20 ^oC.

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

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).

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

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

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.

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).

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).

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 ^oC water bath (5.4) for 20 minutes.

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

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).

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 ^oC water bath (5.4) for 20 minutes.

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).

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).

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 ^oC 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).

7.2.3.3.Construction of the calibration curve:

dihydrogen phosphate solution (ml)	0	2	4	6	8	10
P2O5 (mg)	0	0,167	0,334	0,501	0,668	0,835

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

where:

110 conversion factor for total P2O5 in g in 100 g of egg yolk

mg P2O5 value established from the calibration curve

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

E weight of the egg-based liqueur in g

40 dilution factor for a 5 ml aliquot of ash solution.

Statistical results of the interlaboratory test:

Year of interlaboratory test:	1998
Number of laboratories:	24
Number of samples:	5
Analyte:	Egg yolk
Samples	A
---	---
Number of laboratories retained after eliminating outliers	19
Number of outliers (laboratories)	3
Number of accepted results	38
Mean value	147,3
Repeatability standard deviation (Sr) g/l	2,44
Repeatability relative standard deviation (RSDr) (%)	1,7
Repeatability limit (r) g/l	6,8
Reproducibility standard deviation (SR) g/l	5,01
Reproducibility relative standard deviation (RSDR) (%)	3,4
Reproducibility limit (R) g/l	14,0

A Advocaat, blind duplicates

B Advocaat, blind duplicates

C Advocaat, blind duplicates

D Advocaat (diluted), split levels (*)

E Advocaat, blind duplicates

X. DETERMINATION OF THE FOLLOWING WOOD COMPOUNDS IN SPIRIT DRINKS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): FURFURAL, 5-HYDROXYMETHYLFURFURAL, 5-METHYLFURFURAL, VANILLIN, SYRINGALDEHYDE, CONIFERALDEHYDE, SINAPALDEHYDE, GALLIC ACID, ELLAGIC ACID, VANILLIC ACID, SYRINGIC ACID AND SCOPOLETIN

1. Scope

The method pertains to the determination of furfural, 5-hydroxymethylfurfural, 5-methylfurfural, vanillin, syringaldehyde, coniferaldehyde, sinapaldehyde, gallic acid, ellagic acid, vanillic acid, syringic acid and scopoletin, by high-performance liquid chromatography.

2. Normative reference

Analytical method recognised by the General Assembly of the International Organisation of Vine and Wine (OIV) and published by OIV under the reference OIV-MA-BS-16: R2009.

Determination by high-performance liquid chromatography (HPLC), with detection by ultraviolet spectrophotometry at several wavelengths and by spectrofluorimetry.

The reagents must be of analytical quality. The water used must be distilled water or water of at least equivalent purity. It is preferable to use microfiltered water with a resistivity of 18,2 M Ω.cm.

4.1. 96 % vol. alcohol.

4.2. HPLC-quality methanol (Solvent B).

4.3. Acetic acid diluted to 0,5 % vol. (Solvent A).

4.4. Mobile phases: (given as an example only).

Solvent A (0,5 % acetic acid) and solvent B (pure methanol). Filter through a membrane (porosity 0,45 μm). Degas in an ultrasonic bath, if necessary.

4.5. Reference standards of 99 % minimum purity: furfural, 5-hydroxymethyl furfural, 5-methylfurfural, vanillin, syringaldehyde, coniferaldehyde, sinapaldehyde, gallic acid, ellagic acid, vanillic acid, syringic acid and scopoletin.

4.6. Reference solution: the standard substances are dissolved in a 50 % vol. aqueous-alcoholic solution. The final concentrations in the reference solution should be of the order of: furfural: 5 mg/l; 5-hydroxymethyl furfural: 10 mg/l; 5-methylfurfural 2 mg/l; vanillin: 5 mg/l; syringaldehyde: 10 mg/l; coniferaldehyde: 5 mg/l; sinapaldehyde: 5 mg/l; gallic acid: 10 mg/l; ellagic acid: 10 mg/l; vanillic acid: 5 mg/l; syringic acid: 5 mg/l; scopoletin: 0,5 mg/l.

