(1)

The table of contents is amended as follows:

(2)

In Chapter III the following part is added:

‘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:

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:

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

TA – FA.

9.2.   Expression in mg of acetic acid per l:

Let:

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).

Samples	A	B	C	D	E	F
Number of laboratories retained after eliminating outliers	16	18	18	14	18	18
Number of outliers (laboratories)	2			4
Number of accepted results	32	36	36	28	36	36
Mean value (mg/L)	272* 241*	30	591* 641*	46	107	492
Repeatability standard deviation, sr (mg/l)	8,0	3,6	15,0	3,7	6,7	8,5
Repeatability relative standard deviation, RSDr (%)	3,1	11,8	2,4	8,0	6,2	1,7
Repeatability limit, r (mg/l)	23	10	42	10	19	24
Reproducibility standard deviation, sR (mg/l)	8,5	8,4	25,0	4,55	13,4	24,4
Reproducibility relative standard deviation, RSDR (%)	3,3	27,8	4,1	9,9	12,5	5,0
Reproducibility limit, R (mg/l)	24	23	70	13	38	68

Sample types:

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The following Chapter VIII is inserted:

‘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:

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):

5.1.7.   Chromatography conditions (example):

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

5.2.   Analytical balance accurate to 0,1 mg.

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

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.

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).

Table 1

Fructose, glucose, maltose

Table 2

Sucrose

Table 3

Total Sugars

(Note: this data was calculated for total sugars, not invert sugar as defined in Section 8 above.)

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The following Chapter X is added:

‘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:

5.2.   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