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.

2. Principle of the method

The wine is treated with one of the following reagents:

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

3. 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.
  2.       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
  2.       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.

4. 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.

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.

2. 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.

3. 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.

4. 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.

5. 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

x_i = 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.

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).

2. Apparatus

Distillation apparatus, with a 100 mL round distillation flask.

3. Reagents

 

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

3.2.  Acetone (propanone).

4. 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.

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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).

2. 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.

3. 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)

4. 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 145 U (e.g. Hofmann La Roche, Mannheim,  Germany ref. Hexo-70-1351)

4.11.        Glycerol purity 98%

4.12.        Glucose purity 99%

4.13.        Reaction buffer pH 8.0 commercial or prepared according to the following method:

In a graduated 100-ml flask (5.2) pour roughly 70 ml (5.3) of water (4.1), and continuously stir (5.5). Add 0.242 g 0.001 g (5.4) of TRIS (4.2), 0.787 g 0.001 g (5.4) of ATP (4.3), 0.494 g 0.001 g (5.4) of sodium phosphate (4.4), 0.009 mg 0.001 g (5.4) of sodium hydroxide (4.5), 0.203 g 0.001 g (5.4) of magnesium chloride (4.6), 2.000 0.001 g (5.4) of Triton X 100 (4.7), 0.820 g 0.001 g (5.4) of potassium chloride (4.8) and 0.010 0.001 g (4.9) of bronopol. Adjust to volume with water (4.1). The final pH must be 8.0 0.1 (5.6), otherwise adjust it with sodium hydroxide or hydrochloric acid. The buffer thus prepared is stable for two months at 4°C.

4.14.        Enzyme solution commercial or prepared according to the following method: Using a graduated pipette (5.7) place 5 ml of glycerol (4.11) into a graduated 10-ml flask, adjust to volume with water (4.1) and homogenize. Dissolve 20 mg 1 mg (5.4) of hexokinase (4.10) and 5 mg of Bronopol (4.9) in 10 ml of the glycerol solution. The activity of the enzyme solution must be 300 U 50 U per ml for the hexokinase. The enzyme solution is stable for 6 months at 4°C.

4.15.        Preparation of the calibration solution (average level) if the supposed content is less than 50 g/L of glucose + fructose)

Place 3.60 g 0.01 g (5.4) of glucose (4.12) (desiccated 12 hours beforehand at 40 °C until constant weight), 0.745 g 0.001 g (5.4) of potassium chloride (4.8) and 0.010 g 0.001 g of bronopol (4.9) in a graduated 100-ml flask (5.2). Add water (4.1). Fully homogenize (5.5). Adjust to volume with water (4.1) after removing the magnetic bar. The final concentration is 36 g/L of glucose. The solution is stable for 6 months at 4 °C.

4.16.        Preparation of the calibration solution (high level) if the supposed content is above 50 g/L of glucose + fructose)

Place 18.0 g 0.01 g (5.4) of glucose (4.12) (desiccated 12 hours beforehand at 40 °C until constant weight), 0.745 g 0.001 g (5.4) of potassium chloride (4.8) and 0.010 g 0.001 g of bronopol (4.9) in a graduated 100-ml flask (5.2). Add water (4.1). Fully homogenize (5.5). Adjust to volume with water (4.1) after removing the magnetic bar. The final concentration is 180 g/L of glucose. The solution is stable for 6 months at 4°C.

5. Apparatus

5.1.  Differential pH-metry apparatus (EUROCHEM CL 10 plus, Microlab EFA or equivalent) see appendix A

5.2.  Graduated 100-ml flask, class A

5.3.  Graduated 100-ml test-tube with sole

5.4.  Precision balance to weigh within 1 mg

5.5.  Magnetic stirrer and magnetic Teflon bar

5.6.  pH-meter

5.7.  Graduated 3-mL, 5-mL pipettes, class A

5.8.  Graduated 10-ml flask, class A

5.9.  Automatic syringe pipettes, 25 and 50 μL

6. Preparation of samples

The samples should not be too charged with suspended matter; in the contrary case, centrifuge or filter them. Sparkling wines must be degassed.

7. Procedure

The operator must respect the instructions for use of the equipment (5.1). Before any use, the instrument must be stabilized in temperature. The circuits must be rinsed with the buffer solution (4.13) after cleaning, if required.

7.1.  Determination of the blank (determination of the enzyme signal)

Fill the electrode compartments (EL₁ and EL₂) of the differential pH-meter (5.1) with the buffer solution (4.13); the potential difference between the two electrodes (D₁) must range between 150 mpH;

Add 24 μL of enzyme solution (4.14) to the reaction vessel (using the micropipette 5.9 or the preparer) and fill electrode EL₂;

Measure the potential difference (D₂) between the two electrodes;

Calculate the difference in pH, for the blank using the following formula:

where

= the difference in pH between two measurements for the blank;

D₁ = the value of the difference in pH between the two electrodes filled with the buffer solution;

D₂ = the value of the difference in pH between the two electrodes, one of which is filled with the buffer solution and the other with the buffer solution and enzyme solution.