Standard laboratory apparatus

5.1. A high-performance liquid chromatograph capable of functioning in binary gradient mode and equipped with:

5.2. Syringes for HPLC.

5.3. Device for membrane-filtration of small volumes.

5.4. Integrator-computer or recorder with performance compatible with the entire apparatus, and in particular, it must have several acquisition channels.

The reference solution and the spirit drink are filtered, if necessary, through a membrane with a maximum pore diameter of 0,45 μm.

6.2. Chromatographic operating conditions: carry out the analysis at ambient temperature by means of the equipment described in (5.1) and using the mobile phases (4.4) with a flow of approximately 0,6 ml per minute following the gradient below (given as an example only) Time: 0 min 50 min 70 min 90 min solvent A (water-acid): 100 % 60 % 100 % 100 % solvent B (methanol): 0 % 40 % 0 % 0 % Note that in certain cases this gradient should be modified to avoid co-elutions.

Express the concentration of each constituent in mg/l.

The following data were obtained in 2009 from an international method-performance study on a variety of spirit drinks, carried out following internationally-agreed procedures (1) (2).

Analyte	Furfural
Samples	Whisky	Brandy	Rum	Cognac 1	Bourbon	Cognac 2
No of laboratories participating	15	15	15	15	15	15
No of results accepted (laboratories)	14	12	13	14	13	13
Mean value (mg/l)	2,9	1,2	1,7	10,6	15,3	13,9
Repeatability standard deviation, sr (mg/l)	0,04	0,05	0,04	0,18	0,23	0,20
Repeatability relative standard deviation, RSDr (%)	1,4	4,5	2,3	1,7	1,5	1,5
Repeatability limit, r (mg/l) (r = 2,8 × sr)	0,1	0,2	0,1	0,5	0,6	0,6
Reproducibility standard deviation, sR (mg/l)	0,24	0,18	0,09	1,4	0,49	0,69
Reproducibility relative standard deviation, RSDR (%)	8	15	5	13	3	5
Reproducibility limit, R (g/l) (R = 2,8 × sR)	0,7	0,5	0,3	3,8	1,4	1,9
Analyte	5-Hydroxymethylfurfural
---	---	---	---	---	---	---
Samples	Whisky	Brandy	Rum	Cognac 1	Bourbon	Cognac 2
No of laboratories participating	16	16	16	16	16	16
No of results accepted (laboratories)	14	14	14	14	14	14
Mean value (mg/l)	5,0	11,1	9,4	33,7	5,8	17,5
Repeatability standard deviation, sr (mg/l)	0,09	0,09	0,09	0,42	0,07	0,13
Repeatability relative standard deviation, RSDr (%)	1,7	0,8	1,0	1,3	1,2	0,8
Repeatability limit, r (mg/l) (r = 2,8 × sr)	0,2	0,3	0,3	1,2	0,2	0,4
Reproducibility standard deviation, sR (mg/l)	0,39	1,01	0,50	4,5	0,4	1,6
Reproducibility relative standard deviation, RSDR (%)	8	9	5	13	7	9
Reproducibility limit, R (g/l) (R = 2,8 × sR)	1,1	2,8	1,4	12,5	1,1	4,6
Analyte	5-Methylfurfural
---	---	---	---	---	---	---
Samples	Whisky	Brandy	Rum	Cognac 1	Bourbon	Cognac 2
