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

The total acidity (TA) expressed in milliequivalents per l of spirit is equal to 0,4 × n1.

The total acidity (TA′) expressed in mg of acetic acid per l of spirit is equal to 24 × n1.

8.2.   Fixed acidity

8.2.1.   Preparation of sample

Evaporate 25 ml of the spirit to dryness:

Pipette 25 ml of the spirit into a flat-bottomed cylindrical evaporating dish 55 mm in diameter. During the first hour of evaporation the evaporating dish is placed on the lid of a boiling water bath so that the liquid will not boil, as this could lead to losses through splattering.

Complete the drying by placing the evaporating dish in a drying oven at 105 °C for two hours. Allow the evaporating dish to cool in a desiccator.

8.2.2.   Titration

Dissolve the residue left after evaporating with cooled boiled distilled water (prepared fresh daily) and make up to a volume to circa 100 ml and add 2-6 drops of the mixed indicator solution (5.2).

Titrate with the 0,01 M sodium hydroxide solution (5.1).

The titration may also be carried out by potentiometry, to pH 7,5.

Let n2 ml be the volume of the 0,01 M sodium hydroxide solution added.

8.2.3.   Calculation

The fixed acidity (FA) expressed in milliequivalents per l of spirit is equal to 0,4 × n2.

The fixed acidity (FA) expressed in mg of acetic acid per l of spirit is equal to 24 × n2.

9. Calculation of volatile acidity

• 9.1. Expression in milliequivalents per l:

Let:

TA	=	total acidity in milliequivalents per l
---	---	---
FA	=	fixed acidity in milliequivalents per l

Volatile acidity, VA, in milliequivalents per l is equal to:

TA – FA.

• 9.2. Expression in mg of acetic acid per l:

Let:

TA′	=	total acidity in mg of acetic acid per l
---	---	---
FA′	=	fixed acidity in mg of acetic acid per l

Volatile acidity, VA, in mg of acetic acid per l is equal to:

TA′ – FA′.

• 9.3. Expression in g of acetic acid per hl of pure 100 % vol. alcohol is equal to:

Image omitted; see official source

where A is the alcoholic strength by volume of the spirit drink.

10. Method performance characteristics (Precision)

10.1.   Statistical results of the interlaboratory test

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	18
Number of samples	6
Samples	A
---	---
Number of laboratories retained after eliminating outliers	16
Number of outliers (laboratories)	2
Number of accepted results	32
Mean valueImage omitted; see official source(mg/L)	272 241
Repeatability standard deviation, sr (mg/l)	8,0
Repeatability relative standard deviation, RSDr (%)	3,1
Repeatability limit, r (mg/l)	23
Reproducibility standard deviation, sR (mg/l)	8,5
Reproducibility relative standard deviation, RSDR (%)	3,3
Reproducibility limit, R (mg/l)	24

Sample types:

• A Plum spirit; split level *

• B Rum I; blind duplicates

• C Rum II; split level *

• D Slivovitz; blind duplicates

• E Brandy; blind duplicates

• F Marc spirit; blind duplicates.

• (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.

V. ANETHOLE. GAS CHROMATOGRAPHIC DETERMINATION OF TRANS-ANETHOLE IN SPIRIT DRINKS

1. Scope

This method is suitable for the determination of trans-anethole in aniseed-flavoured spirit drinks using capillary gas chromatography.

2. Normative references

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

3. Principle

The trans-anethole concentration of the spirit is determined by gas chromatography (GC). The same quantity of an internal standard, e.g. 4-allylanisole (estragole) when estragole is not naturally present in the sample, is added to the test sample and to a trans-anethole reference solution of known concentration, both of which are then diluted with a 45 % ethanol solution and injected directly into the GC system. An extraction is necessary before sample preparation and analysis for liqueurs that contain large amounts of sugars.

4. Reagents and materials

During the analysis, use only reagents of a purity of at least 98 %. Water of at least grade 3 as defined by ISO 3696 should be used.

Reference chemicals should be stored cold (at 4 ^oC), away from light, in aluminium containers or in tinted (amber) glass reagent bottles. The stoppers should preferably be fitted with an aluminium seal. Trans-anethole will need to be ‘thawed’ from its crystalline state before use, but in this case its temperature should never exceed 35 ^oC.

• 4.1. Ethanol 96 % vol. (CAS 64-17-5)

• 4.2. 1-methoxy-4-(1-propenyl) benzene; (trans-anethole) (CAS 4180-23-8)

• 4.3. 4-allylanisole, (estragole) (CAS 140-67-0), suggested internal standard (IS)

• 4.4. Ethanol 45 % vol.