The value of ΔpH_o is used to check the state of the electrodes during titration as well as their possible drift over time; it must lie between –30 and 0 mpH and 1.5 mpH between two consecutive readings. If not, check the quality of the buffer pH and the cleanliness of the hydraulic system and electrodes, clean if necessary and then repeat the blank.

7.2  Calibration

7.2.1  Average level

Fill the electrode compartments (EL₁ and EL₂) with the buffer solution (4.13);

Add 25 μL (with the micropipette 5.9 or the preparer) of the standard glucose solution (4.15) to the reaction vessel;

Fill the electrodes EL₁ and EL₂ with the buffer + standard solution;

Measure the potential difference (D₃) between the two electrodes;

Add 24 μL of enzyme solution (4.14) and fill electrode EL₂ with the buffer + standard solution + enzyme;

After the time necessary for the enzymatic reaction, measure the potential difference (D₄) between the two electrodes;

Calculate the difference in pH, ΔpH_C for the calibration sample using the following formula:

ΔpHC =( ΔpHo

where

ΔpH_C = the difference between two measurements D₃ and D₄ for the calibration sample minus the difference obtained for the blank;

D₃ = the value of the difference in pH between the two electrodes filled with the reference buffer/solution mixture;

D₄ = the value of the difference in pH between the two electrodes, one of which is filled with the reference buffer/solution and the other with the buffer/ enzyme / reference solution.

Calculate the slope of the calibration line:

where

• is the concentration of glucose in the standard solution expressed in g/L.

Check the validity of the calibration by analysing 25 μL of standard solution (ML) of glucose (4.15) according to the procedure (7.3). The result must range between 2% of the reference value. If not, repeat the calibration procedure.

7.2.  High level

Fill the electrode compartments (EL₁ and EL₂) with the buffer (4.13);

Add 10 μL (with the micropipette 5.9 or the preparer) of standard solution (HL) of glucose (4.16) to the reaction vessel;

Fill the electrodes EL₁ and EL₂ with the buffer + standard solution mixture;

Measure the potential difference (D₃) between the two electrodes;

Add 24 μL of enzyme solution (4.14) and fill electrode EL₂ with

• the buffer + standard solution + enzyme mixture;

After the time required for the enzymatic reaction, measure the potential difference (D₄) between the two electrodes;

Calculate the difference in pH, for the calibration sample using the following formula:

where

= the difference in pH between two measurements D₃ and D₄ for the calibration sample minus the difference obtained for the blank;

D₃ = the value of the difference in pH between the two electrodes filled with the buffer/ reference solution mixture;

D₄ = the value of the difference in pH between the two electrodes, one of which is filled with the buffer/ reference solution and the other with the buffer/ reference solution /enzyme.

Calculate the slope of the calibration line:

where

• C_u is the concentration of glucose in the standard solution expressed in g/L.

Check the validity of the calibration by analysing 10 µL of standard solution of glucose (4.16) in accordance with procedure (7.3). The result must range between 2% of the reference value. If not, repeat the calibration procedure.

7.3.  Quantification

Fill the electrode compartments (EL₁ and EL₂) with the buffer solution (4.13)

Add 10 μL (high level) or 25 μL (mean level) (with the micropipette 5.9 or the preparer) of the sample solution to the reaction vessel;

Fill electrodes EL₁ and EL₂with the buffer + sample mixture;

Measure the potential difference (D₅) between the two electrodes;

Add 24 μL of the enzyme solution (4.14) and fill electrode EL₂ with the buffer mixture + sample + enzyme;

Measure the potential difference (D6) between the two electrodes;

Calculate the quantity of aqueous solution in the sample using the following formula:

where

w = the quantity of aqueous solution in the sample (in g/L);

S is the slope determined by the calibration line;

ΔpH_o = the difference in pH between two measurements for the blank;

D₅ = the value of the difference in pH between the two electrodes filled with the sample/ reference solution;

D₆ = the value of the difference in pH between the two electrodes, one of which is filled with the buffer/sample and the other with the buffer/ sample /enzyme.

8. Expression of results

The results are expressed in g/L of glucose + fructose with one significant figure after the decimal point.

9. Precision

The details of the interlaboratory test on the precision of the method are summarized in appendix B.

9.1.  Repeatability

The absolute difference between two individual results obtained in an identical matter tested by an operator using the same apparatus, in the shortest interval of time possible, shall not exceed the repeatability value r in 95% of the cases.

