SECTION 3.1.1 - SUGARS

Codified File

Reducing substances (Type-IV)

OIV-MA-AS311-01A Reducing substances

Type IV method

 

  1. Definition

 

Reducing substances comprise all the sugars exhibiting ketonic and aldehydic functions and are determined by their reducing action on an alkaline solution of a copper salt.

  1. Principle of the method

The wine is treated with one of the following reagents:

  • neutral lead acetate,
  • zinc ferrocyanide (II).
  1. Clarification

 

The sugar content of the liquid in which sugar is to be determined must lie between 0.5 and 5 g/L.

Dry wines should not be diluted during clarification; sweet wines should be diluted during clarification in order to bring the sugar level to within the limits prescribed in the following table.

Description

Sugar content (g/L)

Density

Dilution

(%)

Musts and mistelles

> 125

> 1.038

1

Sweet wines, whether fortified or not

25 to 125

1.005 to 1.038

4

Semi‑sweet wines

5 to 25

0.997 to 1.005

20

Dry wines

< 5

< 0.997

No dilution

3.1.  Clarification by neutral lead acetate.

3.1.1.      Reagents

  • Neutral lead acetate solution (approximately saturated)
  • Neutral lead acetate, Pb (250 g
  • Very hot water to 500 mL
  • Stir until dissolved.
  • Sodium hydroxide solution, 1 M
  • Calcium carbonate.
    1.       Procedure
  • Dry wines.

Place 50 mL of the wine in a 100 mL volumetric flask; add 0.5 (n - 0.5) mL sodium hydroxide solution, 1 M, (where n is the volume of sodium hydroxide solution, 0.1 M, used to determine the total acidity in 10 mL of wine). Add, while stirring, 2.5 mL of saturated lead acetate solution and 0.5 g calcium carbonate. Shake several times and allow to stand for at least 15 minutes. Make up to the mark with water. Filter.

1 mL of the filtrate corresponds to 0.5 mL of the wine.

  • Musts, mistelles, sweet and semi‑sweet wines

Into a 100 mL volumetric flask, place the following volumes of wine (or must or mistelle), the dilutions being given for guidance:

  • Case 1 - Musts and mistelles: prepare a 10% (v/v) solution of the liquid to be analyzed and take 10 mL of the diluted sample.
  • Case 2 - Sweet wines, whether fortified or not, having a density between 1.005 and 1.038: prepare a 20% (v/v) solution of the liquid to be analyzed and take 20 mL of the diluted sample.
  • Case 3 - Semi‑sweet wines having a density between 0.997 and 1.005: take 20 mL of the undiluted wine.

Add 0.5 g calcium carbonate, about 60 mL water and 0.5, 1 or 2 mL of saturated lead acetate solution.  Stir and leave to stand for at least 15 minutes, stirring occasionally.  Make up to the mark with water. Filter.

Note:

  • Case 1: 1 mL of filtrate contains 0.01 mL of must or mistelle.
  • Case 2: 1 mL of filtrate contains 0.04 mL of sweet wine.
  • Case 3: 1 mL of filtrate contains 0.20 mL of semi‑sweet wine.

3.2.  Clarification by zinc ferrocyanide (II)

This clarification process should be used only for white wines, lightly colored sweet wines and musts.

3.2.1.      Reagents

Solution I: potassium ferrocyanide (II):

  • Potassium ferrocyanide (II),: 150 g
  • Water to: 1000 mL

Solution II: zinc sulfate:

  • Zinc sulfate, Zn: 300 g
  • Water to 1000 mL
    1.       Procedure

Into a 100 mL volumetric flask, place the following volumes of wine (or must or mistelle), the dilutions being given for guidance:

  • Case 1 - Musts and mistelles.  Prepare a 10% (v/v) solution of the liquid to be analyzed and take 10 mL of the diluted sample.
  • Case 2 - Sweet wines, whether fortified or not, having a density between 1.005 and 1.038: prepare a 20% (v/v) solution of the liquid to be analyzed and take 20 mL of the diluted sample.
  • Case 3 - Semi‑sweet wines having a density at 20°C between 0.997 and 1.005: take 20 mL of the undiluted wine.
  • Case 4 - Dry wines: take 50 mL of undiluted wine.

Add 5 mL of solution I and 5 mL of solution II. Stir. Make up to the mark with water. Filter.

Note:

Case 1: 1 mL of filtrate contains 0.01 mL of must or mistelle.

Case 2: 1 mL of filtrate contains 0.04 mL of sweet wine.

Case 3: 1 mL of filtrate contains 0.20 mL of semi‑sweet wine.

Case 4: 1 mL of filtrate contains 0.50 mL of dry wine.

  1. Determination of sugars

4.1.  Reagents

Alkaline copper salt solution:

  • Copper sulfate, pure, Cu: 25 g
  • Citric acid monohydrate: 50 g
  • Crystalline sodium carbonate,: 388 g
  • Water to: 1000 mL

Dissolve the copper sulfate in 100 mL of water, the citric acid in 300 mL of water and the sodium carbonate in 300 to 400 mL of hot water.  Mix the citric acid and sodium carbonate solutions. Add the copper sulfate solution and make up to one liter.

Potassium iodide solution, 30% (m/v):

  • Potassium iodide, KI: 30 g
  • Water to : 100 mL

Store in a colored glass bottle.

Sulfuric acid, 25% (m/v):

  • Concentrated sulfuric acid, , ρ20 = 1.84 g/Ml 25 g
  • Water to 100 mL

Add the acid slowly to the water, allow to cool and make up to 100 mL with water.

Starch solution, 5 g/L:

  • Mix 5 g of starch in with about 500 mL of water. Bring to boil, stirring all the time, and boil for 10 minutes. Add 200 g of sodium chloride, NaCl. Allow to cool and then make up to one liter with water.
  • Sodium thiosulfate solution, 0.1 M.

Invert sugar solution, 5 g/L, to be used for checking the method of     determination.

Place the following into a 200 mL volumetric flask:

  • Pure dry sucrose: 4.75 g
  • Water, approximately: 100 mL
  • Conc. hydrochloric acid (ρ= 1.16 – 1.19 g/mL): 5 mL

Heat the flask in a water‑bath maintained at 60°C until the temperature of the solution reaches 50°C; then keep the flask and solution at 50°C for 15 minutes. Allow the flask to cool naturally for 30 minutes and then immerse it in a cold water‑bath. Transfer the solution to a one‑liter volumetric flask and make up to one liter.  This solution keeps satisfactorily for a month.  Immediately before use, neutralize the test sample (the solution being approximately 0.06 M acid) with sodium hydroxide solution.

4.2.  Procedure

Mix 25 mL of the alkaline copper salt solution, 15 mL water and 10 mL of the clarified solution in a 300 mL conical flask. This volume of sugar solution must not contain more than 60 mg of invert sugar.

Add a few small pieces of pumice stone. Fit a reflux condenser to the flask and bring the mixture to the boil within two minutes. Keep the mixture boiling for exactly 10 minutes.

Cool the flask immediately in cold running water.  When completely cool, add 10 mL potassium iodide solution, 30% (m/v); 25 mL sulfuric acid, 25% (m/v), and 2 mL starch solution.

Titrate with sodium thiosulfate solution, 0.1 M. Let n be the number of mL used. Also carry out a blank titration in which the 25 mL of sugar solution is replaced by 25 mL of distilled water. Let n' be the number of mL of sodium thiosulfate used.

4.3.  Expression of results

4.3.1.      Calculations

The quantity of sugar, expressed as invert sugar, contained in the test sample is given in the table below as a function of the number (n' ‑ n) of mL of sodium thiosulfate used.

The sugar content of the wine is to be expressed in grams of invert sugar per liter to one decimal place, account being taken of the dilution made during clarification and of the volume of the test sample.

Table giving the relationship between the volume of sodium thiosulfate solution:

(n'‑n) mL, and the quantity of reducing sugar in mg.

(ml 0.1 M)

Reducing sugars

(mg)

Diff.

(ml 0.1 M)

Reducing sugars

(mg)

Diff.

1

2.4

2.4

13

33.0

2.7

2

4.8

2.4

14

35.7

2.8

3

7.2

2.5

15

38.5

2.8

4

9.7

2.5

16

41.3

2.9

5

12.2

2.5

17

44.2

2.9

6

14.7

2.6

18

47.2

2.9

7

17.2

2.6

19

50.0

3.0

8

19.8

2.6

20

53.0

3.0

9

22.4

2.6

21

56.0

3.1

10

25.0

2.6

22

59.1

3.1

11

27.6

2.7

23

62.2

12

30.3

2.7

Bibliography

  • JAULMES P., Analyses des vins, 1951, 170, Montpellier.
  • JAULMES P., BRUN Mme S., ROQUES Mme J., Trav. Soc. Pharm., 1963, 23, 19.
  • SCHNEYDER J., VLECK G., Mitt. Klosterneuburg, Rebe und Wein, 1961, sér. A, 135.

