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ANNEX

DESCRIPTION OF ANALYTICAL REFERENCE METHODS
	I.
	II.
	III.
	III.1.
	III.2.
	III.3.
	IV.
	V.
	VI.
	VII.
	VIII.
	IX.
▼M2
	X.
▼M3
	XI.
▼B

I.   DETERMINATION OF ALCOHOLIC STRENGTH BY VOLUME OF SPIRIT DRINKS

Introduction

The reference method includes two Appendices:

Appendix I : Preparation of distillate

Appendix II : Measurement of density of distillate

1.   Scope

The method is suitable for the determination of the real alcoholic strength by volume of spirit drinks.

2.   Normative References

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

3.   Terms and Definitions

3.1.

Reference temperature: The reference temperature for the determination of alcoholic strength by volume, density and specific gravity of spirit drinks is 20 °C. Note 1: The term ‘at t °C’ is reserved for all determinations (of density or alcoholic strength by volume) expressed at a temperature other than the reference temperature of 20 °C.

3.2.	Density: The density is the mass per unit volume in vacuo of spirit drinks at 20 °C. It is expressed in kilograms per cubic metre and its symbol is ρ20 °C or ρ20.

3.3.

Specific gravity: The specific gravity is the ratio, expressed as a decimal number, of the density of spirit drinks at 20 °C to the density of water at the same temperature. It is denoted by the symbol d20 °C/20 °C or d20/20, or simply d when there is no possibility of confusion. The characteristic that was measured must be specified on the assay certificate using the above-defined symbols only. Note 2: It is possible to obtain the specific gravity from the density ρ20 at 20 °C: or where 998,203 is the density of water at 20 °C.

3.4.

Real alcoholic strength by volume: The real alcoholic strength by volume of spirit drinks is equal to the number of litres of ethyl alcohol contained in 100 l of a water-alcohol mixture having the same density as the alcohol or spirit after distillation. The reference values for alcoholic strength by volume (% vol) at 20 °C versus density at 20 °C for different water-alcohol mixtures that are to be used are those given in the international table adopted by the International Legal Metrology Organisation in its Recommendation No 22. The general equation relating the alcoholic strength by volume and density of a water-alcohol mixture at a given temperature is given on page 40 in Chapter 3 ‘Alcoholic strength by volume’ of the Annex to Commission Regulation (EEC) No 2676/90 (OJ L 272, 3.10.1990, p. 1) or in the manual of analysis methods of the OIV (1994) (p. 17). Note 3: For liqueurs and crèmes for which it is very difficult to measure volume accurately the sample must be weighed and the alcoholic strength is calculated first by mass. Conversion formula: where ASM = alcoholic strength by mass, ρ20 (alcohol) = 789,24 kg/m3

4.   Principle

Following distillation, the alcoholic strength by volume of the distillate is determined by pycnometry, electronic densimetry, or densimetry using a hydrostatic balance.

APPENDIX I: PREPARATION OF DISTILLATE

1.   Scope

The method is suitable for the preparation of distillates to be used to determine the real alcoholic strength by volume of spirit drinks.

2.   Principle

The spirits are distilled to separate the ethyl alcohol and other volatile compounds from the extractive matter (substances which do not distil).

3.   Reagents and Materials

4.   Apparatus and equipment

Usual laboratory apparatus and in particular the following.

Note:

The apparatus described above is intended for a sample of least 200 ml. However, a smaller sample size (100 ml) can be distilled by using a smaller distillation flask, provided a splashhead or some other device to prevent entrainment is used.

5.   Storage of test samples

Samples are stored at room temperature prior to analysis.

6.   Procedure

Preliminary remark:

6.1.	Distillation apparatus verification. The apparatus used must be capable of the following: The distillation of 200 ml of a water-alcohol solution with known concentration close to 50 % vol must not cause a loss of alcohol of more than 0,1 % vol.

6.2.