No of laboratories participating	11	11	11	11	11	11
No of results accepted (laboratories)	11	11	8	11	10	11
Mean value (mg/l)	0,1	0,2	0,1	0,5	1,7	0,8
Repeatability standard deviation, sr (mg/l)	0,01	0,01	0,02	0,02	0,03	0,07
Repeatability relative standard deviation, RSDr (%)	10,7	6,1	13,6	4,7	2,0	10,0
Repeatability limit, r (mg/l) (r = 2,8 × sr)	0,0	0,0	0,1	0,1	0,1	0,2
Reproducibility standard deviation, sR (mg/l)	0,03	0,04	0,03	0,18	0,20	0,26
Reproducibility relative standard deviation, RSDR (%)	35	18	22	39	12	35
Reproducibility limit, R (g/l) (R = 2,8 × sR)	0,1	0,1	0,1	0,5	0,6	0,7
Analyte	Vanillin
---	---	---	---	---	---	---
Samples	Whisky	Brandy	Rum	Cognac 1	Bourbon	Cognac 2
No of laboratories participating	16	15	16	16	16	16
No of results accepted (laboratories)	16	15	16	16	16	16
Mean value (mg/l)	0,5	0,2	1,2	1,2	3,2	3,9
Repeatability standard deviation, sr (mg/l)	0,03	0,02	0,06	0,11	0,11	0,09
Repeatability relative standard deviation, RSDr (%)	6,8	9,6	4,6	8,9	3,5	2,3
Repeatability limit, r (mg/l) (r = 2,8 × sr)	0,1	0,1	0,2	0,3	0,3	0,3
Reproducibility standard deviation, sR (mg/l)	0,09	0,06	0,18	0,27	0,41	0,62
Reproducibility relative standard deviation, RSDR (%)	19	25	15	22	13	16
Reproducibility limit, R (g/l) (R = 2,8 × sR)	0,3	0,2	0,5	0,8	1,2	1,7
Analyte	Syringaldehyde
---	---	---	---	---	---	---
Samples	Whisky	Brandy	Rum	Cognac 1	Bourbon	Cognac 2
No of laboratories participating	16	15	16	16	16	16
No of results accepted (laboratories)	13	13	13	12	14	13
Mean value (mg/l)	1,0	0,2	4,8	3,2	10,5	9,7
Repeatability standard deviation, sr (mg/l)	0,03	0,02	0,04	0,08	0,10	0,09
Repeatability relative standard deviation, RSDr (%)	2,6	8,1	0,8	2,6	0,9	0,9
Repeatability limit, r (mg/l) (r = 2,8 × sr)	0,1	0,1	0,1	0,2	0,3	0,3
Reproducibility standard deviation, sR (mg/l)	0,08	0,07	0,23	0,19	0,39	0,43
Reproducibility relative standard deviation, RSDR (%)	8	33	5	6	4	4
Reproducibility limit, R (g/l) (R = 2,8 × sR)	0,2	0,2	0,7	0,5	1,1	1,2
Analyte	Coniferaldehyde
---	---	---	---	---	---	---
Samples	Whisky	Brandy	Rum	Cognac 1	Bourbon	Cognac 2
No of laboratories participating	13	12	13	12	13	13
No of results accepted (laboratories)	12	12	13	12	13	13
Mean value (mg/l)	0,2	0,2	0,6	0,8	4,6	1,3
Repeatability standard deviation, sr (mg/l)	0,02	0,02	0,03	0,03	0,09	0,06
Repeatability relative standard deviation, RSDr (%)	9,2	9,8	4,6	4,3	1,9	4,5
Repeatability limit, r (mg/l) (r = 2,8 × sr)	0,04	0,04	0,07	0,09	0,24	0,16
Reproducibility standard deviation, sR (mg/l)	0,04	0,04	0,11	0,18	0,38	0,25
Reproducibility relative standard deviation, RSDR (%)	23	27	21	23	8	19
Reproducibility limit, R (g/l) (R = 2,8 × sR)	0,1	0,1	0,3	0,5	1,1	0,7
Analyte	Sinapaldehyde
---	---	---	---	---	---	---