Glucose and fructose (enzymatic method) (Type-II)

OIV-MA-AS311-02 Glucose and fructose

Type II method

  1. Definition

 

Glucose and fructose may be determined individually by an enzymatic method, with the sole aim of calculating the glucose/fructose ratio.

  1. Principle

Glucose and fructose are phosphorylated by adenosine triphosphate (ATP) during an enzymatic reaction catalyzed by hexokinase (HK), to produce glucose-6‑phosphate (G6P) and fructose-6‑phosphate (F6P):

The glucose-6‑phosphate is first oxidized to gluconate-6‑phosphate by nicotinamide adenine dinucleotide phosphate (NADP) in the presence of the enzyme glucose-6‑phosphate dehydrogenase (G6PDH). The quantity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) produced corresponds to that of glucose-6‑phosphate and thus to that of glucose.

The reduced nicotinamide adenine dinucleotide phosphate is determined from its absorption at 340 nm.

At the end of this reaction, the fructose-6‑phosphate is converted into glucose-6‑phosphate by the action of phosphoglucose isomerase (PGI):

The glucose-6‑phosphate again reacts with the nicotinamide adenine dinucleotide phosphate to give gluconate-6‑phosphate and reduced nicotinamide adenine dinucleotide phosphate, and the latter is then determined.

  1. Apparatus

 

A spectrophotometer enabling measurements to be made at 340 nm, the wavelength at which absorption by NADPH is at a maximum. Absolute measurements are involved (i.e. calibration plots are not used but standardization is made using the extinction coefficient of NADPH), so that the wavelength scales of, and absorbance values obtained from, the apparatus must be checked.

If not available, a spectrophotometer using a source with a discontinuous spectrum that enables measurements to be made at 334 nm or at 365 nm may be used.

Glass cells with optical path lengths of 1 cm or single‑use cells.

Pipettes for use with enzymatic test solutions, 0.02, 0.05, 0.1, 0.2 mL.

  1. Reagents

 

Solution 1: buffer solution (0.3 M triethanolamine, pH 7.6, 0.004 M Mg2+): dissolve 11.2g triethanolamine hydrochloride, (N.HCl, and 0.2 g magnesium sulfate,, in 150 mL of double-distilled water, add about 4 mL 5 M sodium hydroxide solution to obtain a pH value of 7.6 and make up to 200 mL.

This buffer solution may be kept for four weeks at approx. + 4°C.

Solution 2: nicotinamide adenine dinucleotide phosphate solution (about 0.0115 M): dissolve 50 mg disodium nicotinamide adenine dinucleotide phosphate in 5 mL of double-distilled water.

This solution may be kept for four weeks at approx. +4°C.

Solution 3: adenosine-5'‑triphosphate solution (approx. 0.081 M): dissolve 250 mg disodium adenosine-5'‑triphosphate and 250 mg sodium hydrogen carbonate, NaHCO3, in 5 mL of double-distilled water.

This solution may be kept for four weeks at approx. +4°C.

Solution 4: hexokinase/glucose‑6‑phosphate‑dehydrogenase: mix 0.5 mL hexo­kinase (2 mg of protein/mL or 280 U/mL with 0.5 mL glucose‑6‑phosphate‑dehydrogenase (1 mg of protein/mL).

This mixture may be kept for a year at approx. +4°C.

Solution 5: phosphoglucose‑isomerase (2 mg of protein/mL or 700 U/mL). The suspension is used undiluted.

This may be kept for a year at approx. +4°C.

Note: All solutions used above are available commercially.

  1. Procedure

5.1.   Preparation of sample

Depending on the estimated amount of glucose + fructose per liter (g/L) dilute the sample as follows:

Measurement at

340 and 344 nm

(g/L)

Measurement

at 365 nm

(g/L)

Dilution

with water

Dilution

factor F

up to 0.4

0.8

-

-

up to 4.0

8.0

1 + 9

10

up to 10.0

20.0

1 + 24

25

up to 20.0

40.0

1 + 49

50

up to 40.0

80.0

1 + 99

100

Above 40.0

80.0

1 + 999

1000

5.2.   Determination

With the spectrophotometer adjusted to the 340 nm wavelength, make measurements using air (no cell in the optical path) or water as reference.

Temperature between 20 and 25°C.

Into two cells with 1 cm optical paths, place the following:

Reference cell

Sample cell

Solution 1 (taken to 20°C)

2.50 mL

2.50 mL

Solution 2

0.10 mL

Solution 3

0.10 mL

Sample to be measured

0.20 mL

Double -distilled water

0.20 mL

Mix, and after three minutes read the absorbance of the solutions (). Start the reaction by adding:

Solution 4

0.02 mL

0.02 mL

Mix, read the absorbance after 15 minutes and after two more minutes check that the reaction has stopped ().

Add immediately:

Solution 5

0.02 mL

0.02 mL

Mix; read the absorbance after 10 minutes and after two more minutes check that the reaction has stopped ().

Calculate the differences in the absorbance between the reference cell and sample cells.:

corresponds to glucose,   corresponds to fructose,

Calculate the differences in absorbance for the reference cells (AT) and the sample cell (AD) and then obtain:

  • for glucose:
  • for fructose:

Note: The time needed for the completion of enzyme activity may vary from one batch to another. The above value is given only for guidance and it is recommended that it be determined for each batch.

5.3.   Expression of results

5.3.1. Calculation

The general formula for calculating the concentrations is:

where:

V = volume of the test solution (mL)

v = volume of the sample (mL)

MW = molecular mass of the substance to be determined

d = optical path in the cell (cm)

ε = absorption coefficient of NADPH at 340 nm = 6.3

(mmole-1 x l  cm-1)

V = 2.92 mL for the determination of glucose

V = 2.94 mL for the determination of fructose

v = 20 mL

PM = 180

d = 1

so that:

For glucose : C(g/L) = 0.417 

For fructose: C(g/L) = 0.420 

If the sample was diluted during its preparation, multiply the result by the dilution factor F.

Note:  If the measurements are made at 334 or 365 nm, then the following expressions are obtained:

  • measurement at 334 nm: ε= 6.2 (mmole -1  absorbance  cm-1)
  • for glucose : C(g/L) = 0.425 
  • for fructose: C(g/L) = 0.428 
  • measurement at 365 nm: ε = 3.4 (mmole-1  absorbance  cm-1)
  • for glucose: C(g/L) = 0.773 
  • for fructose: C(g/L) = 0.778 

5.3.2. Repeatability (r):

r  = 0.056 xi

xi = the concentration of glucose or fructose in g/L

5.3.3.      Reproducibility (R):

R  = 0.12 + 0.076 xi

xi = the concentration of glucose or fructose in g/L

Bibliography

 

  • BERGMEYER H.U., BERNT E., SCHMIDT F. and STORK H., Méthodes d'analyse enzymatique by BERGMEYER H.U., 2e éd., p. 1163, Verlag‑Chemie Weinheim/Bergstraße, 1970.
  • BOEHRINGER Mannheim, Méthodes d'analyse enzymatique en chimie alimentaire, documentation technique.
  • JUNGE Ch., F.V., O.I.V., 1973, No 438.

Dosage of sugars by HPLC (Type-II)

OIV-MA-AS311-03 Dosage of sugars in wine by HPLC

Type II method

  1. Scope of application

This method is applicable to the direct quantification of sugars in musts and wines up to 20 g/L and, after dilution, beyond.

Glycerol (between 0.5 and 15 g/L) and sucrose (between 1 and 40 g/L) may also be quantified in the same way.

  1. Principle

Sugars and glycerol are separated by HPLC using an alkylamine column and detected by refractometer.

  1. Reagents

3.1.   Demineralised Type I water (ISO 3696) or equivalent (HPLC grade);

3.2.   acetonitrile [75-05-8] (minimal transmission at 200 nm - purity 99%);

3.3.   fructose [57-48-7] (purity 99%);

3.4.   glucose [492-62-6] (purity 99%);

3.5.   sucrose [57-50-1] (purity 99%);

3.6.   glycerol [56-81-5] (purity 99%).

Preparation of reagent solutions

3.7.   Demineralised water (3.1): filtered through a 0.45 µm cellulose membrane;

3.8.   eluent: acetonitrile (3.2)/water (3.9) with a respective ratio of 80/20.

