Commission Regulation (EC) No 606/2009 (repealed)Show full title

ANNEX IV SPECIAL COMMUNITY ANALYSIS METHODS

A. ALLYL ISOTHIOCYANATE

1. Principle of the method

Any allyl isothiocyanate present in the wine is collected by distillation and identified by gas chromatography.

2. Reagents

2.1. Ethanol, absolute.

2.2. Standard solution: solution of allyl isothiocyanate in absolute alcohol containing 15 mg of allyl isothiocyanate per litre.

2.3. Freezing mixture consisting of ethanol and dry ice (temperature -60 °C).

3. Apparatus

3.1. Distillation apparatus as shown in the figure. A stream of nitrogen is passed continuously through the apparatus.

3.2. Heating mantle, thermostatically controlled.

3.3. Flowmeter.

3.4. Gas chromatograph fitted with a flame spectrophotometer detector equipped with a selective filter for sulphur compounds (wavelength = 394 nm) or any other suitable detector.

3.5. Stainless steel chromatograph column of internal diameter 3 mm and length 3 m filled with Carbowax 20M at 10 % on Chromosorb WHP, 80 to 100 mesh.

3.6. Microsyringe, 10µl.

4. Procedure

Put two litres of wine into the distillation flask, introduce a few millilitres of ethanol (paragraph 2.1) into the two collecting tubes so that the porous parts of the gas dispersion rods are completely immersed. Cool the two tubes externally with the freezing mixture. Connect the flask to the collecting tubes and begin to flush the apparatus with nitrogen at a rate of three litres per hour. Heat the wine to 80 °C with the heating mantle, distil and collect 45 to 50 ml of the distillate.

Stabilize the chromatograph. It is recommended that the following conditions are used:

• injector temperature: 200 °C,

• column temperature: 130 °C,

• helium carrier gas flow rate: 20 ml per minute.

With the microsyringe, introduce a volume of the standard solution such that the peak corresponding to the allyl isothiocyanate can easily be identified on the gas chromatogram.

Similarly introduce an aliquot of the distillate into the chromatograph. Check that the retention time of the peak obtained corresponds with that of the peak of allyl isothiocyanate.

Under the conditions described above, compounds naturally present in the wine will not produce interfering peaks on the chromatogram of the sample solution.

Apparatus for distillation under a current of nitrogen

B. SPECIAL ANALYSIS METHODS FOR RECTIFIED CONCENTRATED GRAPE MUST

(a) Total cations

1. Principle

The test sample is treated by a strongly acid cation exchanger. The cations are exchanged with H⁺. Total cations are expressed by the difference between the total acidity of the effluent and that of the test sample.

2. Apparatus

2.1. Glass column of internal diameter 10 to 11 mm and length approximately 300 mm, fitted with a drain tap.

2.2. pH meter with a scale graduated at least in 0,1 pH units.

2.3. Electrodes:

• glass electrode, kept in distilled water,

• calomel/saturated potassium chloride reference electrode, kept in a saturated solution of potassium chloride, or

• a combined electrode, kept in distilled water,

3. Reagents

3.1. Strongly acid cation exchange resin in H + form pre-swollen by soaking in water overnight.

3.2. Sodium hydroxide solution, 0,1 M.

3.3. Paper pH indicator.

4. Procedure

4.1. Preparation of sample

Use the solution obtained by diluting the rectified concentrated must to 40 % (m/v): introduce 200 g of accurately weighed rectified concentrated must into a 500 ml volumetric flask. Make up to the mark with water and homogenise.

4.2. Preparation of the ion exchange column

Introduce into the column approximately 10 ml pre-swollen ion exchanger in H + form. Rinse the column with distilled water until all acidity has been removed, using the paper indicator to monitor this.

4.3. Ion exchange

Pass 100 ml of the rectified concentrated must solution prepared as in paragraph 4.1 through the column at the rate of one drop every second. Collect the effluent in a beaker. Rinse the column with 50 ml of distilled water. Titrate the acidity in the effluent (including the rinse water) with the 0,1 M sodium hydroxide solution until the pH is 7 at 20 °C. The alkaline solution should be added slowly and the solution continuously shaken. Let n ml be the volume of 0,1 M sodium hydroxide solution used.

5. Expression of the results

The total cations are expressed in milliequivalents per kilogram of total sugar to one decimal place.

5.1. Calculations

• Acidity of the effluent expressed in milliequivalents per kilogram of rectified concentrated must:

  Where E = The free sulphur dioxide in milligrams per litre is 2,5 n.