Add 560 g of distilled water to 378 g of ethanol 96 % vol.

• 4.5. Preparation of standard solutions

All standard solutions should be stored at room temperature (15 to 35 ^oC) away from light in aluminium containers or in tinted (amber) glass reagent bottles. The stopper should preferably be fitted with an aluminium seal.

Trans-anethole and 4-allylanisole are practically insoluble in water, and it is therefore necessary to dissolve the trans-anethole and 4-allylanisole in some 96 % ethanol (4.1) before the addition of 45 % ethanol (4.4).

The stock solutions must be freshly prepared each week.

4.5.1.   Standard solution A

Stock solution of trans-anethole (concentration: 2 g/l)

Weigh 40 mg of trans-anethole (4.2) in a 20 ml volumetric flask (or 400 mg in 200 ml, etc.). Add some 96 % ethanol (4.1) and make up to volume with 45 % vol. ethanol (4.4), mix thoroughly.

4.5.2.   Internal standard solution B

Stock solution of internal standard, e.g. estragole (concentration: 2 g/l)

Weigh 40 mg of estragole (4.3) in a 20 ml volumetric flask (400 mg in 200 ml etc.). Add some 96 % vol. ethanol (4.1) make up to volume with 45 % vol. ethanol (4.4), mix thoroughly.

4.5.3.   Solutions used to check the linearity response of the flame ionisation detector (FID)

The linearity response of the FID must be checked for the analysis taking into account a range of concentrations of trans-anethole in spirits from 0 g/l up to 2.5 g/l. In the procedure of analysis, the unknown samples of spirits to be analysed are diluted 10 times (8.3). For the conditions of the analysis described in the method, stock solutions corresponding to concentrations of 0, 0,05, 0,1, 0,15, 0,2, and 0,25 g/l of trans-anethole in the sample to be analysed are prepared as follows: take 0,5, 1, 1,5, 2, and 2,5 ml of stock solution A (4.5.1) and pipette in separate 20 ml volumetric flasks; pipette into each flask 2 ml of internal standard solution B (4.5.2) and make up to volume with 45 % vol. ethanol (4.4), mix thoroughly.

The blank solutions (8.4) is used as the 0 g/l solution.

4.5.4.   Standard solution C

Take 2 ml of standard solution A (4.5.1) and pipette into a 20 ml volumetric flask, then add 2 ml of internal standard solution B (4.5.2) and make up to volume with 45 % vol. ethanol (4.4), mix thoroughly.

5. Apparatus and equipment

• 5.1.A capillary gas chromatograph fitted with a flame ionisation detector (FID) and integrator or other data handling system capable of measuring peak heights or areas, and with an automatic sampler or the necessary equipment for manual sample injection.

• 5.2.Split/splitless injector

• 5.3.Capillary column, for example:

Length: 50 m

Internal diameter: 0,32 mm

Film thickness: 0,2 μm

Stationary phase: FFAP — modified TPA polyethylene glycol cross-linked porous polymer.

• 5.4.Common laboratory equipment: A grade volumetric glassware, analytical balance (precision: ± 0,1 mg).

6. Chromatography conditions

The column type and dimensions, and the GC conditions, should be such that anethole and the internal standard are separated from each other and from any interfering substances. Typical conditions for the column given as an example in 5.3 are:

• 6.1. Carrier gas: analytical helium

• 6.2. Flow rate: 2 ml/min

• 6.3. Injector temperature: 250 ^oC

• 6.4. Detector temperature: 250 ^oC

• 6.5. Oven temperature conditions: isothermal, 180 ^oC, run time 10 minutes

• 6.6. Injection volume: 1 μl, split 1:40.

7. Samples

Samples should be stored at room temperature, away from light and cold.

8. Procedure

8.1.   Sample screening for estragole

To ensure that there is no estragole naturally present in the sample, a blank analysis should be carried out without the addition of any internal standard. If estragole is naturally present then another internal standard must be chosen (for instance menthol).

Pipette 2 ml sample into a 20 ml volumetric flask and make up to volume with 45 % vol. ethanol (4.4), mix thoroughly.

8.2.   Preparation of unknown samples

Pipette 2 ml sample into a 20 ml volumetric flask then add 2 ml of internal standard solution B (4.5.2) and make up to volume with 45 % vol. ethanol (4.4), mix thoroughly.

8.3.   Blank

Pipette 2 ml of internal standard solution B (4.5.2) into a 20 ml volumetric flask and make up to volume with 45 % vol. ethanol (4.4), mix thoroughly.