Note 2: the water/acetonitrile ratio may be adapted according to the objectives.

  1. Apparatus

4.1.   0.45 μm Cellulose filtration membrane;

4.2.   silica-based, octadecyl-bonded filter cartridge (e.g. Sep-Pak );

4.3.   common apparatus for high-performance liquid chromatography;

4.4.   alkylamine column (5 μm, 250 x 4.6 mm);

Note 3: columns of different lengths, internal diameter and particle size may be used but the type II method refers to the dimensions provided.

4.5.   refractometric index detector (RID);

4.6.   common laboratory apparatus.

  1. Sampling

The samples are degassed beforehand if necessary (e.g. with nitrogen or helium, or in an ultrasonic bath).

  1. Procedure

 

6.1.   Preparation of the sample

6.1.1. Dilution

Wines containing less than 20 g/L of (glucose + fructose) are analysed undiluted. Musts and wines containing more than 20 g/L have to be diluted to be within the range of calibration.

6.1.2. Filtration

The samples must be filtered using a 0.45 µm membrane (4.1) before analysis.

6.1.3. Elimination of phenolic compounds (if necessary)

For a must or wine, pass over a C18 cartridge (4.2).

6.2.   Analyses

6.2.1. Analytical conditions

Note 4: The following instructions are mandatory for the type II method.

Note 5: Conditions may be adapted by the laboratory with the loss of the type II reference.

HPLC system (4.3) equipped with column (4.4) and RID (4.5).

Mobile phase: isocratic acetonitrile/water eluent (3.10).

Flow: 1 mL/min.

Injected volume: between 10 and 50 μL, to be adapted according to the material used.

Examples of chromatograms are shown in Annex B (Figures 1 and 2).

The fructose-glucose resolution is recommended to be 2.

6.2.2. External calibration

The calibration solution that applies to all compounds described in this procedure may contain the following:

  • 10 g/L glycerol (3.6) 0.01 g/L,
  • 10 g/L fructose (3.3) 0.01 g/L,
  • 10 g/L glucose (3.4) 0.01 g/L,
  • 10 g/L sucrose (3.5) 0.01 g/L.

Note 6: if quantifying only one of these compounds, a solution that contains only the one required can be prepared.

6.3.   Calculation of response factors for external calibration used in routine analyses

RFi= area i/Ci

where

  • area i = peak area of the product in the calibration solution
  • and Ci = quantity of product present in the calibration solution.

It is also possible to use a calibration curve.

  1. Expression of results
    1.    Calculation of concentrations

Ce = areae /RFi

Where:

areae = peak area of product present in the sample.

The results are expressed in g/L.

Note 7: the results are indicated to a maximum of one decimal place.

  1. Quality assurance and control

Traceable to the international references through mass, volume and temperature.

Synthetic mixtures or samples coming, for instance, from proficiency ring test are used as internal quality control. A control chart may be used

  1. Performance of the method

No known compound co-elutes with fructose, glucose or sucrose.

Robustness: the analysis is sensitive to slight variations in temperature. Columns should be protected from temperature variations.

  1. Precision

(See Annex B.3)

10.1.         Glucose (content 3 g/L)

  • Repeatability limit reproducibility limit = 13%
    1.          Fructose (content 2 g/L)
  • Repeatability limit = 7%
  • Reproducibility limit = 10%
    1.          Glucose + fructose (content 5 g/L)
  • Repeatability limit Reproducibility limit = 10%

Figure 1 Chromatogram of a calibration solution (sugars and glycerol at 10 g/l

Figure 2 Chromatogram of a rosé wine

Glycerol (GY), fructose (FR), glucose (GL), saccharose (SA)

fructose (FR), glucose (GL), saccharose (SA) Glycerol (GY),

Figure 3 - Measure of pitches of noise after enlargement of chromatogram

RT1: retention time of fructose; RT2: retention time of glucose

W1/2: width of peak at mid-height; Yi: pitch of noise at point i

Annex B

(informative)

Precision data

B.1 - Samples in the interlaboratory test trial

This study was carried out by the Interregional Laboratory of the Répression de Fraudes in Bordeaux. The test trial involved 6 samples in blind duplicates (12 samples in total), identified as A to J (4 white wines and 4 red wines; 2 white Port wines and 2 red Port wines), containing glucose and fructose and whose content of each sugar was between 2 and 65 g/L. The wines from the region of Bordeaux were supplemented with glucose and fructose and stabilised with 100 mg/L of (TRICARD and MEDINA, 2003).

B.2 - Chromatographic conditions

Considering the response factors of these two sugars and the scales of the chromatograms, the noise corresponds to a concentration of 0.04 g/L for fructose and of 0.06 g/L for glucose (see Figure A3).

The limits of detection (3 times the noise) and of quantification (10 times the noise) are then obtained:

These results are compliant with those determined by TUSSEAU and BOUNIOL (1986) and are repeatable on other chromatograms.

B.3 - Precision

Nine laboratories participated in the interlaboratory study:

Istituto Sperimentale per l'Enologia, Asti, Italy;

Laboratoire de la DGCCRF de Montpellier, France;

Laboratoire LARA, Toulouse, France;

Instituto do vinho do Porto, Porto, Portugal;

Instituto da Vinha e do Vinho, Unhos, Portugal;

Estación de Viticultura y Enología, Vilafranca del Penedés, Spain;

Comité Interprofessionnel du vin de Champagne, Epernay, France;

Station fédérale de Changins, Switzerland;

Laboratoire de la DGCCRF de Talence, France.

The analyses of 3 points of the set of calibration solutions and the 12 samples were carried out successively by applying the method of analysis given.

The results were analysed according to the OIV protocol (Validation protocol of methods of analysis – Resolution OENO 6/1999).

This protocol does not require the analyses to be repeated, whereas 4 laboratories gave results of analyses repeated 3 times. A single series was chosen (the first one) for the analysis of the results, in compliance with the OIV protocol.

The calculations of repeatability according to Youden, reproducibility and Cochran and Grubbs tests were performed.

Data on the repetitions made it possible to work out the standard deviations of repeatability in another way (according to ISO 5725).

B.3.1 – GLUCOSE

Glucose by HPLC (g/L)

Number of laboratories

9

9

9

9

9

9

Number of samples

2

2

2

2

2

2

Average value

2.9

2.9

12.6

12.4

44.6

67.5

Repeatability standard deviation

0.44

0.17

0.67

0.34

1.05

3.31

Repeatability limit

1.42

0.55

2.15

1.07

3.35

10.58

Reproducibility standard deviation

0.78

0.30

0.90

0.52

1.43

3.28

Reproducibility limit

2.32

0.90

2.68

1.55

4.28

9.78

Horrat value

5.7*

2.1

1.84

1.08

1.01

1.62

* not taken into account for the expression of precision

 

Correlation between r and R and the concentration for glucose (ISO 5725)

B.3.2 – FRUCTOSE

Fructose by HPLC (g/L)

Number of laboratories

9

9

9

9

9

9

Number of samples

2

2

2

2

2

2

Average value

1.9

5.2

10.0

13.0

62.6

73.0

Repeatability standard deviation

0.09

0.24

0.32

0.16

3.20

2.10

Repeatability limit

0.27

0.79

1.03

0.51

3.20

6.72

Reproducibility standard deviation

0.25

0.25

0.32

0.43

2.91

1.93

Reproducibility limit

0.75

0.75

0.96

1.30

8.68

5.77

Horrat value

2.54

1.09

0.81

0.87

1.53

0.89

Correlation between r and R and the concentration for fructose (ISO 5725)

B.3.3 – GLUCOSE + FRUCTOSE

Glucose + fructose by HPLC (g/L)

Number of laboratories

9

9

9

9

9

9

Number of samples

2

2

2

2

2

2

Average value

4.7

8.1

22.6

25.4

107.3

140.5

Repeatability standard deviation

0.48

0.38

1.06

0.46

1.92

5.30

Repeatability limit

1.52

1.21

3.07

1.48

6.13

17.0

Reproducibility standard deviation

0.89

0.46

1.06

0.64

3.47

4.74

Reproducibility limit

2.64

1.38

3.17

1.90

10.34

14.15

Horrat value

4.17*

1.39

1.33

0.72

1.15

1.26

* not taken into account for the expression of precision

Correlation between r and R and the concentration for glucose + fructose (ISO 5725)

Bibliography

  • TRICARD, C. and MEDINA, B., ‘Essai inter laboratoire OIV – Dosage des sucres dans les vins par HPLC’, FV 1143, 2003, 8 pages.
  • TUSSEAU, D. and BOUNIOL, C., Sc. Alim., No. 6, 1986, pp. 559-577.
  • TUSSEAU, D., ‘Limite de détection - limite de quantification’, FV OIV 1000, 1996.
  • ‘Protocol for the design, conduct and interpretation of collaborative studies’, Resolution OIV-OENO 6-2000.
  • ‘Application of statistics - Accuracy (trueness and precision) of measurement methods and results -...’, ISO Standard 5725, 1994.