Spirit drinks with alcoholic strength below 50 % vol. Measure out 200 ml of the spirit into a volumetric flask. Record the temperature of this liquid, or maintain at standard temperature (20 °C). Pour the sample into the round-bottomed flask of the distillation apparatus and rinse the volumetric flask with three aliquots each of approximately 20 ml of distilled water. Add each rinse water aliquot to the contents of the distillation flask. Note: This 60-ml dilution is sufficient for spirits containing less than 250 g of dry extract per litre. Otherwise, to prevent pyrolysis, the volume of rinse water must be at least 70 ml if the dry extract concentration is 300 g/l, 85 ml for 400 g/l dry extract, and 100 ml for 500 g/l dry extract (some fruit liqueurs or crèmes). Adjust these volumes proportionally for different sample volumes. Add a few anti-bumping granules (3.1) (and antifoam for crème liqueurs). Pour 20 ml of distilled water into the original 200 ml volumetric flask that will be used to hold the distillate. This flask must then be placed in a cold water bath (4.1) (10 to 15 °C for aniseed-flavoured spirit drinks). Distil, avoiding entrainment and charring, occasionally agitating the contents of the flask, until the level of distillate is a few millimetres below the calibration mark of the volumetric flask. When the temperature of this distillate has been brought down to within 0,5 °C of the liquid's initial temperature, make up to the mark with distilled water and mix thoroughly. This distillate is used for the determination of alcoholic strength by volume (Appendix II)

6.3.

Spirit drinks with alcoholic strength above 50 % vol. Measure out 100 ml of the spirit drink into a 100-ml volumetric flask and pour into the round bottomed flask of the distillation apparatus. Rinse the volumetric flask several times with distilled water and add the washings to the contents of the round-bottomed distillation flask. Use enough water to bring the flask's contents up to approximately 230 ml. Pour 20 ml of distilled water into a 200-ml volumetric flask that will be used to hold the distillate. This flask must then be placed in a cold water bath (4.1) (10 to 15 °C for aniseed-flavoured spirits). Distil, agitating the contents occasionally, until the level of distillate is a few millimetres below the calibration mark of the 200-ml volumetric flask. When the temperature of this distillate has been brought down to within 0,5 °C of the liquid's initial temperature, make up to the mark with distilled water and mix thoroughly. This distillate is used for the determination of alcoholic strength by volume (Appendix II) Note: The alcoholic strength by volume of the spirit drink is twice the alcoholic strength of the distillate.

APPENDIX II: MEASUREMENT OF DENSITY OF DISTILLATE

METHOD A:   DETERMINATION OF REAL ALCOHOLIC STRENGTH BY VOLUME OF SPIRIT DRINKS — MEASUREMENT BY PYCNOMETRY

A.1.   Principle

The alcoholic strength by volume is obtained from the density of the distillate measured by pycnometry.

A.2.   Reagents and materials

During the analysis, unless otherwise is stated, use only reagents of recognised analytical grade and water of at least grade 3 as defined in ISO 3696:1987.

A.2.1.

Sodium chloride solution (2 % w/v) To prepare 1 litre, weigh out 20 g of sodium chloride and dissolve to 1 litre using water.

A.3.   Apparatus and Equipment

Usual laboratory apparatus and in particular the following:

Note 1:

The method for determining the densities in vacuo of spirits calls for the use of a twin-pan balance, a pycnometer and a tare bottle of the same outside external volume to cancel out the effect of air buoyancy at any given moment. This simple technique may be applied using a single-pan balance provided that the tare bottle is weighed again to monitor changes in air buoyancy over time.

A.4.   Procedure

Preliminary remarks:

A.4.1.   Calibration of pycnometer

The pycnometer is calibrated by determining the following parameters:

A.4.1.1.

Calibration using a single-pan balance: Determine: —  the mass of the clean, dry pycnometer (P), —  the mass of the water-filled pycnometer at t °C (P1), —  the mass of the tare bottle (T0). A.4.1.1.1. Weigh the clean, dry pycnometer (P). A.4.1.1.2. Fill the pycnometer carefully with distilled water at ambient temperature and fit the thermometer. Carefully wipe the pycnometer dry and place it in the thermally-insulated jacket. Agitate by inverting the container until the thermometer's temperature reading is constant. Set the pycnometer flush with the upper rim of the side tube. Read the temperature t °C carefully and if necessary correct for any inaccuracies in the temperature scale. Weigh the water-filled pycnometer (P1). A.4.1.1.3. Weigh the tare bottle (T0). A.4.1.1.4. Calculation —  Tare of the empty pycnometer = P – m where m is the mass of air in the pycnometer. m = 0,0012 × (P1 – P) Note 2: 0,0012 is the density of dry air at 20 °C at a pressure of 760 mm Hg —  Volume of the pycnometer at 20 °C: where Ft is the factor for temperature t °C taken from Table I of Chapter 1 ‘Density and specific gravity’ of the Annex to Regulation (EEC) No 2676/90 (p. 10). V20 °C must be known to the nearest 0,001 ml. —  Mass of water in the pycnometer at 20 °C: where 0,998203 is the density of water at 20 °C. Note 3: If necessary, the value 0,99715 of the density in air can be used and the alcoholic strength calculated with reference to the corresponding density in HM Customs and Excise tables in air.