8.4.   Linearity test

Prior to the commencement of the analysis the linearity of the response of the FID should be checked by successively analysing in triplicate each of the linearity standard solutions (4.5.3).

From the integrator peak areas or peak heights for each injection plot a graph of their mother solution concentration in g/l versus the ratio R for each.

R = trans-anethole peak height or area divided by the estragole peak height or area.

A linear plot should be obtained.

8.5.   Determination

Inject the blank solution (8.3), followed by standard solution C (4.5.4), followed by one of the linearity standards (4.5.3) which will act as a quality control sample (this may be chosen with reference to the probable concentration of trans-anethole in the unknown), followed by five unknowns (8.2); insert a linearity (quality control) sample after every five unknown samples, to ensure analytical stability.

9. Calculation of response factor

Measure either peak areas (using an integrator or other data system) or peak heights (manual integration) for trans-anethole and internal standard peaks.

9.1.   Response factor (RFi) calculation

The response factor is calculated as follows

Image omitted; see official source

where:

• Ci is the concentration of trans-anethole in the standard solution A (4.5.1)

• Cis is the concentration of internal standard in the standard solution B (4.5.2)

• areai is the area (or height) of the trans-anethole peak

• areais the area (or height) of the internal standard peak

RFi is calculated from the five samples of solution C (4.5.4).

9.2.   Analysis of the linearity response test solutions

Inject the linearity response test solutions (4.5.3).

9.3.   Analysis of the sample

Inject the unknown sample solution (8.2).

10. Calculation of results

The formula for the calculation of the concentration of trans-anethole is the following:

Image omitted; see official source

where:

• ci is the unknown trans-anethole concentration

• Cis is the concentration of internal standard in the unknown (4.5.2)

• Area or heighti is the area or height of the trans-anethole peak

• Area or heightis the area or height of the internal standard peak

• RFi is the response coefficient (calculated as in 9.1)

The trans-anethole concentration is expressed as grams per litre, to one decimal place.

11. Quality assurance and control

The chromatograms should be such that anethole and the internal standard are separated from each other and from any interfering substances. The RFi value is calculated from the results for the five injections of solution C (4.5.4). If the coefficient of variation (CV % = (standard deviation/mean)*100)) is within plus or minus 1 %, the RFi average value is acceptable.

The calculation above should be used to calculate the concentration of trans-anethole in the sample selected for the quality control from the linearity control solutions (4.5.3).

If the mean calculated results from analysis of the linearity solution selected for internal quality control sample (IQC) are within plus or minus 2,5 % of their theoretical value, then the results for the unknown samples can be accepted.

12. Treatment of spirits sample containing large amount of sugar and of liqueur sample prior to GC analysis

Extraction of alcohol from spirit drink containing a large amount of sugar, in order to be able to determine the trans-anethole concentration using capillary gas chromatography.

12.1.   Principle

An aliquot of the liqueur sample is taken and to this is added the internal standard, at a concentration similar to that of the analyte (trans-anethole) in the liqueur. To this are added sodium phosphate dodecahydrate and anhydrous ammonium sulphate. The resulting mixture is well shaken and chilled, two layers develop, and the upper alcohol layer is removed. An aliquot of this alcohol layer is taken and diluted with 45 % ethanol solution (4.4) (Note: no internal standard is added at this stage, because it has already been added). The resulting solution is analysed in gas chromatography.

12.2.   Reagents and materials

During the extraction use only reagents of a purity greater than 99 %.

• 12.2.1.Ammonium sulphate, anhydrous, (CAS 7783-20-2).

• 12.2.2.Sodium phosphate, dibasic, dodecahydrate, (CAS 10039-32-4).

12.3.   Apparatus and equipment

Conical flasks, separating flasks, refrigerator.

12.4.   Procedure

12.4.1.   Sample screening for estragole

To ensure that there is no estragole naturally present in the sample, a blank extraction (12.6.2) and analysis should be carried out without the addition of any internal standard. If estragole is naturally present then another internal standard must be chosen.

12.4.2.   Extraction

Pipette 5 ml of 96 % ethanol (4.1) into a conical flask, weigh into this flask 50 mg of internal standard (4.3), and add 50 ml of the sample. Add 12 g of ammonium sulphate, anhydrous (12.2.1), and 8.6 g of dibasic sodium phosphate, dodecahydrate (12.2.2). Stopper the conical flask.

Shake the flask for at least 30 minutes. A mechanical shaking device may be used, but not a Teflon coated magnetic stirring bar, as the Teflon will absorb some of the analyte. Note that the added salts will not dissolve completely.

Place the stoppered flask in a refrigerator (T < 5 C) for at least two hours.