Stabilisation of musts to detect Addition of sucrose

OIV-MA-AS311-04 Stabilization of musts to detect the addition of sucrose

  1. Principle of the method

The sample is brought to pH 7 with a sodium hydroxide solution and an equal volume of acetone is added.

The acetone is removed by distillation prior to determination of sucrose by TLC (thin‑layer chromatography) and HPLC (high‑performance liquid chromatography) (see Sucrose Chapter).

  1. Apparatus

Distillation apparatus, with a 100 mL round distillation flask.

  1. Reagents

 

3.1.  Sodium hydroxide solution, 20% (m/v)

3.2.  Acetone (propanone).

  1. Method

 

4.1.  Stabilizing the samples

20 mL of must is placed in a 100 mL strong‑walled flask and brought to pH 7 with the 20% sodium hydroxide solution (m/V) (six to twelve drops). 20 mL of acetone are added. Stopper and store at low temperature.

WARNING: Acetone has high vapour pressure and is highly inflammable.

4.2.  Preparing the sample to determine sucrose by TLC or HPLC.

Place the contents of the flask in the 100 mL round flask of the distillation apparatus.  Distil and collect approximately 20 mL of distillate, which is discarded. Add 20 mL of water to the contents of the distilling flask and distil again, collecting about 25 mL of distillate, which is discarded.

Transfer the contents of the distillation flask to a graduated 20 mL volumetric flask and make up to the mark with the rinsing water from the round flask.  Filter.  Analyze the filtrate and (if detected) measure the sucrose using TLC or HPLC.

Bibliography

  • TERCERO C., F.V., O.I.V., 1972, No. 420 and 421.

Determination of the deuterium distribution in ethanol derived from fermentation of grape musts, concentrated grape musts, grape sugar (rectified concentrated grape musts) and wines by application of nuclear magnetic resonance (SNIF-NMR/RMN-FINS)

OIV-MA-AS311-05 Determination of the deuterium distribution in ethanol derived from fermentation of grape musts, concentrated grape musts, grape sugar (rectified concentrated grape musts) and wines by application of nuclear magnetic resonance (SNIF-NMR/RMN-FINS)[1]

Type II method

 

  1. Introduction

The deuterium contained in the sugars and the water in grape must is redistributed after fermentation in molecules I, II, III and IV of the wine:

HOD

I

II

III

IV
  1. Scope

 

The method enables measurement of the Deuterium isotope ratios (D/H) in wine ethanol and ethanol obtained by fermentation of products of the vine (musts, concentrated musts, rectified concentrated musts).

  1. Definitions

: Isotope ratio associated with molecule I

: Isotope ratio associated with molecule II

: Isotope ratio of the water in the wine (or in fermented products)

R expresses the relative distribution of deuterium in molecules I and II; R is measured directly from the intensities h (peak heights) of the signals and then R =

  1. Principle

The above defined parameters R, (D/H)I and (D/H)II are determined by nuclear magnetic resonance of the deuterium in the ethanol extracted from the wine or from the fermentation products of the must, the concentrated must or the grape sugar (rectified concentrated must) obtained under given conditions.

  1. Reagents and materials
    1.   reagents:
      1.      reagents for the determination of water by the Karl Fischer method (when this method is used for the measurement of the alcohol grade of the distillate).
      2.      Hexafluorobenzene (C6F6) used as lock substance
      3.      Trifluoroacetic acid (TFA, CAS: 76-05-1) or alternatively trifluoroacetic anhydride (TFAA, CAS: 407-25-0)
    2.   Reference Materials (available from the Institute for Reference Materials and Measurements IRMM in Geel (B)):
      1.      Sealed NMR tubes CRM-123, used to check the calibration of the NMR instrumentation
      2.      Standard N,N-tetramethyl urea (TMU); standard TMU with a calibrated isotope ratio D/H.
      3.      Other CRMs available used to check the distillation and preparation steps:

CRM

  

Parameter

Certified value

Uncertainty

CRM-656

Ethanol from wine, 96% vol.

      
   

(ethanol) in % w/w

94.61

0.05
   

C (ethanol) in  ‰ VPDB

-26.91

0.07
   

(ethanol) in ppm

102.84

0.20
   

(ethanol) in ppm

132.07

0.30
   

R (ethanol)

2.570

0.005

CRM-660

hydro alcoholic solution, 12% vol.

      
   

(ethanol) in % vol.

11.96

0.06
   

C (ethanol) in  ‰ VPDB

-26.72

0.09
   

(ethanol) in ppm

102.90

0.16
   

(ethanol) in ppm

131.95

0.23
   

R

2.567

0.005
   

(D/H)w (water) in ppm

148.68

0.14

5.3.  Apparatus

5.3.1.     NMR spectrometer fitted with a specific 'deuterium' probe tuned to the characteristic frequency o of the field Bo (e.g. for Bo = 7.05 T, o = 46.05 MHz and for Bo = 9.4 T, o = 61.4 MHz) having a proton decoupling channel (B2) and field-frequency stabilization channel (lock) at the fluorine frequency. The NMR instrument can possibly be equipped with an automatic sample changer and additional data-processing software for the evaluation of the spectra and computation of the results. The performance of the NMR spectrometer can be checked using the Certified Reference Materials (sealed tubes CRM 123).

5.3.2.     10 mm NMR sample tubes

5.3.3.     Distillation apparatus

Note: Any method for ethanol extraction can be used as long as the alcohol in the wine is recovered without isotopic fractionation.

 The Cadiot column shown in figure 1 is an example of a manual distillation system that allows to extract 96 to 98.5% of the ethanol of a wine without isotopic fractionation and obtain a distillate with an alcohol grade of 92 to 93 in % w/w (95% vol.).

Such a system is composed of:

  • Electric heating mantle with voltage regulator,
  • One‑liter round‑bottom flask with ground glass neck joint,
  • Cadiot column with rotating band (moving part in Teflon),
  • conical flasks with ground glass neck joints, for collection of the distillate

Automatic distillation systems are also available.

The performance of the distillation system may be checked periodically for both the yield of extraction as well as for accuracy for the isotopic determination. This control can be done by distillation and measurement of CRM -660.

5.3.4.     The following common laboratory equipment and consumables is needed:

  • micropipette with appropriate tips,
  • balance with 0.1 mg accuracy or better,
  • balance with 0.1g accuracy or better
  • single use syringe for transfer of liquids,
  • precise graduated flasks (50ml, 100 ml, 250ml,…)
  • flasks equipped with airtight closing systems and inert septa (for storage of aliquots of wines, distillates and residues until measurement)
  • equipment and consumables as specified in the other methods referred to herein.

The laboratory equipment and consumables indicated in the above lists are examples and may be replaced by other equipment of equivalent performance.

  1. Sampling (Preparation of the sample)

6.1.  If not yet available, determine the alcoholic strength of the wine or of the fermented product (tv) to better than the nearest 0.05 % vol. (eg. using the OIV method MA-F-AS312-01-TALVOL).

6.2.  Extraction of the ethanol

Using the appropriate graduated flask, introduce a homogeneous sample of a suitable volume V ml of the wine or the fermented product into the round-bottom flask of the distillation apparatus. Place a ground conical flask to receive the distillate. Heat the product to be distilled to obtain a constant reflux ratio at the level of the condenser. Start the collection of the distillate when a stable temperature of the vapours typical of the ethanol-water azeotrope (78°C) is reached and stop the collection when the temperature increases. The collection of distillate should be continued until the ethanol-water azeotrope is completely recovered.

When using manually a Cadiot column (Figure 1) the following procedure can be applied:

Collect the boiling liquid corresponding to the ethanol-water azeotrope, when the temperature increases, discontinue collection for five minutes. When the temperature returns to 78 °C, recommence collecting the distillate until the temperature of the vapours increases again. Repeat this operation until the temperature, after discontinuing collection, does not return to 78 °C.

Alternatively, commercially available distillation systems can be used.

The weight of distillate collected is weighed to better than 0.1g.

In order to avoid isotopic fractionation, the distillate should be kept in a tight vial preventing any evaporation until further use for determination of the alcoholic strength (6.3) and preparation of the NMR tube (7.1).