A.4.1.2.

Calibration method using a twin-pan balance: A.4.1.2.1.  Place the tare bottle on the left-hand pan and the clean, dry pycnometer with its collecting stopper on the right-hand pan. Balance them by placing weights on the pycnometer side: p grams. A.4.1.2.2.  Fill the pycnometer carefully with distilled water at ambient temperature and fit the thermometer; carefully wipe the pycnometer dry and place it in the thermally insulated jacket; agitate by inverting the container until the thermometer's temperature reading is constant. Accurately adjust the level to the upper rim of the side tube. Clean the side tube, fit the collecting stopper; read the temperature t °C carefully and if necessary correct for any inaccuracies in the temperature scale. Weigh the water-filled pycnometer, with p′ the weight in grams making up the equilibrium. A.4.1.2.3.  Calculation —  Tare of the empty pycnometer = p + m where m is the mass of air in the pycnometer. m = 0,0012 × (p – p′) —  Volume of the pycnometer at 20 °C: where Ft is the factor for temperature t °C taken from Table I of Chapter 1 ‘Density and specific gravity’ of the Annex to Regulation (EEC) No 2676/90 (p. 10). V20 °C must be known to the nearest 0,001 ml. —  Mass of water in the pycnometer at 20 °C: where 0,998203 is the density of water at 20 °C.

A.4.2.   Determination of alcoholic strength of test sample

A.5.   Method performance characteristics (precision)

A.5.1.   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].

METHOD B:   DETERMINATION OF REAL ALCOHOLIC STRENGTH BY VOLUME OF SPIRIT DRINKS — MEASUREMENT BY ELECTRONIC DENSIMETRY (BASED ON THE RESONANT FREQUENCY OSCILLATION OF A SAMPLE IN AN OSCILLATION CELL)

B.1.   Principle

The liquid's density is determined by electronic measurement of the oscillations of a vibrating U-tube. To perform this measurement, the sample is added to an oscillating system, whose specific oscillation frequency is thus modified by the added mass.

B.2.   Reagents and materials

During the analysis, unless otherwise is stated, use only reagents of recognised analytical grade and water of at least grade 3 as defined in ISO 3696:1987.

B.3.   Apparatus and equipment

Usual laboratory apparatus and in particular the following:

B.4.   Procedure

B.5.   Method performance characteristics (precision)

B.5.1.   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].

METHOD C:   DETERMINATION OF REAL ALCOHOLIC STRENGTH BY VOLUME OF SPIRIT DRINKS — MEASUREMENT BY DENSIMETRY USING HYDROSTATIC BALANCE

C.1.   Principle

The alcoholic strength of spirits can be measured by densimetry using a hydrostatic balance based on Archimedes' principle according to which a body immersed in a liquid receives a vertical upward thrust from the liquid equal to the weight of liquid displaced.

C.2.   Reagents and materials

During the analysis, unless otherwise is stated, use only reagents of recognised analytical grade and water of at least grade 3 as defined in ISO 3696:1987.

C.2.1.   Float cleaning solution (sodium hydroxide, 30 % w/v)

To prepare 100 ml, weigh 30 g of sodium hydroxide and make up to volume using 96 % volume ethanol.

C.3.   Apparatus and Equipment

Usual laboratory apparatus and in particular the following:

Note 1:

Use of a twin-pan balance is also possible; the principle is described in Chapter 1 ‘Density and specific gravity’ of the Annex to Regulation (EEC) No 2676/90 (p. 7).

C.4.   Procedure

The float and measuring cylinder must be cleaned between each measurement with distilled water, dried with soft laboratory paper which does not shed fibres and rinsed with the solution whose density is to be determined. Measurements must be made as soon as the apparatus has reached stability so as to restrict alcohol loss by evaporation.

C.5.   Method performance characteristics (precision)

C.5.1.   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].

Figure 1. Distillation apparatus for measuring the real alcoholic strength by volume of spirits
1.1-litre round-bottomed flask with standardised spherical ground-glass joint.
2.20-cm Vigreux rectifying column.
3.10-cm straight-rimmed West condenser.
4.40-cm cooling coil.

II.   DETERMINATION OF TOTAL DRY EXTRACT OF SPIRIT DRINKS BY GRAVIMETRY

1.   Scope

Regulation (EEC) No 1576/89 provides for this method only for aquavit for which the dry extract is limited to 15 g/l.

2.   Normative References

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

3.   Definition

The total dry extract or total dry matter includes all matter that is non-volatile under specified physical conditions.