After this time, there should be two distinct liquid layers and a solid residue. The alcohol layer should be clear; if not, replace in the refrigerator until a clear separation is achieved.

When the alcohol layer is clear, carefully take an aliquot (e.g. 10 ml), without disturbing the aqueous layer, place in an amber vial and close securely.

12.4.3.   Preparation of the extracted sample to be analysed

Allow extract (12.4.2) to reach room temperature.

Take 2 ml of the alcohol layer of the attemperated extracted sample and pipette into a 20 ml volumetric flask, make up to volume with 45 % ethanol (4.4), mix thoroughly.

12.5.   Determination

Follow the procedure as outlined in 8.5.

12.6.   Calculation of results

Use the following formula to calculate the results:

Image omitted; see official source

where:

• mis is the weight of internal standard (4.3) taken (12.4.2) (in milligrams)

• V is the volume of unknown sample (50 ml)

• RFi is the response factor (9.1)

• areai is the area of the trans-anethole peak

• areais is the area of the internal standard peak

The results are expressed in grams per litre, to one decimal place.

12.7.   Quality control and assurance

Follow the procedure as outlined in 11 above.

13. Method performance characteristics (precision)

Statistical results of the interlaboratory test:

the following tables give the values for anethole.

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	16
Number of samples	10
Analyte	anethole
Samples	A
---	---
Number of laboratories retained after eliminating outliers	15
Number of outliers (laboratories)	1
Number of accepted results	30
Mean value g/l	1,477
Repeatability standard deviation (Sr) g/l	0,022
Repeatability relative standard deviation (RSDr) (%)	1,5
Repeatability limit (r) g/l	0,062
Reproducibility standard deviation (SR) g/l	0,034
Reproducibility relative standard deviation (RSDR) (%)	2,3
Reproducibility limit (R) g/l	0,094

Sample types:

• A pastis, blind duplicates

• B pastis, blind duplicates

• C pastis, blind duplicates

• D pastis, blind duplicates

• E pastis, single duplicates

• F pastis, single duplicates

Samples	G	H	I	J
Number of laboratories retained after eliminating outliers	16	14	14	14
Number of outliers (Laboratories)	—	2	1	1
Number of accepted results	32	28	28	28
Mean value g/l	0,778 0,530 (*)	1,742	0,351	0,599
Repeatability standard deviation (Sr) g/l	0,020	0,012	0,013	0,014
Repeatability relative standard deviation (RSDr) (%)	3,1	0,7	3,8	2,3
Repeatability limit (r) g/l	0,056	0,033	0,038	0,038
Reproducibility standard deviation (SR) g/l	0,031	0,029	0,021	0,030
Repeatability relative standard deviation (RSDR) (%)	4,8	1,6	5,9	5,0
Reproducibility limit (R) g/l	0,088	0,080	0,058	0,084

Sample types:

• G ouzo, split levels (*)

• H anis, blind duplicates

• I aniseed-flavoured liqueur, duplicates

• J aniseed-flavoured liqueur, duplicates.

VI.   GLYCYRRHIZIC ACID. DETERMINATION OF GLYCYRRHIZIC ACID USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

1. Scope

This method is suitable for the determination of glycyrrhizic acid in aniseed-flavoured spirit drinks using high performance liquid chromatography (HPLC). Regulation (EEC) No 1576/89 specifies that any aniseed-flavoured spirit called ‘pastis’ must contain between 0,05 and 0,5 g of glycyrrhizic acid per litre.

2. Normative references

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

3. Principle

The glycyrrhizic acid concentration is determined using high-performance liquid chromatography (HPLC) with UV detection. A standard solution and the test sample are filtered and they are separately injected directly into the HPLC system.

4. Reagents and materials

During the analysis, use only reagents of HPLC grade, absolute ethanol and water of grade 3 as defined by ISO 3696.

• 4.1. Ethanol 96 % vol. (CAS 64-17-5).

• 4.2. Ammonium glycyrrhizinate, C42H62O16.NH3 (Glycyrrhizic acid ammonium salt)

(Mol. Wt.: 839,98)(CAS 53956-04-0): purity at least 90 %

(Mol. Wt.: glycyrrhizic acid 822,94).

• 4.3. Glacial acetic acid, CH3COOH, (CAS 64-19-7).

• 4.4. Methanol, CH3OH (CAS 67-56-1).

• 4.5. Ethanol 50 % vol.

For 1 000  ml at 20 ^oC:

• — 96 % vol. ethanol (4.1): 521 ml

• — Water (2.0): 511 ml.