An aliquot of a few ml of the residues is kept. Its isotope ratio may be determined if required.

6.3.  Determination of the alcoholic strength of the distillate

The alcoholic strength (%w/w) of the distillate must be determined with a precision better than 0.1%.

The water content of the distillate (' g) can be determined by the Karl Fischer method using a sample of about 0.5 ml of alcohol of exactly known mass  g .The alcohol strength by mass of the distillate is then given by:

Alternatively the alcoholic strength can be determined by densimetry for instance using a electronic densimeter.

6.4.  Yield of distillation

The yield of distillation is estimated using the following formula:

Yield of dist.% = 100 /(V.tv)

Given the uncertainty on each term and especially on tv, the yield of distillation is estimated at 0.5% (in the case of a wine of 10%v/v).

When using the Cadiot column, no significant isotope fractionation effect is expected for yield of extraction higher than 96%. In any case the operator may use a sufficient volume Vml of wine or fermented product for the distillation to ensure that the yield of extraction is sufficient. Typically aliquots of 750, 500, 400 and 300ml of wine sample should be sufficient to obtain a 96% yield when carrying out the above distillation procedure with the Cadiot column on wines or fermented products of respectively tv = 4, 6, 8 and 10% vol.

6.5.  Fermentation of musts, concentrated musts and rectified concentrated musts

Prior to use, the yeast can be reactivated in a small volume of must. The fermentation vessel is equipped with a device to keep it airtight and to avoid loss of ethanol.

6.5.1.     Musts

Place about one litre of must, whose concentration of fermentable sugars has been previously determined, in the fermentation vessel. Add about 1 g of dry yeast eventually reactivated beforehand. Insert device to keep it airtight. Allow fermentation to proceed until the sugar is used up. The fermented product can then be distilled following the procedure already described for wine in 6.1 to 6.4

Note: Musts preserved by addition of sulphur dioxide have to be de-sulphited by bubbling nitrogen through the must in a water bath at 70 to 80 °C under reflux in order to prevent isotope fractionation through evaporation of water. Alternatively, the sulphur dioxide can be removed by a small addition of a solution of hydrogen peroxide ().

6.5.2.     Concentrated musts

Place V ml of concentrated must containing a known amount of sugar (approximately 170 g) into the fermentation vessel. Top up to one litre with (1000 - V) ml of water. Add dry yeasts (1 g) and 3 g of Bacto Yeast Nitrogen Base without amino acids. Homogenize and proceed as described in 6.5.1.

6.5.3.     Grape sugar (Rectified concentrated musts)

Proceed as described in 6.5.2, topping up to one litre with (1000 - V) ml of water also containing 3 g of dissolved tartaric acid.

Note: Concentrated musts and rectified concentrated musts are diluted in local water having a (D/H) isotope concentration different of that of the original must. By convention, the (D/H)I and (D/H)II  parameters measured on ethanol have to be normalised  as if the must had fermented in water having the same deuterium concentration as V-SMOW ( 155.76 ppm). 

This normalisation of the data is performed by using the following equations (Martin et al., 1996, J. AOAC, 79, 62-72):

Given the uncertainty on each term and especially on tv, the yield of distillation is estimated at 0.5% (in the case of a wine of 10%v/v).

When using the Cadiot column, no significant isotope fractionation effect is expected for yield of extraction higher than 96%. In any case the operator may use a sufficient volume Vml of wine or fermented product for the distillation to ensure that the yield of extraction is sufficient. Typically aliquots of 750, 500, 400 and 300ml of wine sample should be sufficient to obtain a 96% yield when carrying out the above distillation procedure with the Cadiot column on wines or fermented products of respectively tv = 4, 6, 8 and 10% vol.

6.6.  Fermentation of musts, concentrated musts and rectified concentrated musts

Where is the deuterium isotope ratio of the diluted must. This value can be computed using the equation of the Global Meteoric Water Line (Craig, 1961):

Where is measured on the diluted must by the method for 18O/16O isotope ratio determination of water in wines and must [OIV-MA-AS2-12].

Retain 50 ml of sample of must or sulphur dioxide treated must or concentrated must or rectified concentrated must with a view to the possible extraction of the water and the determination of its isotope ratio

  1. Procedure

7.1.  Preparation of alcohol sample for NMR measurement

10 mm diameter NMR probe: in a previously weighed bottle, collect 3.2 ml of distillate as described in section 6.2 and weigh it to the nearest 0.1 mg (mA); then take 1.3 ml sample of the internal standard TMU (5.2.2) and weigh to the nearest 0.1 mg (mST).

Depending on the type of spectrometer and probe used, add a sufficient quantity of hexafluorobenzene (5.1.2) as a field-frequency stabilization substance (lock):

Spectrometer

10 mm probe

7.05 T

150 μl

9.4 T

35 μl

These figures are indicative and the actual volume to be used should be adjusted to the sensitivity of the NMR instrument. While preparing the tube and until the NMR measurement, the operator should take care to avoid any evaporation of ethanol and TMU since this would cause isotopic fractionation, errors in the weights (and) of the components and erroneous NMR results.

The correcteness of the procedure of measurement including this preparation step can be checked using the CRM 656.

Note: the hexafluorobenzene can be added with 10% (v/v) of trifluoroacetic acid (5.1.3) in order to catalyze the fast hydrogen exchange on hydroxyle bond resulting in a single NMR peak for both the hydroxyle and residual water signals.

7.2.  Recording of ²H NMR spectra of the alcohol

7.2.1.     The homogeneity of the magnetic field B0 in the sample is optimized through the “shimming” procedure maximizing the 19F NMR lock signal observed the hexafluorobenzene. Modern NMR spectrometers can perform automatically and efficiently this “shimming” procedure provided that the initial settings are close enough to the optimal magnetic field homogeneity for a given sample as is generally the case for a batch of ethanol samples prepared as described in 7.1. The efficiency of this procedure can be checked through the resolution measured on the spectrum obtained without exponential multiplication (i.e. LB = 0) (Figure 2b) and expressed by the half-width of the methyl and methylene signals of ethanol and the methyl signal of TMU, which must be less than 0.5 Hz in the best conditions. The sensitivity, measured with an exponential multiplying factor LB equal to 2 (Figure 2a) must be greater than or equal to 150 for the methyl signal of ethanol of alcoholic strength 95 % vol (93.5 % mas).

7.2.2.     Checking the instrumental settings

Carry out customary standardization for homogeneity and sensitivity according to the manufacturer's specifications.

Use the sealed tubes CRM123 (H: High , M: Medium, L: Low).

Following the procedure described below in 9.3, determine the isotope values of these alcohols, denoting them Hmeas, Mmeas, Lmeas .

Compare them with the given corresponding standard values, denoted by a superscript Hst, Mst, Lst.

Typically, as an indication the standard deviation obtained for 10 repetitions of each spectrum should be of the order of 0.01 for the ratio R and 0.5 ppm for and 1 ppm for .

The average values obtained for the various isotopic parameters (R, , must be within the corresponding standard deviation of repeatability given for those parameters for the CRM123. If they are not, carry out the checks again.

Once the settings have been optimized also other CRM materials can be used to monitor the quality of measurements in routine analysis.

7.3.  Conditions for obtaining NMR spectra

Place a sample of alcohol prepared as in 7.1 in a 10 mm tube and introduce it into the probe.

Suggested conditions for obtaining NMR spectra are as follows:

  • a constant probe temperature, set to better less than 0.5°K variation in the range 302 K to 306 K depending on the heating power generated by the  decoupling;
  • acquisition time of at least 6.8 s for 1200 Hz spectral width (16K memory) (i.e. about 20 ppm at 61.4 MHz or 27 ppm at 46.1 MHz);
  • 90° pulse;
  • parabolic detection: fix the offset 01 between the OD and CHD reference signals for ethanol and between the HOD and TMU reference signals for water;
  • determine the value of the decoupling offset 02 from the proton spectrum measured by the decoupling coil on the same tube. Good decoupling is obtained when 02 is located in the middle of the frequency interval existing between the CH3- and CH2- groups. Use the wide band decoupling mode or composite pulse sequences (eg. WALTZ16) to ensure homogeneous decoupling on the whole spectrum.

For each spectrum, carry out a number of accumulations NS sufficient to obtain the signal-to-noise ratio indicated as sensitivity in 7.2 and repeat NE times this set of NS accumulations. The values of NS depend on the types of spectrometer and probe used. Examples of the possible choices are:

Spectrometer

10 mm probe

7.05 T

NS = 304

9.4 T

NS = 200

The number of repetitions NE should be statistically meaningful and sufficient to achieve the performance and precision of the method as reported below in §9.