4.   Principle

Weighing of the residue left by evaporation of the spirit on a boiling water bath and drying in a drying oven.

5.   Apparatus and Equipment

6.   Sampling and samples

Samples are stored at room temperature prior to analysis.

7.   Procedure

8.   Calculation

The mass of the residue multiplied by 40 is equal to the dry extract contained in the spirit and it must be expressed in g/l to one decimal place.

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].

III.   DETERMINATION OF VOLATILE SUBSTANCES AND METHANOL OF SPIRIT DRINKS

III.1.   GENERAL REMARKS

1.   Definitions

Regulation (EEC) No 1576/89 sets minimum levels of volatile compounds other than ethanol and methanol for a series of spirit drinks (rum, spirits of viticultural origin, fruit spirits, etc.). For this series of drinks only, these levels are conventionally considered to be equivalent to the sum of the concentrations of:

The following are the conventional methods for measuring volatile compounds:

2.   Gas chromatographic analysis of volatile compounds

Gas chromatographic assays of volatile compounds other than those set out above may prove particularly interesting as a means of determining both the origin of the raw material used in the distillation and the actual conditions of distillation.

Some spirits contain other volatile components, such as aromatic compounds, which are characteristic of the raw materials used to obtain the alcohol, of the aroma of the spirit drink and of the special features of the preparation of the spirit. These compounds are important for evaluating the requirements set out in Regulation (EEC) No 1576/89.

III.2.   GAS CHROMATOGRAPHIC DETERMINATION OF VOLATILE CONGENERS: ALDEHYDES, HIGHER ALCOHOLS, ETHYL ACETATE AND METHANOL

1.   Scope

This method is suitable for use for the determination of 1,1-diethoxyethane (acetal), 2-methylbutan-1-ol (active amyl alcohol), 3-methylbutan-1-ol (isoamyl alcohol), methanol (methyl alcohol), ethyl ethanoate (ethyl acetate), butan-1-ol (n-butanol), butan-2-ol (sec-butanol), 2-methylpropan-1-ol (isobutyl alcohol), propan-1-ol (n-propanol) and ethanal (acetaldehyde) in spirit drinks using gas chromatography. The method uses an internal standard, for example pentan-3-ol. The concentrations of the analytes are expressed as grams per 100 litres of absolute alcohol; the alcoholic strength of the product must be determined prior to analysis. The spirit drinks that can be analysed using this method include whisky, brandy, rum, wine spirit, fruit spirit and grape marc spirit.

2.   Normative References

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

3.   Definition

Congeners are volatile substances formed along with ethanol during fermentation, distillation and maturation of spirit drinks.

4.   Principle

Congeners in spirit drinks are determined by direct injection of the spirit drink, or appropriately diluted spirit drink, into a gas chromatography (GC) system. A suitable internal standard is added to the spirit drink prior to injection. The congeners are separated by temperature programming on a suitable column and are detected using a flame ionisation detector (FID). The concentration of each congener is determined with respect to the internal standard from response factors, which are obtained during calibration under the same chromatographic conditions as those of the spirit drink analysis.

5.   Reagents and materials

Unless otherwise stated, use only reagents of a purity greater than 97 %, purchased from an ISO-accredited supplier with a certificate of purity, free from other congeners at test dilution (this may be confirmed by injection of individual congener standards at the test dilution using GC conditions as in 6.4) and only water of at least grade 3 as defined in ISO 3696. Acetal and acetaldehyde must be stored in the dark at < 5 °C, all other reagents may be stored at room temperature.

▼M3

▼B

6.   Apparatus and equipment

7.   Sampling and samples.

8.   Procedure (used for the validated method)

9.   Calculation

An automated system of data handling can be used, provided the data can be checked using the principles described in the method below.

Measure either peak areas or peak heights for congener and internal standard peaks.

10.   Quality assurance and control (used for the validated method)

Using equation (2) above, calculate the concentration of each congener in the quality control standard solutions prepared by following the procedure as in 8.1.1 to 8.1.4. Using equation (3), calculate the percentage recovery of the target value. If the analysed results are within ± 10 % of their theoretical values for each congener, analysis may proceed. If not, an investigation should be made to find the cause of the inaccuracy and remedial action taken as appropriate.

11.   Method performance characteristics (precision)

Statistical results of the interlaboratory test: the following tables give the values for the following compounds: ethanal, ethyl acetate, acetal, total ethanal, methanol, butan-2-ol, propan-1-ol, butan-1-ol, 2-methyl-propan-1-ol, 2 methyl-butan-1-ol, 3 methyl-butan-1-ol.