• 4.6. Preparation of the HPLC elution solutions

4.6.1.   Elution solvent A (example)

80 parts (by volume) of water (2.0)

20 parts (by volume) of acetic acid (4.3).

Degas the elution solvent for five minutes.

Note:

If the water being used has not been microfiltered, it is advisable to filter the prepared elution solvent on a filter for organic solvents with a pore size less than or equal to 0,45 μm.

4.6.2.   Elution solvent B

Methanol (4.4).

• 4.7. Preparation of standard solutions

All standard solutions must be freshly prepared after two months.

4.7.1.   Reference solution C

Weigh to the nearest 0,1 mg, 25 mg of ammonium glycyrrhizinate (4.2) in a 100 ml volumetric flask. Add some 50 % vol. ethanol (4.5) and dissolve the ammonium glycyrrhizinate. When it has dissolved make up to the mark with 50 % vol. ethanol (4.5).

Filter through a filter for organic solvents.

4.7.2.   Standard solutions used to check the linearity of the response of the instrumentation

A 1,0 g/l stock solution is prepared by weighing, to the nearest 0,1 mg, 100 mg of ammonium glycyrrhizinate in a 100 ml volumetric flask. Add some 50 % vol. ethanol (4.5) and dissolve the ammonium glycyrrhizinate. When it has dissolved make up to the mark with 50 % vol. ethanol (4.5).

At least four other solutions corresponding to 0,05, 0,1, 0,25 and 0,5 g/l of ammonium glycyrrhizinate are prepared by pipetting respectively 5 ml, 10 ml, 25 ml and 50 ml of the 1,0 g/l stock solution in separate 100 ml volumetric flasks. Then make up to the mark with 50 % vol. ethanol (4.5) and mix up thoroughly.

Filter all solutions though a filter for organic solvents.

5. Apparatus and equipment

5.1.   Separation system

• 5.1.1.High-performance liquid chromatograph.

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

• 5.1.3.UV spectrophotometric detection system: to be set at 254 nm.

• 5.1.4.Solvent degassing system.

• 5.2. Computational integrator or recorder, the performance of which is compatible with the rest of the system.

• 5.3. Column (example):

Material: stainless steel or glass

Internal diameter: 4 to 5 mm

Length: 100 to 250 mm

Stationary phase: cross-linked silica with a (preferably spherical) octadecyl functional group (C18), maximum particle size: 5 μm.

• 5.4. Laboratory equipment

• 5.4.1.Analytical balance with a precision of 0,1 mg

• 5.4.2.A-grade volumetric glassware

• 5.4.3.Micromembrane filtration arrangement for small volumes.

6. Chromatography conditions

• 6.1.Elution characteristics: (example)

• — flow rate: 1 ml/minute,

• — solvent A = 30 %,

• — solvent B = 70 %.

• 6.2.Detection:

• — UV = 254 nm

7. Procedure

7.1.   Preparation of the spirit sample

Filter, if necessary, through a filter for organic solvents (pore diameter: 0,45 μm).

7.2.   Determination

Once the chromatography conditions have stabilised,

• — inject 20 μl of the reference solution C (4.7.1),

• — inject 20 μl of the sample solution,

• — compare the two chromatograms. Identify the glycyrrhizic acid peaks from their retention times. Measure their areas (or heights) and calculate the concentration in g/l to two decimal figures using the following equation: Image omitted; see official source where: c is the concentration in grams per litre of glycyrrhizic acid in the spirit being analysed C is the concentration in grams per litre of ammonium glycyrrhizinate in the reference solution h is the area (or height) of the glycyrrhizic acid peak of the spirit being analysed H is the area (or height) of the glycyrrhizic acid peak of the reference solution P is the purity of the reference ammonium glycyrrhizinate (in %) 823 is the mass of one mole of glycyrrhizic acid 840 is the mass of one mole of ammonium glycyrrhizinate.

8. Method performance characteristics (precision)

Statistical results of the interlaboratory test:

the following table give the values for glycyrrhizic acid.

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	16
Number of samples	5
Analyte	glycyrrhizic acid
Samples	A
---	---
Number of laboratories retained after eliminating outliers	13
Number of outliers (laboratories)	3
Number of accepted results	26
Mean value g/l	0,046
Repeatability standard deviation (Sr) g/l	0,001
Repeatability relative standard deviation (RSDr) (%)	1,5
Repeatability limit (r) g/l	0,002
Reproducibility standard deviation (SR) g/l	0,004
Reproducibility relative standard deviation (RSDR) (%)	8,6
Reproducibility limit (R) g/l	0,011

Sample types:

• A pastis, blind duplicates

• B pastis, split levels (*)

• C pastis, blind duplicates

• D pastis, blind duplicates

• E pastis, blind duplicates

VII.   CHALCONES. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR VERIFYING THE PRESENCE OF CHALCONES IN PASTIS

1. Scope

This method is suitable for determining whether chalcones are present in aniseed-flavoured drinks or not. Chalcones are natural colorants of the flavonoid family that are present in liquorice root (Glycyrrhiza glabra).