In the case that two NMR sample tubes have been prepared following the procedure described in 7.1, five repetitions of NMR spectra (NE=5) can be recorded on each tube. The final result for each isotopic parameter corresponds to the mean value of the measurements obtained on the two NMR sample tubes. In that case, the acceptance criteria for validation of the results obtained with these two tubes are:
  1. Expression of results

For each of the NE spectra (see NMR spectrum for ethanol, Figure 2a), determine:

With

  • and , see 7.1
  • see 6.3
  • = isotope ratio of internal standard (TMU) indicated on certificate delivered by IRMM.

The use of peak heights instead of peak area, which is less precise, supposes that peak width at half height is identical and is a reasonable approximation if applicable (Figure 2b).

For each of the isotope parameters, calculate the average and the confidence interval for the number of repeated spectra acquired on a given sample.

Optional softwares enable such calculations to be carried out on-line.
 

  1. Precision

The repeatability and Reproducibility of the SNIF-NMR method has been studied through collaborative studies on fruit juices as reported in the bibliography here below. However these studies considered only the parameter (D/H)I. In the case of wine data from in-house studies carried out by several laboratories can be considered for establishing the standard deviation of repeatability and the limit of repeatability as presented in Annex I. The results of proficiency testing reported in Annex II provide data that can be used to compute the standard deviation of Reproducibility and the limit of Reproducibility for wines.

These figures can be summarised as follows:

 

(D/H)I

(D/H)II

R

Sr

0.26

0.30

0.005

r

0.72

0.84

0.015

SR

0.35

0.62

0.006

R

0.99

1.75

0.017

with

  • : standard deviation of repeatability 
  • r : limit of repeatability
  • : standard deviation of reproducibility
  • R : limit of Reproducibility

References

  • Martin G.J., Martin M.L., MABON F., Anal. Chem., 1982, 54, 2380-2382.
  • Martin G.J., Martin M.L., J. Chim. Phys., 1983, 80, 294-297.
  • Martin G.J., Guillou C., NAULET N., BRUN S., Tep Y., Cabanis J.C.,
  • Cabanis M.T., Sudraud P., Sci. Alim., 1986, 6, 385-405.
  • Martin G.J., Zhang B.L., NAULET N. and MARTIN M.L., J. Amer. Chem. Soc., 1986, 108, 5116-5122.
  • Martin G.J., Guillou C., Martin M.L., Cabanis M.T., TEP Y. et AERNY J., J. Agric. Food Chem., 1988, 36, 316.
  • MARTIN G. G., WOOD  R., MARTIN, G. J.,  J. AOAC Int., 1996 , 79 (4), 917-928.
  • MARTIN G.G., HANOTE V., LEES M., MARTIN Y-L.,.  J. Assoc Off Anal Chem, 1996, 79, 62-72
  • CRAIG H., Science , 1961,  133,. 1702 – 1703

Description: Description: Description: Untitled2

Figure 1 - Apparatus for extracting ethanol

 

Figure 2a

2H NMR spectrum of an ethanol from wine with an internal standard (TMU: N, N-tetramethylurea)

Figure 2b

2H spectrum of ethanol taken under the same conditions as those of Figure 2a, but without exponential multiplication (LB = 0)

 

Annex I:  Estimation of the repeatability from in-house repeatability studies

The in-house repeatability studies performed in 4 laboratories provide data that allows the estimation of the repeatability of the SNIF-NMR method.

These in-house repeatability studies have been performed by duplicate distillations and measurements of 10, 9 or 15 different wine samples by the laboratories 1, 2 and 3.

Alternatively the laboratory 4 performed 16 distillations and measurements on the same wine in condition of repeatability on a short period of time.

Table I-1: lab 1 : 10 wines analysed in duplicates

       

(D/H)I

  

(D/H)II

  

R

  
       

abs ((D/H)I)

Squares

abs

((D/H)II)

Squares

abs

((R))

Squares

Sample

(D/H)I

(D/H)II

R

 

  

 

  

 

  

1

103.97

130.11

2.503

0.55

0.302

0.68

0.462

0.000

0.00000
 

104.52

130.79

2.503

            

2

103.53

130.89

2.529

0.41

0.168

0.32

0.102

0.016

0.00026
 

103.94

130.57

2.513

            

3

102.72

130.00

2.531

0.32

0.102

0.20

0.040

0.004

0.00002
 

103.04

130.20

2.527

            

4

105.38

132.39

2.513

0.14

0.020

0.20

0.040

0.000

0.00000
 

105.52

132.59

2.513

            

5

101.59

127.94

2.519

0.48

0.230

0.20

0.040

0.016

0.00026
 

101.11

128.14

2.535

            

6

103.23

132.14

2.560

0.30

0.090

0.36

0.130

0.001

0.00000
 

102.93

131.78

2.561

            

7

103.68

130.95

2.526

0.15

0.023

0.75

0.563

0.011

0.00012
 

103.53

130.20

2.515

            

8

101.76

128.86

2.533

0.24

0.058

0.42

0.176

0.003

0.00001
 

101.52

128.44

2.530

            

9

103.05

129.59

2.515

0.04

0.002

0.44

0.194

0.007

0.00005
 

103.01

129.15

2.508

            

10

101.47

132.63

2.614

0.50

0.250

0.18

0.032

0.010

0.00010
 

100.97

132.45

2.624

            
       

Sum of squares:

1.245

  

1.779

  

0.00081
       

Sr

0.25

 

0.30

 

0.006
       

r

0.71

 

0.84

 

0.018
                           

 

Table I-2: lab 2 : 9 wines analysed in duplicates

       

(D/H)I

  

(D/H)II

  

R

  
       

abs ((D/H)I)

Squares

abs

((D/H)II)

Squares

abs

((R))

Squares

Sample

(D/H)I

(D/H)II

R

 

 

 

 

 

 

1

105.02

133.78

2.548

0.26

0.068

0.10

0.010

0.008

0.00007
 

104.76

133.88

2.556

            

2

102.38

130.00

2.540

0.73

0.533

0.40

0.160

0.010

0.00011
 

101.65

129.60

2.550

            

3

100.26

126.08

2.515

0.84

0.706

0.64

0.410

0.008

0.00007
 

99.42

125.44

2.523

            

4

101.17

128.83

2.547

0.51

0.260

0.45

0.203

0.004

0.00002
 

100.66

128.38

2.551

            

5

101.47

128.78

2.538

0.00

0.000

0.26

0.068

0.005

0.00003
 

101.47

128.52

2.533

            

6

106.14

134.37

2.532

0.12

0.014

0.04

0.002

0.002

0.00000
 

106.26

134.41

2.530

            

7

103.62

130.55

2.520

0.05

0.003

0.11

0.012

0.003

0.00001
 

103.57

130.66

2.523

            

8

103.66

129.88

2.506

0.28

0.078

0.55

0.302

0.004

0.00001
 

103.38

129.33

2.502

            

9

103.50

129.66

2.506

0.43

0.185

0.22

0.048

0.015

0.00021
 

103.93

129.44

2.491

            
       

Sum of squares:

1.846

  

1.214

  

0.00053
                   
       

Sr

0.32

 

0.26

 

0.005
       

r

0.91

 

0.74

 

0.015

Table I-3: lab 3 : 15 wines analysed in duplicates

       

(D/H)I

  

(D/H)II

  

R

  
       

abs

((D/H)I)

Squares

abs

((D/H)II)

Squares

abs

((R))

Squares

Sample

(D/H)I

(D/H)II

R

 

 

 

 

 

 

1

101.63

125.87

2.477

0.06

0.004

0.46

0.212

0.007

0.00005
 

101.57

125.41

2.470

            

2

99.24

124.41

2.507

0.05

0.002

0.04

0.002

0.001

0.00000
 

99.19

124.37

2.508

            

3

101.23

125.07

2.471

0.06

0.004

0.16

0.026

0.005

0.00002
 

101.17

125.23

2.476

            

4

100.71

125.29

2.488

0.07

0.005

1.16

1.346

0.024

0.00058
 

100.78

124.13

2.464

            

5

99.89

124.02

2.483

0.18

0.032

0.56

0.314

0.007

0.00005
 

99.71

123.46

2.476

            

6

100.60

124.14

2.468

0.19

0.036

0.66

0.436

0.018

0.00032
 

100.41

124.80

2.486

            

7

101.47

125.60

2.476

0.23

0.053

0.14

0.020

0.003

0.00001
 

101.70

125.74

2.473

            