• Total acidity of the rectified concentrated must in milliequivalents per kilogram: a.

• Total cations in milliequivalents per kilogram of total sugars:

  ((2,5n-a)/(P)) × 100

  P = percentage concentration (m/m) of total sugars.

(b) Conductivity

1. Principle

The electrical conductivity of a column of liquid defined by two parallel platinum electrodes at its ends is measured by incorporating it in one arm of a Wheatstone bridge.

The conductivity varies with temperature and it is therefore expressed at 20 °C.

2. Apparatus

2.1. Conductivity meter enabling measurements of conductivity to be made over a range from 1 to 1 000 microsiemens per cm (µS cm^–1).

2.2. Waterbath for bringing the temperature of samples to be analysed to approximately 20 °C (20 ± 2 °C).

3. Reagents

3.1. Demineralised water with specific conductivity below 2 µS cm^–1 at 20 °C.

3.2. Reference solution of potassium chloride

Dissolve 0,581 g of potassium chloride, KCl, previously dried to constant mass at a temperature of 105 °C, in demineralised water (paragraph 3.1). Make up to one litre with demineralised water (paragraph 3.1). This solution has a conductivity of 1 000 μS cm^–1 at 20 °C. It should not be kept for more than three months.

4. Procedure

4.1. Preparation of the sample to be analysed

Use the solution with a total sugar concentration of 25 % (m/m) (25° Brix): weigh a mass equal to 2 500/P and make up to 100 g with water (paragraph 3.1), where P = percentage (m/m) concentration of total sugars in the rectified concentrated must.

4.2. Determination of conductivity

Bring the sample to be analysed to 20 °C by immersion in a waterbath. Maintain the temperature to within ± 0,1 °C.

Rinse the conductivity cell twice with the solution to be examined.

Measure the conductivity and express the result in µS cm^–1.

5. Expression of the results

The result is expressed in microsiemens per cm (µScm^–1) at 20 °C to the nearest whole number for the 25 % (m/m) (25° Brix) solution of rectified concentrated must.

5.1. Calculations

If the apparatus does not have temperature compensation, correct the measured conductivity using Table I. If the temperature is below 20 °C, add the correction; if the temperature is above 20 °C, subtract the correction.

Table I

Corrections to be made to the conductivity for temperatures different from 20 °C (µS cm^–1)

a Subtract the correction.
b Add the correction.
Conductivity	Temperature (°C)
	20,219,8	20,419,6	20,619,4	20,819,2	21,019,0	21,218,8	21,418,6	21,618,4	21,818,2	22,0a18,0b
0	0	0	0	0	0	0	0	0	0	0
50	0	0	1	1	1	1	1	2	2	2
100	0	1	1	2	2	3	3	3	4	4
150	1	1	2	3	3	4	5	5	6	7
200	1	2	3	3	4	5	6	7	8	9
250	1	2	3	4	6	7	8	9	10	11
300	1	3	4	5	7	8	9	11	12	13
350	1	3	5	6	8	9	11	12	14	15
400	2	3	5	7	9	11	12	14	16	18
450	2	3	6	8	10	12	14	16	18	20
500	2	4	7	9	11	13	15	18	20	22
550	2	5	7	10	12	14	17	19	22	24
600	3	5	8	11	13	16	18	21	24	26

(c) Hydroxymethylfurfural (HMF)

1. Principle of the methods

1.1. Colorimetric method

Aldehydes derived from furan, the main one being hydroxymethylfurfural, react with barbituric acid and paratoluidine to give a red compound which is determined by colorimetry at 550 nm.

1.2. High-performance liquid chromatography (HPLC)

Separation through a column by reversed-phase chromatography and determination at 280 nm.

2. Colorimetric method

2.1. Apparatus

2.1.1. Spectrophotometer for making measurements between 300 and 700 nm.

2.1.2. Glass cells with optical paths of 1 cm.

2.2. Reagents

2.2.1. Barbituric acid, 0,5 % solution (m/v).

Dissolve 500 mg of barbituric acid, C₄O₃N₂H₄, in distilled water and heat slightly over a waterbath at 100 °C. Make up to 100 ml with distilled water. The solution keeps for about a week.

2.2.2. Paratoluidine solution, 10 % (m/v).

Place 10 g of paratoluidine, C₆H₄(CH₃) NH₂, in a 100 ml volumetric flask; add 50 ml of isopropanol, CH₃CH(OH)CH₃, and 10 ml of glacial acetic acid, CH₃COOH (ρ₂₀ = 1,05 g/ml). Make up to 100 ml with isopropanol. This solution should be renewed daily.