For an aniseed-flavoured spirit to be called ‘pastis’, it must contain chalcones (Regulation (EEC) No 1576/89).

2. Normative references

ISO 3696: 1987, Water for analytical laboratory use — Specifications and test methods.

3. Principle

A reference liquorice extract solution is prepared. The presence or absence of chalcones is determined using high-performance liquid chromatography (HPLC) with UV detection.

4. Reagents and materials

During the analysis, use only reagents of HPLC grade. The ethanol should be 96 % vol. Only water of grade 3 as defined by ISO 3696 should be used.

• 4.1. Ethanol 96 % vol. (CAS 64-17-5)

• 4.2. Acetonitrile, CH3CN, (CAS 75-05-8)

• 4.3. Reference substance: Glycyrrhiza glabra: liquorice, ‘sweet root’

Coarsely ground liquorice roots (Glycyrrhiza glabra). Average dimensions of the rodlike particles: length: 10 to 15 mm, thickness: 1 to 3 mm.

• 4.4. Sodium acetate, CH3COONa, (CAS 127-09-3)

• 4.5. Glacial acetic acid, CH3COOH, (CAS 64-19-7)

• 4.6. Preparation of solutions

4.6.1.   Ethanol 50 % volume

For 1 000  ml at 20 °C:

• — 96 % vol. ethanol (4.1): 521 ml,

• — Water (2.0): 511 ml.

4.6.2.   Solvent A: acetonitrile

Acetonitrile (4.2) of HPLC analytical purity.

Degas

4.6.3.   Solvent B: 0,1 M sodium acetate buffer solution, pH 4,66.

Weigh 8,203 g of sodium acetate (4.4), add 6,005 g of glacial acetic acid (4.5) and make up to 1 000  ml with water (2) in a volumetric flask.

5. Preparation of the reference extract from Glycyrrhiza glabra (4.3)

• 5.1. Weigh 10 g of ground liquorice root (Glycyrrhiza glabra) (4.3) and place in a round-bottomed distillation flask

• — add 100 ml of 50 % vol. ethanol (4.6.1),

• — boil under reflux for one hour,

• — filter,

• — set the filtrate aside for later use.

• 5.2. Recover the liquorice extract from the filter

• — place in a round-bottomed distillation flask,

• — add 100 ml of 50 % vol. ethanol (4.6.1),

• — boil under reflux for one hour,

• — filter. Set aside the filtrate for later use.

• 5.3. The liquorice root extraction must be performed three times in succession.

• 5.4. Combine the three filtrates.

• 5.5. Evaporate the solvent phase (of 5.4) on a rotary evaporator.

• 5.6. Take up the residual extract (of 5.5) with 100 ml 50 % vol. ethanol (4.6.1).

6. Apparatus and equipment

6.1.   Separation system.

• 6.1.1.High-performance liquid chromatograph.

• 6.1.2.Pumping system capable of achieving and maintaining a constant or programmed rate of flow at high pressure.

• 6.1.3.UV/visible spectrophotometric detection system that can be set at 254 and 370 nm.

• 6.1.4.Solvent degassing system:

• 6.1.5.Column oven that can be set at a temperature of 40 ± 0,1 °C.

• 6.2. Computational integrator or recorder, the performance of which is compatible with the rest of the separation system.

• 6.3. Column

Material: stainless steel or glass

Internal diameter: 4 to 5 mm

Stationary phase: cross-linked silica with an octadecyl derived functional group (C18), particle size: 5 μm at most (cross-linked phase).

• 6.4. Common laboratory equipment, including:

• 6.4.1. analytical balance. (precision: ± 0,1 mg);

• 6.4.2. distillation apparatus with a reflux condenser, comprising, for example:
• — a 250 ml round-bottomed flask with a standardised ground-glass joint,
• — a 30 cm long reflux condenser, and
• — a heat source (any pyrogenic reaction involving the extractive matter must be avoided by using an appropriate arrangement).

• 6.4.3. Rotary evaporation apparatus.

• 6.4.4. Filtration set-up (i.e. Buchner funnel).

• 6.5. Chromatography conditions (example).