8

102.02

124.00

2.431

0.13

0.017

0.07

0.005

0.005

0.00002
 

102.15

123.93

2.426

            

9

99.69

124.60

2.500

0.40

0.160

0.53

0.281

0.000

0.00000
 

100.09

125.13

2.500

            

10

99.17

123.71

2.495

0.30

0.090

0.19

0.036

0.004

0.00002
 

99.47

123.90

2.491

            

11

100.60

123.89

2.463

0.40

0.160

0.54

0.292

0.001

0.00000
 

101.00

124.43

2.464

            

12

99.38

124.88

2.513

0.33

0.109

0.55

0.302

0.002

0.00000
 

99.05

124.33

2.511

            

13

99.51

125.24

2.517

0.44

0.194

0.01

0.000

0.011

0.00012
 

99.95

125.25

2.506

            

15

101.34

124.68

2.460

0.43

0.185

0.41

0.168

0.002

0.00000
 

101.77

125.09

2.458

            
     

Sum of squares:

1.050

  

3.437

  

0.00120
       

Sr

0.19

 

0.34

 

0.006
       

r

0.53

 

0.96

 

0.018

Table I-4: one wine analysed 16 times

:

                    

 

            
                     

Repetition

(D/H)I

(D/H)II

R

 

  

(D/H)I

  

(D/H)II

  

R

1

101.38

126.87

2.503

  

Variance:

0.0703

  

0.0840

  

0.000013

2

101.30

126.22

2.492

              

3

100.98

125.86

2.493

  

Sr

0.27

  

0.29

  

0.004

4

100.94

126.00

2.497

  

 

 

  

 

  

 

5

100.71

125.79

2.498

  

r

0.75

  

0.82

  

0.010

6

100.95

126.05

2.497

              

7

101.17

126.30

2.497

              

8

101.22

126.22

2.494

              

9

100.99

125.91

2.494

              

10

101.29

126.24

2.493

              

11

100.78

126.07

2.502

              

12

100.65

125.65

2.497

              

13

101.01

126.17

2.498

              

14

100.89

126.05

2.499

              

15

101.66

126.52

2.489

              

16

100.98

126.11

2.498

              
                     

The pooled data for the standard deviation of repeatability and for the limit of repeatability can thus be estimated as:

R

Sr

0.26

0.30

0.005

limit of repeatability

0.72

0.84

0.015

Data of in-house repeatability studies were provided by (in alphabetic order):

  • Bundesinstitut für Risikobewertung,

Thielallee 88-92 PF 330013 D-14195 BERLIN – GERMANY

  • Fondazione E. Mach-Istituto Agrario di San Michele all'Adige,

Via E. Mach, 1 - 38010 San Michele all'Adige (TN), ITALY

  • Joint Research Centre - Institute for Health and Consumer Protection,

I-21020 ISPRA (VA) – ITALY

  • Laboratorio Arbitral Agroalimentario, Carretera de la Coruña, km 10,7

E-28023 MADRID –SPAIN

 

Annex II: Evaluation of the Reproducibility from proficiency testing data

 

Since December 1994 international proficiency testing exercises on the determination of isotopic parameters on wine and various other food matrices have been regularly organised. These proficiency testing exercises allow participating laboratories to assess their performance and the quality of their analyses. The statistical exploitation of these results obtained on a large number of samples over a long period of time allows the appreciation of the variability of the measurements under conditions of reproductibility. This enables a good estimation of the variance parameters and of the reproducibility limit of the method. The results of 40 rounds of proficiency testing since 1994 until 2010 for various type of wine (red, white, rosé, dry, sweet and sparkling) are summarised in the table II-1 here below.

For and   the  pooled SR can thus be calculated using the following equation:

with Ni ,and SR,i the number of values and the standard deviation of reproducibility of the ith round, and K the number of rounds.

Considering the definition of the intramolecular ratio R, and applying the standard error propagation rules assuming that (D/H)I and (D/H)II are uncorrelated (the covariance terms are then zero), one can also estimate the standard deviation of Reproducibility for this parameter.

The following figures can thus be calculated:

R

0.35

0.62

0.006

limit of repeatability R

0.99

1.75

0.01

Table II-1: FIT Proficiency Testing- Summary of statiscal values observed on wines samples

         

(D/H)I

      

(D/H)II

  

Sample

Year

Round

  

N

Mean

SR

  

N

Mean

SR

Red wine

1994

R1

  

10

102.50

0.362

  

10

130.72

0.33

Rosé wine

1995

R1

  

10

102.27

0.333

  

10

128.61

0.35

Red wine

1995

R2

  

11

101.45

0.389

  

11

127.00

0.55

Red wine

1996

R1

  

11

101.57

0.289

  

11

132.23

0.34

Rosé wine

1996

R2

  

12

102.81

0.322

  

12

128.20

0.60

White wine

1996

R3

  

15

103.42

0.362

  

15

127.97

0.51

Red wine

1996

R4

  

15

102.02

0.377

  

13

131.28

0.30

Rosé wine

1997

R1

  

16

103.36

0.247

  

16

126.33

0.44

White wine

1997

R2

  

16

103.42

0.444

  

15

127.96

0.53

Sweet White Wine

1997

R2

  

14

99.16

0.419

  

15

130.02

0.88

Wine

1997

R3

  

13

101.87

0.258

  

15

132.03

0.61

Sweet Wine

1997

R3

  

12

102.66

0.214

  

12

128.48

0.48

Rosé wine

1997

R4

  

16

102.29

0.324

  

16

129.29

0.63

Sweet Wine

1997

R4

  

15

102.04

0.269

  

13

131.27

0.30

White wine

1998

R1

  

16

105.15

0.302

  

16

127.59

0.59

Sweet Wine

1998

R3

  

16

102.17

0.326

  

16

129.60

0.56

Red wine

1998

R4

  

17

102.44

0.306

  

17

131.60

0.47

  

White wine

1999

R1

  

14

102.93

0.404

  

13

129.64

0.46

  

Sweet Wine

2000

R2

  

15

103.19

0.315

  

14

129.43

0.60

  

Wine

2001

R1

  

12

105.28

0.264

  

16

131.32

0.68

  

Sweet Wine

2001

R2

  

14

101.96

0.249

  

15

128.99

1.05

  

Wine

2002

R1

  

17

101.01

0.365

  

16

129.02

0.74

  

Wine

2002

R2

  

17

101.30

0.531

  

17

129.28

0.93

  

Wine

2003

R1

  

18

100.08

0.335

  

18

128.98

0.77

  

Sweet Wine

2003

R2

  

17

100.51

0.399

  

18

128.31

0.80

  

Wine

2004

R1

  

18

102.88

0.485

  

19

128.06

0.81

  

Sweet Wine

2004

R3

  

16

101.47

0.423

  

16

130.10

0.71

  

Wine

2005

R1

  

19

101.33

0.447

  

19

129.88

0.76

  

Sweet wine

2005

R2

  

15

102.53

0.395

  

15

131.36

0.38

  

Dry wine

2006

R1

  

18

101.55

0.348

  

18

131.30

0.51

  

Sweet wine

2006

R2

  

18

100.31

0.299

  

18

127.79

0.55

  

Wine

2007

R1

  

18

103.36

0.403

  

18

130.90

0.90

  

Sweet wine

2007

R2

  

19

102.78

0.437

  

19

130.72

0.55

  

Wine

2008

R1

  

24

103.20

0.261

  

23

131.29

0.59

  

Sweet wine

2008

R2

  

20

101.79

0.265

  

19

129.73

0.34

  

Dry wine

2009

R1

  

24

102.96

0.280

  

23

130.25

0.49

  

Sweet wine

2009

R2

  

21

101.31

0.310

  

21

127.07

0.50

  

Dry wine

2010

R1

  

21

101.80

0.350

  

20

129.65

0.40

  

Sparkling wine

2010

R1

  

11

101.51

0.310

  

11

129.09

0.68

  

Dry wine

2010

R2

  

20

104.05

0.290

  

19

133.31

0.58

  
                               

 

[1] Specific Natural Isotope Fractionation studied by Nuclear Magnetic Resonance). Patent: France, 8122710; Europe, 824022099; USA, 854550082; Japan 57123249.

Polyols derived from sugars (Type-IV)

OIV-MA-AS311-06 Determination of polyols derived from sugars and residual sugars found in dry wines by means of gas chromatography

Type IV method

 

  1. Scope

Simultaneous determination of the erythritol, arabitol, mannitol, sorbitol and meso-inositol content of wines.