2.2.3. Ethanal (acetaldehyde), CH₃CHO, 1 % (m/v) aqueous solution.

Prepare just before use.

2.2.4. Hydroxymethylfurfural, C₆O₃H₆, 1 g/l aqueous solution.

Prepare successive dilutions containing 5, 10, 20, 30 and 40 mg/l. The 1 g/l and the diluted solutions must be freshly prepared.

2.3. Procedure

2.3.1. Preparation of sample

Use the solution obtained by diluting the rectified concentrated must to 40 % (m/v): introduce 200 g of accurately weighed rectified concentrated must into a 500 ml volumetric flask. Make up to the mark with water and homogenise. Carry out the determination on 2 ml of this solution.

2.3.2. Colorimetric determination

Into each of two 25 ml flasks a and b fitted with ground glass stoppers place 2 ml of the sample prepared as in paragraph 2.3.1. Place in each flask 5 ml of paratoluidine solution (paragraph 2.2.2); mix. Add 1 ml of distilled water to flask b (control) and 1 ml barbituric acid solution (paragraph 2.2.1) to flask a. Shake to homogenize. Transfer the contents of the flasks into spectrophotometer cells with optical paths of 1 cm. Zero the absorbence scale using the contents of flask b for a wavelength of 550 nm. Follow the variation in the absorbence of the contents of flask a; record the maximum value A, which is reached after two to five minutes.

Samples with hydroxymethylfurfural concentrations above 30 mg/l must be diluted before the analysis.

2.3.3. Preparation of the calibration curve

Place 2 ml of each of the hydroxymethylfurfural solutions with 5, 10, 20, 30 and 40 mg/l (paragraph 2.2.4) into two sets of 25 ml flasks a and b and treat them as described in paragraph 2.3.2.

The graph representing the variation of absorbence with the hydroxymethylfurfural concentration in mg/l is a straight line passing through the origin.

2.4. Expression of results

The hydroxymethylfurfural concentration in rectified concentrated musts is expressed in milligrams per kilogram of total sugars.

2.4.1. Method of calculation

The hydroxymethylfurfural concentration C mg/l in the sample to be analysed is that concentration on the calibration curve corresponding to the absorbence A measured on the sample.

The hydroxymethylfurfural concentration in milligrams per kilogram of total sugars is given by:

250 × ((C)/(P))

P = percentage (m/m) concentration of total sugars in the rectified concentrated must.

3. High-performance liquid chromatography

3.1. Apparatus

3.1.1. High-performance liquid chromatograph equipped with:

• a loop injector, 5 or 10 μl,

• spectrophotometer detector for making measurements at 280 nm,

• column of octadecyl-bonded silica (e.g.: Bondapak C₁₈ — Corasil, Waters Ass.),

• a recorder and, if required, an integrator,

Flow rate of mobile phase: 1,5 ml/minute.

3.1.2. Membrane filtration apparatus, pore diameter 0,45 µm.

3.2. Reagents

3.2.1. Doubly distilled water.

3.2.2. Methanol, CH₃OH, distilled or HPLC quality.

3.2.3. Acetic acid, CH₃COOH, (ρ₂₀ = 1,05 g/ml).

3.2.4. Mobile phase: water-methanol (paragraph 3.2.2)-acetic acid (paragraph 3.2.3) previously filtered through a membrane filter (0,45 µm), (40:9:1 v/v).

This mobile phase must be prepared daily and outgassed before use.

3.2.5. Reference solution of hydroxymethylfurfural, 25 mg/l (v/v).

Into a 100 ml volumetric flask, place 25 mg of hydroxymethylfurfural, C₆H₃O₆, accurately weighed, and make up to the mark with methanol (paragraph 3.2.2). Dilute this solution 1/10^e with methanol (paragraph 3.2.2) and filter through a membrane filter (0,45 µm).

If kept in a hermetically sealed brown glass bottle in a refrigerator, this solution will keep for two to three months.

3.3. Procedure

3.3.1. Preparation of sample

Use the solution obtained by diluting the rectified concentrated must to 40 % (m/v) (introduce 200 g of accurately weighed rectified concentrated must into a 500 ml volumetric flask. Make up to the mark with water and homogenise) and filter it through a membrane filter (0,45 µm).

3.3.2. Chromatographic determination

Inject 5 (or 10) µl of the sample prepared as described in paragraph 3.3.1. and 5 (or 10) µl of the reference hydroxymethylfurfural solution (paragraph 3.2.5) into the chromatograph. Record the chromatogram.