Samples	Whisky	Brandy	Rum	Cognac 1	Bourbon	Cognac 2
No of laboratories participating	14	14	14	14	15	14
No of results accepted (laboratories)	14	13	12	13	13	12
Mean value (mg/l)	0,3	0,2	0,2	1,6	8,3	0,3
Repeatability standard deviation, sr (mg/l)	0,02	0,01	0,02	0,06	0,14	0,03
Repeatability relative standard deviation, RSDr (%)	7,5	4,6	11,2	3,7	1,6	11,4
Repeatability limit, r (mg/l) (r = 2,8 × sr)	0,06	0,03	0,06	0,17	0,38	0,08
Reproducibility standard deviation, sR (mg/l)	0,09	0,05	0,08	0,20	0,81	0,18
Reproducibility relative standard deviation, RSDR (%)	31	27	46	13	10	73
Reproducibility limit, R (g/l) (R = 2,8 × sR)	0,2	0,2	0,2	0,6	2,3	0,5
Analyte	Gallic acid
---	---	---	---	---	---	---
Sample	Whisky	Brandy	Rum	Cognac 1	Bourbon	Cognac 2
No of laboratories participating	16	15	16	16	16	16
No of results accepted (laboratories)	15	14	16	16	16	16
Mean value (mg/l)	1,2	0,4	2,0	6,1	7,3	21,8
Repeatability standard deviation, sr (mg/l)	0,07	0,04	0,06	0,18	0,18	0,60
Repeatability relative standard deviation, RSDr (%)	6,1	8,1	2,9	3,0	2,4	2,8
Repeatability limit, r (mg/l) (r = 2,8 × sr)	0,2	0,1	0,2	0,5	0,5	1,7
Reproducibility standard deviation, sR (mg/l)	0,43	0,20	0,62	3,3	2,2	7,7
Reproducibility relative standard deviation, RSDR (%)	36	47	31	53	30	35
Reproducibility limit, R (g/l) (R = 2,8 × sR)	1,2	0,6	1,7	9,1	6,2	21,7
Analyte	Ellagic acid
---	---	---	---	---	---	---
Samples	Whisky	Brandy	Rum	Cognac 1	Bourbon	Cognac 2
No of laboratories participating	7	7	7	7	7	7
No of results accepted (laboratories)	7	7	7	7	7	6
Mean value (mg/l)	3,2	1,0	9,5	13	13	36
Repeatability standard deviation, sr (mg/l)	0,20	0,16	0,30	0,41	0,95	0,34
Repeatability relative standard deviation, RSDr (%)	6,3	16	3,2	3,2	7,4	1,0
Repeatability limit, r (mg/l) (r = 2,8 × sr)	0,6	0,4	0,9	1,1	2,7	1,0
Reproducibility standard deviation, sR (mg/l)	1,41	0,42	4,0	5,0	4,9	14
Reproducibility relative standard deviation, RSDR (%)	44	43	42	39	39	40
Reproducibility limit, R (g/l) (R = 2,8 × sR)	4,0	1,2	11	14	14	40
Analyte	Vanillic acid
---	---	---	---	---	---	---
Samples	Whisky	Brandy	Rum	Cognac 1	Bourbon	Cognac 2
No of laboratories participating	15	15	15	15	15	15
No of results accepted (laboratories)	12	11	14	14	15	14
Mean value (mg/l)	0,2	0,2	1,5	0,8	2,4	2,7
Repeatability standard deviation, sr (mg/l)	0,03	0,04	0,03	0,10	0,13	0,21
Repeatability relative standard deviation, RSDr (%)	14,2	16,5	2,3	12,6	5,3	7,7
Repeatability limit, r (mg/l) (r = 2,8 × sr)	0,1	0,1	0,1	0,3	0,4	0,6
Reproducibility standard deviation, sR (mg/l)	0,06	0,05	0,51	0,2	1,22	0,70
Reproducibility relative standard deviation, RSDR (%)	28	20	35	31	51	26
Reproducibility limit, R (g/l) (R = 2,8 × sR)	0,2	0,1	1,4	0,7	3,4	2,0