• 6.5.1.Elution characteristics of solvents A (4.6.2) and B (4.6.3):

• — shift from 20/80 (v/v) to 50/50 (v/v) gradient in 15 minutes,

• — shift from 50/50 (v/v) to 75/25 (v/v) gradient in five minutes,

• — equal strength at 75/25 (v/v) for five minutes,

• — stabilisation of the column between injections,

• — equal strength at 20/80 (v/v) for five minutes.

• 6.5.2.Flow rate: 1 ml/minute.

• 6.5.3.UV detector settings:

the detector must be set at 370 nm to detect the presence of chalcones and then at 254 nm to detect glycyrrhizic acid.

Note:

the change of wavelength (from 370 nm to 254 nm) must be carried out 30 seconds before the beginning of the peak of elution of glycyrrhizic acid.

7. Procedure

7.1.   Preparation of the spirit sample

Filter through a filter for organic solvents (pore diameter: 0,45 μm).

7.2.   Preparation of the residual liquorice extract (5.6)

Make a one in ten dilution with 50 % vol. ethanol (4.6.1) before analysis.

7.3.   Determination

• 7.3.1.Inject 20 μl of the prepared liquorice extract (7.2). Perform the analysis using the chromatography conditions described above (6.5).

• 7.3.2.Inject 20 μl of the sample (7.1) (aniseed-flavoured spirit sample). Perform the analysis using the chromatography conditions described above (6.5).

• 7.3.3.Compare the two chromatograms. There must be a great similarity between the two chromatograms in the chalcone exit zone (during the detection at 370 nm under the analysis conditions described above) (see Figure 1).

8. Characteristic chromatogram for a pastis

Image omitted; see official source

Figure 1

Chromatogram obtained by the method described above, showing the presence of chalcones in a ‘pastis’. Peaks 1 to 8 are chalcones and peak 9 is glycyrrhizic acid.

9. Method performance characteristics (precision)

Results of the interlaboratory test:

the following table gives the performance for recognition of presence or absence of chalcones in pastis and aniseed-flavoured spirits.

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	14
Number of samples	11
Analyte	chalcones
Samples	A
---	---
Number of laboratories retained after eliminating outliers	14
Number of outliers (laboratories)	—
Number of accepted results	28
Number of results for presence of chalcones	28
Number of results for absence of chalcones	0
Percentage of correct results (%)	100

Inconsistent results between the two duplicates, attributed to a sampling error

Samples	G	H	I	J	K
Number of laboratories retained after eliminating outliers	14	14	14	14	14
Number of outliers (laboratories)	—	—	—	—	—
Number of accepted results	28	14	14	28	28
Number of results for presence of chalcones	0	0	0	0	0
Number of results for absence of chalcone	28	14	14	28	28
Percentage of correct results (%)	100	100	100	100	100

Sample types:

• A pastis, blind duplicates

• B pastis, single sample

• C pastis, single sample

• D ‘pastis’ (not containing chalcones), blind duplicates

• E ‘pastis’ (not containing chalcones), blind duplicates

• F aniseed-flavoured liqueur (not containing chalcones), blind duplicates

• G aniseed-flavoured liqueur (not containing chalcones), blind duplicates

• H ouzo (not containing chalcones), single sample

• I ouzo (not containing chalcones), single sample

• J anis (not containing chalcones), blind duplicates

• K ‘pastis’ (not containing chalcones), blind duplicates.

VIII.   TOTAL SUGARS

1. Scope

The HPLC–RI method is applicable for the determination of total sugars (expressed as invert sugar) in spirit drinks, with the exclusion of liqueurs containing egg and milk products.

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.

2. Normative references

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

3. Principle

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. Reagents and materials

• 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. Solvents (example)

The elution solvent is composed of:

75 parts by volume of acetonitrile (4.6),

25 parts by volume of distilled water (4.7).

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. Preparation of stock standard solution (20 g/l)

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. Preparation of working standard solutions (2,5, 5,0, 7,5, 10,0 and 20,0 g/L)

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. Apparatus and Equipment

5.1.   HPLC system capable of achieving baseline resolution of all of the sugars.

• 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.

• 5.1.7. Chromatography conditions (example):

Elution solvent (4.8), flow rate: 1 ml/minute.

Detection: Differential refractometry.

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.

6. Sample storage

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

7. Procedure

7.1.   PART A: Sample preparation

• 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).

7.2.   PART B: HPLC

7.2.1.   Determination

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.

Invert sugar (g/l)	=	glucose (g/l) + fructose (g/l) + maltose (g/l) + lactose (g/l) + (sucrose (g/l) × 1,05).
---	---	---
1,05	=	(molecular weight of fructose + molecular weight of glucose)/molecular weight of sucrose.