The retention time of hydroxymethylfurfural is approximately six to seven minutes.

3.4. Expression of results

The hydroxymethylfurfural concentration in rectified concentrated musts is expressed in milligrams per kilogram of total sugars.

3.4.1. Method of calculation

Let the hydroxymethylfurfural concentration in the 40 % (m/v) solution of the rectified concentrated must be C mg/l.

The hydroxymethylfurfural concentration in milligrams per kilogram of total sugars is given by:

250 × ((C)/(P))

P = percentage (m/m) concentration of total sugars in the rectified concentrated must.

(d) Heavy metals

1. Principle

I. Rapid method for evaluation of heavy metals

Heavy metals are revealed in the suitably diluted rectified concentrated must by the coloration produced by the formation of sulphides. They are assessed by comparison with a standard lead solution corresponding to the maximum admissible concentration.

II. Determination of lead content by atomic absorption spectrophotometry

The chelate given by lead with ammonium pyrrolidinedithiocarbamate is extracted with methylisobutylketone and the absorbence measured at 283,3 nm. The lead content is determined by using known additional amounts of lead in a set of reference solutions.

2. Rapid method for evaluation of heavy metals

2.1. Reagents

2.1.1. Dilute hydrochloric acid, 70 % (m/v).

Take 70 g of hydrochloric acid, HCl (ρ₂₀ = 1,16 to 1,19 g/ml), and make up to 100 ml with water.

2.1.2. Dilute hydrochloric acid, 20 % (m/v).

Take 20 g of hydrochloric acid, HCl (ρ₂₀ = 1,16 to 1,19 g/ml), and make up to 100 ml with water.

2.1.3. Dilute ammonia.

Take 14 g of ammonia, NH₃ (ρ₂₀ = 0,931 to 0,934 g/ml) and make up to 100 ml with water.

2.1.4. pH 3,5 buffer solution.

Dissolve 25 g of ammonium acetate (CH₃COONH₄), in 25 ml of water and add 38 ml of dilute hydrochloric acid (paragraph 2.1.1). Adjust the pH if necessary with the dilute hydrochloric acid (paragraph 2.1.2) or the dilute ammonia (paragraph 2.1.3) and make up to 100 ml with water.

2.1.5. Thioacetamide solution, (C₂H₅NS), 4 % (m/v).

2.1.6. Glycerol solution, (C₃H₈O₃, 85 % (m/v)

(n_D ^20 °C = 1,449 to 1,455).

2.1.7. Thioacetamide reagent.

To 0,2 ml of thioacetamide solution (paragraph 2.1.5) add 1 ml of a mixture of 5 ml of water, 15 ml of 1 M sodium hydroxide solution and 20 ml of glycerol (paragraph 2.1.6). Heat over a waterbath at 100 °C for 20 seconds. Prepare just before use.

2.1.8. Solution containing 0,002 g/l of lead.

Prepare a 1 g/l lead solution by dissolving 0,400 g of lead nitrate, Pb (NO₃)₂, in water and making up to 250 ml with water. At the time of use, dilute this solution with water to two parts in 1 000 (v/v) in order to obtain a 0,002 g/l solution.

2.2. Procedure

Dissolve a test sample of 10 g of the rectified concentrated must in 10 ml of water. Add 2 ml of the pH 3,5 buffer solution (paragraph 2.1.4); mix. Add 1,2 ml of the thioacetamide reagent (paragraph 2.1.7). Mix at once. Prepare the control under the same conditions by using 10 ml of the 0,002 g/l lead solution (paragraph 2.1.8).

After two minutes, any brown coloration of the rectified concentrated must solution should not be more intense than that of the control.

2.3. Calculations

Under the conditions of the above procedure, the control sample corresponds to a maximum admissible heavy metal concentration expressed as lead of 2 mg/kg of rectified concentrated must.

3. Determination of lead content by atomic absorption spectrophotometry

3.1. Apparatus

3.1.1. Atomic absorption spectrophotometer equipped with an air-acetylene burner.

3.1.2. Lead hollow cathode lamp.

3.2. Reagents

3.2.1. Dilute acetic acid.

Take 12 g of glacial acetic acid (ρ₂₀ = 1,05 g/ml) and make up to 100 ml with water.