| Analyte | Syringic acid | | | | | |

| --- | --- | --- | --- | --- | --- | --- | | Samples | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 | | No of laboratories participating | 16 | 15 | 16 | 16 | 16 | 16 | | No of results accepted (laboratories) | 16 | 15 | 15 | 15 | 16 | 15 | | Mean value (mg/l) | 0,4 | 0,2 | 2,5 | 1,4 | 3,4 | 4,8 | | Repeatability standard deviation, sr (mg/l) | 0,03 | 0,02 | 0,06 | 0,13 | 0,08 | 0,11 | | Repeatability relative standard deviation, RSDr (%) | 6,7 | 12,6 | 2,3 | 9,0 | 2,3 | 2,3 | | Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,1 | 0,1 | 0,2 | 0,4 | 0,2 | 0,3 | | Reproducibility standard deviation, sR (mg/l) | 0,08 | 0,05 | 0,29 | 0,26 | 0,43 | 0,67 | | Reproducibility relative standard deviation, RSDR (%) | 19 | 29 | 11 | 18 | 13 | 14 | | Reproducibility limit, R (g/l) (R = 2,8 × sR) | 0,2 | 0,1 | 0,8 | 0,7 | 1,2 | 1,9 |

| Analyte | Scopoletin | | | | | |

| --- | --- | --- | --- | --- | --- | --- | | Samples | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 | | No of laboratories participating | 10 | 10 | 10 | 10 | 10 | 10 | | No of results accepted (laboratories) | 9 | 8 | 9 | 8 | 8 | 8 | | Mean value (mg/l) | 0,09 | 0,04 | 0,11 | 0,04 | 0,65 | 0,15 | | Repeatability standard deviation, sr (mg/l) | 0,0024 | 0,0008 | 0,0018 | 0,0014 | 0,0054 | 0,0040 | | Repeatability relative standard deviation, RSDr (%) | 2,6 | 2,2 | 1,6 | 3,3 | 0,8 | 2,7 | | Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,007 | 0,002 | 0,005 | 0,004 | 0,015 | 0,011 | | Reproducibility standard deviation, sR (mg/l) | 0,01 | 0,01 | 0,03 | 0,01 | 0,09 | 0,02 | | Reproducibility relative standard deviation, RSDR (%) | 15 | 16 | 23 | 17 | 15 | 15 | | Reproducibility limit, R (g/l) (R = 2,8 × sR) | 0,04 | 0,02 | 0,07 | 0,02 | 0,26 | 0,06 |

(1)‘Protocol for the design, conduct and interpretation of method-performance studies’, Horwitz, W. (1995) Pure and Applied Chemistry, 67, 332-343.

(2)Horwitz, W. (1982) Analytical Chemistry, 54, 67A-76A.

XI.   DETERMINATION OF ¹⁴C CONTENT IN ETHANOL

Determination of the ¹⁴C content in ethanol permits a distinction to be made between alcohol from fossil fuels (synthesis alcohol) and alcohol from recent raw materials (fermentation alcohol).

The ¹⁴C content of ethanol shall be considered as the ¹⁴C content determined using the method described here or the method described in standard EN 16640 Method C.

The natural ¹⁴C content in the atmosphere (the reference value), which is absorbed by living vegetation by assimilation, is not a constant value. The reference value is therefore determined on ethanol from raw materials of the most recent vegetation period. This annual reference value is determined according to standard EN 16640. However, another reference value can be accepted where it is certified by an accredited body.

The ¹⁴C content of samples containing alcohol with at least 85 % mass ethanol is determined directly by liquid scintillation count.

5,0 g 2,5-diphenyloxazole (PPO)

0,5 g p-bis-[4-methyl-5-phenyloxazolyl(2)]-benzene (dimethyl-POPOP) in 1 litre analytical grade toluene.

Commercial, ready-to-use toluene scintillators of this composition may also be used.

n-Hexadecane ¹⁴C with an activity of about 1 × 10⁶ dpm/g (approximately 1,67 × 10⁶ cBq/g) and a guaranteed accuracy of determined activity of ± 2 % rel.

Synthesis alcohol from raw materials of fossil origin with at least 85 % mass ethanol, to determine the background.

4.4.Alcohol from recent raw materials of the most recent vegetation period with at least 85 % mass ethanol as reference material.

5.1.Multi-channel liquid scintillation spectrometer with processor and automatic external standardisation and display of the external standard/channel ratio (usual design: three meter channels and two external standard channels).

5.2.Low-potassium counter tubes suitable for the spectrometer, with dark screw-tops containing a polyethylene insert.

5.3.Volumetric pipettes, 10 ml.

5.4.Automatic dosing device 10 ml.

5.5.250 ml round-bottom flask with ground-glass stopper.