Because the determination of sugars by gas chromatography (GC) is long and complicated, it is reserved for the determination of traces of sugars and, especially, of sugars for which no other routine enzyme method exists –(Arabinose, Rhamnose, Mannose and Galactose) although it is also applicable to glucose and fructose, the advantage being that it is possible to simultaneously determine all sugar monomers, dimers and even trimers.

Comment 1 - It is not possible to determine sugars once they have been reduced to alditol form because of the presence of corresponding polyols.

Comment 2 - In the form of trimethylsilylated derivatives (TMS), sugars give 2 α and β forms and occasionally 3 or 4 (Gamma…) corresponding to the different anomers present in wines.

Comment 3 - Without prior dilution, it is difficult to determine glucose and fructose content using this method when it exceeds 5 g/l.

  1. Principle

Residual sugars in dry wines can be determined by gas chromatography after the formation of their trimethylsilylated derivatives.

The internal standard is pentaerythritol.

  1. Reagents

 

Silane mixture for example purposes:

3.1.  Pure hexamethyldesilazane (HMDS)

3.2.  Pure trifluoroacetic anhydride (TFA)

3.3.  Pure pyridine

3.4.  Pure pentaerythritol

3.5.  Distilled water

3.6.  10 g/l pentaerythritol (internal standard solution): dissolve 0.15 g of pentaerythritol (3.4) in 100 ml of water (3.5)

3.7.  Pure products that may be used to prepare control solutions, notably glucose, fructose, arabinose, mannitol and sorbitol (non-exhaustive list)

3.8.  Control solutions of pure products at 200 mg/l: dissolve 20 mg of each of the products to be determined (3.6) in 100 ml of water.

Comment – Sugar solutions should be prepared immediately prior to use.

  1. Apparatus and Equipment

4.1.  1-ml pipettes, with 1/10th ml graduations

4.2.  Propipette™ bulbs

4.3.  100- μl syringe

4.4.  5-ml tubes with screw stoppers fitted with a Teflon-coated sealing cap.

4.5.  Rotary vacuum evaporator capable of housing screw-cap test tubes (4.4) in order to evaporate samples to dryness

4.6.  Gas chromatograph fitted with a flame ionisation detector x g, and an injector operating in "split" mode - 1/30th to 1/50th division of the injected volume (1 μl)

4.7.  Non-polar capillary column (SE-30, CPSil-5, HP-1, etc.) 50 m x 0.25 mm, 15 mμ stationary phase film thickness (as an example).

4.8.  10- μl injection syringe

4.9.  Data acquisition system

4.10.        Ultra-sonic bath

4.11.        Laboratory fume cupboard

  1. Preparation of samples

5.1.  Addition of the internal standard: 1 ml of wine (pipette, 4.1) or of 200 mg/l control solution (3.6) is placed in the screw-cap test tube (4.4)

Note: It is possible to operate with lower volumes of wine especially in high content sugar environments.

50 μl of the 10 g/l pentaerythritol solution (3.5) is added by means of the syringe (4.3)

5.2.  Obtaining dry solid matter:

The screw-cap test tube is placed on the rotary evaporator, with a water bath kept below 40°C. Evaporation continues until all traces of liquid have disappeared.

5.3.  Addition of reagents

5.3.1.      Place the tubes containing the dry solid matter and reagents 3.1, 3.2 and 3.3 in the fume cupboard (4.11) and switch on the ventilation.

5.3.2.      Using the pipettes (4.1) and Propipette™ bulbs (4.2), add 0.20 ml of pyridine (3.3), 0.7 ml of HMDS (3.1) and 0.1 ml of TFA (3.2) to the test tube one after the other.

5.3.3.      Seal the test tube with its stopper.

5.3.4.      Put the test tube in the ultra-sonic bath (4.10) for 5 minutes until the dry solid matter has completely dispersed.

5.3.5.      Place the test tube in a laboratory kiln at 60°C for two hours in order to obtain the total substitution of the hydroxyl or acid hydrogen by the trimethylsilyl groups (TMS).

Comment: a single phase only should remain after heating (if not, water would be left in the test tube). Likewise, there should be no brownish deposit, which would indicate an excess of non-derived sugar.

  1. Chromatographic assay

6.1.  Place the cooled test tube in the ventilated fume cupboard (4.11), remove 1 μl

 with the syringe (4.8) and inject into the chromatograph in "split" mode (permanent split).

Treat the wine-derived and control sample in the same way.

6.2.  Programme the kiln temperature, for example from 60°C to 240°C at a rate of 3°C per minute, such that the complete assay lasts, for example, one hour for complete mannitol and sorbitol separation (resolution higher than 1.5).

  1. Calculations

Example: calculation of sorbitol concentration

If 

s = the peak area of the sorbitol in the wine

S = the peak area of the sorbitol in the control solution

i = the peak area of the internal standard in the wine

I = the peak area of the internal standard in the control solution

The sorbitol content of the wine (ts) will be 

The same logic makes it possible to calculate the glucose content (tg)

when g is the sum of the areas of the two peaks of glucose in the wine and G is the sum of the areas of the two peaks of glucose in the control solution.

  1. Characteristics of the method

 

Detection threshold approximately 5 mg/l for a polyol (a single chromatographic peak). Average repeatability in the region of 10% for a sugar or polyol concentration in the region of 100 mg/l.

Table 1 Repeatability of the determination of a number of substances found in the dry solid matter of wine after TMS derivatization.

Tartaric acid

Fructose

Glucose

Mannitol

Sorbitol

Dulcitol

Meso-inositol

Average (mg/l)

2013

1238

255

164

58

31

456

Typical variance(mg/l)

184

118

27

8

2

2

28

CV (%)

9

10

11

5

3

8

6

Bibliography

  • RIBEREAU-GAYON P. and BERTRAND A. 1972, Nouvelles applications de la chromatographie en phase gazeuse à l’analyse des vins et au contrôle de leur qualité, Vitis, 10, 318-322.
  • BERTRAND A. (1974), Dosage des principaux acides du vin par chromatographie en phase gazeuse. FV OIV 717—718, 253—274.
  • DUBERNET M.0. (1974), Application de la chromatographie en phase gazeuse à l’étude des sucres et polyols du vin: thèse 3° Cycle, Bordeaux.

Figure 1 Chromatogram of a white wine following silylation. CPSil-5CB 50 m x 0.25 mm x 0.15 μm column. Split injection, 60°C, 3°C/min, 240°C. Magnification below.

 

Identification of peaks: 1 : reactive mixture; 2 and 3: unknown acids; 4: pentaerythriol; 5 and 6: unknown; 7: tartaric acid and arabinose; 8, 10 and 11: rhamnose; 9: arabinose; 12: xylitol; 13: arabitol; 14, 15 and 16: fructose; 17: galactose and unknown; 18: glucose α; 19: galactose and galacturonic acid; 20 and 21: unknown; 22: mannitol; 23: sorbitol; 24: glucose β; 25 and 27: unknown; 26: galacturonic acid; 28 and 30: galactonolactone; 29: mucic acid; 31: meso-inositol.

Chromatogram of a white wine following silylation. CPSil-5CB 50 m x 0.25 mm x 0.15 μm column. Split injection, 60°C, 3°C/min, 240°C. Magnificat


Glucose and fructose (pHmetry) (Type-III)

OIV-MA-AS311-07 Joint determination of the glucose and fructose content in wines by differential ph-metry

Type III method

  1. Scope

This method is applicable to the analysis of glucose and fructose in wines between 0 and 60 g/L (average level) or 50 and 270 g/L (high level).

  1. Principle

The joint determination of glucose and fructose content by differential pH-metry consists in the phosphorylation of the glucose and fructose by hexokinase. The ions generated stoechiometrically in relation to the quantities of glucose and fructose are then quantified.

  1. Reactions

The glucose and fructose present are phosphorylated by adenosine triphosphate (ATP) during an enzymatic reaction catalysed by hexokinase (HK) (EC. 2.7.1.1)

  1. Reagents

4.1.  Demineralised Water (18 M) or bi-distilled

4.2.  2-Amino-2-(hydroxymethyl)propane-1,3-diol (TRIS) purity 99%

4.3.  Disodic adenosine triphosphate (ATP, 2Na) purity 99%

4.4.  Trisodium phosphate with twelve water molecules (Na3PO4∙12H2O) purity 99%

4.5.  Sodium hydroxide (NaOH) purity 98%

4.6.  Magnesium chloride with six water molecules (Mg∙ purity 99%

4.7.  Triton X 100

4.8.  Potassium chloride (KCl) purity 99%

4.9.  2-Bromo-2-nitropropane-1,3-diol (Bronopol) ()

4.10.        Hexokinase (EC. 2.7.1.1) 1 mg