3.2.2. Solution of ammonium pyrrolidinedithiocarbamate, C₅H₁₂N₂S₂, 1 % (m/v).

3.2.3. Methylisobutylketone, (CH₃)₂ CHCH₂COCH₃.

3.2.4. Solution containing 0,010 g/l of lead.

Dilute the 1 g/l lead solution (paragraph 2.1.8) to 1 % (v/v).

3.3. Procedure

3.3.1. Preparation of solution to be examined

Dissolve 10 g of rectified concentrated must in a mixture of equal volumes of dilute acetic acid (paragraph 3.2.1) and water, and make up to 100 ml with this mixture.

Add 2 ml of ammonium pyrrolidinedithiocarbamate solution (paragraph 3.2.2) and 10 ml of methylisobutylketone (paragraph 3.2.3). Shake for 30 seconds while protected from bright light. Leave the two layers to separate. Use the methylisobutylketone layer.

3.3.2. Preparation of reference solutions

Prepare three reference solutions containing, in addition to 10 g of rectified concentrated must, 1, 2 and 3 ml respectively of the solution containing 0,010 g/l of lead (paragraph 3.2.4). Treat these in the same way as the solution to be examined.

3.3.3. Control

Prepare a control by proceeding under the same conditions as in paragraph 3.3.1, but without the addition of the rectified concentrated must.

3.3.4. Determination

Set the wavelength to 283,3 nm.

Atomise the methylisobutylketone from the control sample in the flame and zero the absorbence scale.

By operating with their respective solvent extracts, determine the absorbences of the solution to be examined and the reference solutions.

3.4. Expression of results

Express the lead content in milligrams per kilogram of rectified concentrated must to one decimal place.

3.4.1. Calculations

Plot the curve giving the variation in absorbence as a function of the lead concentration added to the reference solutions, zero concentration corresponding to the solution to be examined.

Extrapolate the straight line joining the points until it cuts the negative part of the concentration axis. The distance of the point of intersection from the origin gives the lead concentration in the solution to be examined.

(e) Chemical determination of ethanol

This method is used for the determination of the alcoholic strength of low-alcohol liquids such as musts, concentrated musts and rectified concentrated musts.

1. Principle

Simple distillation of the liquid. Oxidation of the ethanol in the distillate by potassium dichromate. Titration of the excess dichromate with an iron (II) solution.

2. Apparatus

2.1. Distillation apparatus used to measure the alcoholic strength

3. Reagents

3.1. Potassium dichromate solution.

Dissolve 33,600 g of potassium dichromate, (K₂Cr₂O₇), in sufficient quantity of water to make one litre of solution at 20 °C.

One millilitre of this solution oxidizes 7,8924 mg of alcohol.

3.2. Iron (II) ammonium sulphate solution.

Dissolve 135 g of iron (II) ammonium sulphate, Fe SO₄, (NH₄)₂SO₄, 6 H₂O in sufficient quantity of water to make one litre of solution and add 20 ml of concentrated sulphuric acid, (H₂SO₄), (ρ₂₀ = 1,84 g/ml). This solution more or less corresponds to half its volume of dichromate solution when just prepared. Subsequently, it oxidizes slowly.

3.3. Potassium permanganate solution.

Dissolve 1,088 g of potassium permanganate, KMnO₄, in a sufficient quantity of water to make one litre of solution.

3.4. Sulphuric acid, diluted 1:2 (v/v).

A little at a time and stirring continuously, add 500 ml of sulphuric acid, (H₂SO₄) (ρ₂0 = 1,84 g/ml) to 500 ml of water.

3.5. Ferrous orthophenanthroline reagent.

Dissolve 0,695 g of ferrous sulphate, FeSO₄, 7 H₂O, in 100 ml of water, and add 1,485 g of orthophenanthroline monohydrate, C₁₂H₈N₂, H₂O. Heat to help the dissolution. This bright red solution keeps well.

4. Procedure

4.1. Distillation

Place 100 g of rectified concentrated must and 100 ml of water in the distillation flask. Collect the distillate in a 100 ml volumetric flask and make up to the mark with water.

4.2. Oxidation

Take a 300 ml flask with a ground glass stopper and with a widened neck enabling the neck to be rinsed without loss. In the flask, place 20 ml of the titrant potassium dichromate solution (paragraph 3.1) and 20 ml of the 1:2 (v/v) dilute sulphuric acid (paragraph 3.4) and shake. Add 20 ml of the distillate. Stopper the flask, shake, and wait at least 30 minutes, shaking occasionally. (This is the ‘measurement’ flask.)