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

▼M2

III.3.   DETERMINATION OF VOLATILE ACIDITY OF SPIRIT DRINKS

1.    Scope

The method has been validated in an interlaboratory study for rum, brandy, marc and fruit spirits, at levels ranging from 30 mg/l to 641 mg/l.

2.    Normative references

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

3.    Definitions

3.1.	Volatile acidity is calculated by deducting the fixed acidity from the total acidity.

3.2.	Total acidity is the sum of titratable acidities.

3.3.	Fixed acidity is the acidity of the residue left after evaporating the spirit to dryness.

4.    Principle

The total acidity and fixed acidity are determined by titration or by potentiometry.

5.    Reagents and materials

During the analysis, unless otherwise stated, use only reagents of recognised analytical grade and water of at least grade 3 as defined in ISO 3696:1987.

5.1.	0,01 M sodium hydroxide solution (NaOH)

5.2.	Mixed indicator solution: Weigh 0,1 g of indigo carmine and 0,1 g of phenol red. Dissolve in 40 ml water and make up to 100 ml with ethanol.

6.    Apparatus and equipment

Indirect laboratory apparatus, grade A glassware and the following:

7.    Sampling and samples

Samples are stored at room temperature prior to analysis.

8.    Procedure

8.1.   Total acidity

8.1.1.   Preparation of sample

The spirit is irradiated with ultrasonic (ultrasonication) or stirred two minutes under a vacuum to rid it of carbon dioxide if required.

8.1.2.   Titration

Pipette 25 ml of the spirit into a 500 ml Erlenmeyer flask.

Add about 200 ml of cooled boiled distilled water (prepared fresh daily) and 2-6 drops of the mixed indicator solution (5.2).

Titrate with the 0,01 M sodium hydroxide solution (5.1) until the yellow-green colour changes to violet in the case of colourless spirits, the yellow-brown colour to red-brown in the case of brown-coloured spirits respectively.

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

Let n1 ml be the volume of the 0,01 M sodium hydroxide solution added.

8.1.3.   Calculation

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:

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: 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).

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.

▼M1

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

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:

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

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:

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 %.

12.3.   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:

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:

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

5.2.	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

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,

8.   Method performance characteristics (precision)

Statistical results of the interlaboratory test:

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.

6.2.	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

8.   Characteristic chromatogram for a pastis

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:

▼M2

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).

▼M1

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

5.   Apparatus and equipment

6.   Samples

Samples are stored at room temperature prior to analysis.

7.   Procedure

7.1.   Sample preparation

7.2.   Photometric phosphate assay

7.2.1.   Comparative solution

7.2.2.   Sample solution

7.2.3.   Calibration curve

8.   Expression of results

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.

9.   Method performance characteristics (precision)

Statistical results of the interlaboratory test:

▼M2

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

8.2.   5-Hydroxymethylfurfural

8.3.   5-Methylfurfural

8.4.   Vanillin

8.5.   Syringaldehyde

8.6.   Coniferaldehyde

8.7.   Sinapaldehyde

8.8.   Gallic acid

8.9.   Ellagic acid

8.10.   Vanillic acid

8.11.   Syringic acid

8.12.   Scopoletin

▼M3

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.

4.3.    14C-free ethanol

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

5.    Apparatus

6.    Procedure

6.1.   Adjusting the equipment

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.

6.2.   Selection of counter tubes

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.

6.3.   Determination of the external standard/channel ratio (ESCR).

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.

6.4.   Preparation of sample

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.    Measurement of samples using external standard

7.2.   Measurement

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.

8.    Measuring samples using internal standard hexadecane14C

8.1.   Procedure

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.

8.2.   Handling the internal standard and counter tubes

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

9.1.   The unit of activity of a radio-active substance is the becquerel; 1 Bq = 1 decay/sec.

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.

9.2.   Expression of results with external standard

9.3.   Expression of results with internal standard

9.4.   Abbreviations

cpmpr

=

the mean sample count rate over the total measuring time.

cpmNE

=

the mean background pulse rate calculated in the same way.

cpmIS

=

count rate of samples, with an internal standard.

dpmIS

=

the quantity of internal standard added (calibration radioactivity dpm).

V

=

the volume of the samples used in ml.

F

=

the content in grammes pure alcohol per ml corresponding to its concentration.

Z

=

the efficiency corresponding to the ESCR value.

1,918

=

the number of grammes alcohol per gramme carbon.

10.    Reliability of the method

10.1.   Repeatability (r)

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

10.2.   Reproducibility (R)

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