9. Method performance characteristics (precision)

9.1.   Statistical results of the interlaboratory test

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.

Table 1

Fructose, glucose, maltose

Analyte	Fructose	Fructose	Glucose	Glucose	Glucose	Maltose	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

Table 2

Sucrose

Analyte	Sucrose	Sucrose	Sucrose	Sucrose	Sucrose	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 [^E0003]	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

split level.

Table 3

Total Sugars

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 [^E0004]	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

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.

3. Principle

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

4. Reagents and materials

• 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:

dissolve 20 g of ammonium molybdate (CAS 12054-85-2), (NH4)6Mo7O24.4H2O in 400 ml water at 50 ^oC;

dissolve, in another vessel, 1 g of ammonium vanadate (CAS 7803-55-6), NH4VO3, 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

• 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.

6. Samples

Samples are stored at room temperature prior to analysis.

7. Procedure

7.1.   Sample preparation

• 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.   Photometric phosphate assay

7.2.1.   Comparative solution

• 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.   Sample 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.   Calibration curve

• 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

8. Expression of results

The egg yolk content in g/l is calculated from the following formula:Image omitted; see official source

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.

9. Method performance characteristics (precision)

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
---	---
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

Sample types

• 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.

3. Principle

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

4. Reagents

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.

5. Apparatus

Standard laboratory apparatus

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

• 5.1.1. A spectrophotometric detector capable of measuring at wavelengths from 260 to 340 nm. It is however preferable to work with a multiple wavelength detector with a diode array or similar, in order to confirm the purity of the peaks.

• 5.1.2. A spectrofluorimetric detector — excitation wavelength: 354 nm, emission wavelength: 446 nm (for the trace determination of scopoletin; which is also detectable at 313 nm by spectrophotometry).

• 5.1.3. An injection device capable of introducing 10 or 20 μl (for example) of the test sample.

• 5.1.4. A high-performance liquid chromatography column, RP C18 type, 5 μm maximum particle size.

• 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.

6. Procedure

6.1.   Preparation of the solution to be injected

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.

• 6.3. Determination

• 6.3.1. Inject the reference standards separately, then mixed.

Adapt the operating conditions so that the resolution factors of the peaks of all the compounds are equal to at least 1.

• 6.3.2. Inject the sample as prepared in 6.1.

• 6.3.3. Measure the area of the peaks in the reference solution and the spirit drink and calculate the concentrations.

7. Expression of results

Express the concentration of each constituent in mg/l.

8. Performance characteristics of the method (precision)

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).

8.1.   Furfural

Analyte	Furfural	Furfural	Furfural	Furfural	Furfural	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

8.2.   5-Hydroxymethylfurfural

Analyte	5-Hydroxymethylfurfural	5-Hydroxymethylfurfural	5-Hydroxymethylfurfural	5-Hydroxymethylfurfural	5-Hydroxymethylfurfural	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

8.3.   5-Methylfurfural

Analyte	5-Methylfurfural	5-Methylfurfural	5-Methylfurfural	5-Methylfurfural	5-Methylfurfural	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

8.4.   Vanillin

Analyte	Vanillin	Vanillin	Vanillin	Vanillin	Vanillin	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

8.5.   Syringaldehyde

Analyte	Syringaldehyde	Syringaldehyde	Syringaldehyde	Syringaldehyde	Syringaldehyde	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

8.6.   Coniferaldehyde

Analyte	Coniferaldehyde	Coniferaldehyde	Coniferaldehyde	Coniferaldehyde	Coniferaldehyde	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

8.7.   Sinapaldehyde

Analyte	Sinapaldehyde	Sinapaldehyde	Sinapaldehyde	Sinapaldehyde	Sinapaldehyde	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

8.8.   Gallic acid

Analyte	Gallic acid	Gallic acid	Gallic acid	Gallic acid	Gallic acid	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

8.9.   Ellagic acid

Analyte	Ellagic acid	Ellagic acid	Ellagic acid	Ellagic acid	Ellagic acid	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

8.10.   Vanillic acid

Analyte	Vanillic acid	Vanillic acid	Vanillic acid	Vanillic acid	Vanillic acid	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

8.11.   Syringic acid

Analyte	Syringic acid	Syringic acid	Syringic acid	Syringic acid	Syringic acid	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

8.12.   Scopoletin

Analyte	Scopoletin	Scopoletin	Scopoletin	Scopoletin	Scopoletin	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

1. Introduction

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).

2. Definition

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.

3. Principle

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

4. Reagents

4.1.   Toluene scintillator

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.

4.2.   ¹⁴C standard

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.