Carry out the titration of the iron (II) ammonium sulphate solution (paragraph 3.2) with respect to the potassium dichromate solution by placing in an identical flask the same quantities of reagents but replacing the 20 ml of distillate by 20 ml of distilled water. (This is the ‘control’ flask.)

4.3. Titration

Add four drops of the orthophenanthroline reagent (paragraph 3.5) to the contents of the ‘measurement’ flask. Titrate the excess dichromate by adding to it the iron (II) ammonium sulphate solution (paragraph 3.2). Stop adding the ferrous solution when the mixture changes from green-blue to brown.

To judge the end-point more precisely, change the colour of the mixture back from brown to green-blue with the potassium permanganate solution (paragraph 3.3). Subtract a tenth of the volume of this solution used from the volume of the iron (II) solution added. Let the difference be n ml.

Proceed in the same way with the ‘control’ flask. Let n′ ml be the difference here.

5. Expression of the results

The ethanol is expressed in grams per kilogram of total sugars and is quoted to one decimal place.

5.1. Method of calculation

n′ ml of ferrous solution reduces 20 ml of dichromate solution which oxidizes 157,85 mg of pure ethanol.

One millilitre of iron (II) solution has the same reducing power as:

((157,85)/(n)) mg of ethanol

n-n′ ml of iron (II) solution have the same reducing power as:

157,85 × ((n′ — n)/(n)) mg of ethanol.

Ethanol concentration in g/kg of rectified concentrated must is given by:

7,892 × ((n′ — n)/(n))

Ethanol concentration in g/kg of total sugars is given by:

789,2 × ((n′ — n)/(n′ × P))

P = percentage (m/m) concentration of total sugars in the rectified concentrated must.

(f) Meso-inositol, scyllo-inositol and sucrose

1. Principle

Gas chromatography of silylated derivatives.

2. Reagents

2.1. Internal standard: xylitol (aqueous solution of about 10 g/l to which a spatula tip of sodium azide is added)

2.2. Bis(trimethylsilyl)trifluoroacetamide — BSTFA — (C₈H₁₈F₃NOSi₂)

2.3. Trimethylchlorosilane (C₃H₉ClSi)

2.4. Pyridine p.A. (C₅H₅N)

2.5. Meso-inositol (C₆H₁₂O₆)

3. Apparatus

3.1. Gas chromatograph equipped with:

3.2. Capillary column (e.g. in fused silica, coated with OV 1, film thickness of 0,15 µ, length 25 m and internal diameter of 0,3 mm).

Operating conditions: carrier gas: hydrogen or helium

• carrier gas flow rate: about 2 ml/minute,

• injector and detector temperature: 300 °C,

• programming of temperature: 1 minute at 160 °C, 4 °C per minute to 260 °C, constant temperature of 260 °C for 15 minutes,

• splitter ratio: about 1:20.

3.3. Integrator.

3.4. Microsyringe, 10 µl.

3.5. Micropipettes, 50, 100 and 200 µl.

3.6. 2 ml flasks with Teflon stopper.

3.7. Oven.

4. Procedure

An accurately weighed sample of about 5 g of rectified concentrated must is placed in a 50 ml flask. 1 ml of standard solution of xylitol (paragraph 2.1) is added and water added to capacity. After mixing, 100 µl of solution is taken and placed in a flask (point 3.6) where it is dried under a gentle stream of air. 100 µl of absolute ethyl alcohol may be added if necessary to facilitate evaporation.

The residue is carefully dissolved in 100 µl of pyridine (paragraph 2.4) and 100 µl of bis(trimethylsilyl)trifluoroacetamide (paragraph 2.2) and 10 µl of trimethylchlorosilane (paragraph 2.3) are added. The flask is closed with the Teflon stopper and heated at 60 °C for one hour.

Draw off 0,5 µl of clear fluid and inject using a heated hollow needle in accordance with the stated splitter ratio.

5. Calculation of results

5.1. A solution is prepared containing:

60 g/l of glucose, 60 g/l of fructose, 1 g/l of meso-inositol and 1 g/l of sucrose.

5 g of the solution is weighed and the procedure at paragraph 4 followed. The results for meso-inositol and sucrose with respect to xylitol are calculated from the chromatogram.

In the case of scyllo-inositol, which is not commercially available and has a retention time lying between the last peak of the anomeric form of glucose and the peak for meso-inositol (see diagram), the same result as for meso-inositol is taken.

6. Expression of the results

6.1. Meso-inositol and scyllo-inositol are expressed in milligrams per kilogram of total sugars.

Sucrose is expressed in grams per kilogram of must.