5.6.Alcohol distillation apparatus with heating mantle, e.g. type Micko.

5.7.Microliter syringe 50 μl.

5.8.Pycnometer funnel, pycnometers, 25 ml and 50 ml. As an alternative, equivalent equipment, such as electronic densimetry, should be allowed.

5.9.Thermostat with a temperature stability of ± 0,01 °C.

The equipment shall be adjusted according to the manufacturer’s instructions. Measuring conditions are optimal when the value E2/B, the quality index, is at its maximum.

E = efficiency

B = background

Only two meter channels are optimised. The third is left fully open for control purposes.

A larger number of counter tubes than will later be needed are each filled with 10 ml of ¹⁴C-free synthesis ethanol and 10 ml toluene scintillator. Each is measured for at least 4 cycles × 100 minutes. Tubes whose backgrounds vary by more than ± 1 % rel. from the mean are discarded. Only tubes new from the factory and from the same batch may be used.

During the process of setting the channels (point 6.1) the ESCR is determined using the appropriate computer program when the efficiency is determined. The external standard used is ¹³⁷caesium, which is already built-in by the manufacturer.

Samples having an ethanol content of at least 85 % mass and free from impurities, which absorb at wavelengths below 450 nm may be measured. The low residue of esters and aldehydes has no disruptive effect. The alcohol content of the sample is previously determined with an approximation of 0,1 %.

7.1.The low absorbance samples as described in point 6.4 with an ESCR value of approximately 1,8 may be measured through the ESCR, which provides a measure of the efficiency ratio.

10 ml each of the samples prepared according to point 6.4 is pipettes into a selected counter tube checked for background and 10 ml of toluene scintillator is added via an automatic dosing device. The samples in the tubes are homogenised by suitable rotary movements; the liquid shall not be allowed to wet the polyethylene insert in the screw-top. A tube containing ¹⁴C-free fossil ethanol is prepared in the same way to measure the background. To check the relevant annual ¹⁴C value a duplicate of recent ethanol from the latest vegetation period is prepared, a tube being mixed with internal standard, see point 8.

The control and background samples are placed at the beginning of the measurement series, which shall contain no more than 10 samples for analysis. Total measuring time per sample is at least 2 × 100 minutes, with the individual samples being measured in part stages of 100 minutes so that any equipment drift or other defect can be detected. (One cycle therefore corresponds to a measuring interval of 100 minutes per sample).

Background and control samples shall be freshly prepared every four weeks.

In the case of slightly extinguished samples (ESCR circa 1,8) the efficiency is only negligibly affected by the change in this value. If the change is within ± 5 % rel. the same efficiency can be expected. For more greatly extinguished samples, such as denatured alcohols, the efficiency may be established via the extinction correction graph. If an appropriate computer program is not available the internal standard shall be used, and this gives an unambiguous result.

Control and background samples (recent and fossil ethanol) and the unknown material are each measured as duplicates. One sample of the duplicate is prepared in a non-selected tube and an accurately dosed quantity (30 μl) of hexadecane¹⁴C is added as internal standard (added activity around 26 269 dpm/gC approximately 43 782 cBq/gC). For the sample preparation and measuring time of the other samples see point 7.2, but the measuring time for the samples with the internal standard may be reduced to about five minutes by presetting at 10⁵ pulses. One duplicate each of background and control samples is used per measuring series; these are placed at the beginning of the measuring series.

To prevent contamination when measuring with the internal standard these shall be stored and handled well away from the area where the samples for analysis are prepared and measured. After measurement the tubes checked for background may be re-used. The screw-tops and tubes containing the internal standard shall be disposed of.

9. Expression of the results

Indication of specific radio-activity is expressed as becquerels relative to one gram carbon = Bq/gC.

To obtain more practical results, these shall be expressed in centi-bequerels = cBq/gC.

The descriptions and formulae used in the literature, based on dpm, may also be used. To obtain corresponding figures in cBq merely multiply the dpm figure by 100/60.

r = 0,632 cBq/g C; S(r) = ± 0,223 cBq/g C

R = 0,821 cBq/g C; S(R) = ± 0,290 cBq/g C.