The value is: r = 0.021x + 0.289 where x is the content in g/L of glucose + fructose

9.2.  Reproducibility

The absolute difference between two individual results obtained with an identical matter tested in two different laboratories, shall not exceed the reproducibility value of R in 95% of the cases.

The value is: R = 0.033x + 0.507 where w is the content in g/L of glucose + fructose

10. Other characteristics of the analysis

10.1.        Detection and quantification limits

10.1.1. Detection limit

The detection limit is determined by using 10 series of three repetitions of an analytical blank and linear regression carried out with the wines of the precision test; it is equal to three standard deviations. In this case, the method gave as a result a detection limit of 0.03 g/L. Tests by successive dilutions confirmed this value.

10.1.2. Quantification limit

The quantification limit is determined by using 10 series of three repetitions of an analytical blank and linear regression carried out with the wines of the precision test; it is equal to ten standard deviations. In this case, the method gave as a result a quantification limit of 0.10 g/L. Tests by successive dilutions confirmed this value. The quantifications of white and red wine carried out by the laboratories that took part in the interlaboratory analysis also confirm these figures.

10.2.        Accuracy

Accuracy is evaluated based on the average coverage rate calculated for the loaded wines analysed double-blind during the interlaboratory test (wines A, B, C, D, F and J). It is equal to 98.9% with a confidence interval of 0.22%.

11. Quality control

Quality controls can be carried out with certified reference materials, wines whose characteristics have been determined by consensus, or loaded wines regularly used in analytical series, and by following the related control charts.

Appendix A Diagram of the differential pH-metry apparatus

A: differential amplifier; B: buffer solution; C: mixing chamber; D: indicator; EL1 and EL2 capillary electrodes; EL: electronics; G: ground; K: keyboard; M: magnetic stirrer; P: printer; P1 to P3: peristaltic pumps; S: injection syringe for the sample and enzyme; W: waste.

Appendix B Statistical data obtained with the interlaboratory test results
 

In accordance with ISO 5725-2:1994, the following parameters were defined during an interlaboratory test. This test was carried out by the laboratory of the Inter-trade Committee for Champagne Wine in Epernay (France).

Year of the interlaboratory test: 2005

Number of laboratories: 13 double blind

Number of samples: 10

	Wine A	Wine B	Wine  C	Wine  D	Wine  E	Wine  F	Wine  G	Wine  H	Wine  I	Wine  J
Average in g/L	8.44	13.33	18.43	23.41	28.03	44.88	86.40	93.34	133.38	226.63
Number of laboratories	13	13	13	13	13	13	13	13	13	13
Number of laboratories after elimination of greatest dispersions	13	13	13	13	13	13	13	13	13	13
Standard deviation of repeatability	0.09	0.13	0.21	0.21	0.29	0.39	0.81	0.85	1.19	1.51
Repeatability limit	0.27	0.38	0.61	0.62	0.86	1.14	2.38	2.51	3.52	4.45
RSDr, 100%	1.08	0.97	1.13	0.91	1.04	0.86	0.94	0.91	0.89	0.67
HORRAT r	0.26	0.25	0.31	0.26	0.30	0.27	0.32	0.32	0.33	0.47
Standard deviation of reproducibility	0.17	0.27	0.37	0.59	0.55	0.45	1.27	1.43	1.74	2.69
Reproducibility limit	0.50	0.79	1.06	1.71	1.60	1.29	3.67	4.13	5.04	7.78
RSDR, 100%	2.05	2.05	1.99	2.54	1.97	1.00	1.47	1.53	1.31	1.19
HORRAT R	0.50	0.54	0.55	0.72	0.58	0.31	0.51	0.53	0.48	0.47

Types of samples:

Wine A: white wine naturally containing sugar, loaded with 2.50 g/L glucose and
of 2.50 g/L of fructose;

Wine B: white wine naturally containing sugar (wine A), loaded with 5.00 g/L glucose and 50 g/L of fructose;

Wine C: white wine naturally containing sugar (wine A), loaded with 7.50 g/L glucose and 7,50 g/L of fructose;

Wine D: white wine naturally containing sugar (wine A), loaded with 10.0 g/L glucose and 10.0 g/L of fructose;

Wine E: aromatised wine;

Wine F: white wine naturally containing less than 0.4 g/L of sugar, loaded with 22.50 g/L glucose and 22.50 g/L of fructose;

Wine G: naturally sweet red wine;

Wine H: sweet white wine;

Wine I: basis wine;

Wine J: white wine naturally containing less than 0.4 g/L of sugar, loaded with 115.00 g/L glucose and 115.00 g/L of fructose;

Bibliography

• LUZZANA M., PERELLA M. and ROSSI-BERNARDI L (1971): Anal. Biochem, 43, 556-563.
• LUZZANA M., AGNELLINI D., CREMONESI P. and CARAMENTI g. (2001): Enzymatic reactions for the determination of sugars in food samples using the differential pH technique. Analyst, 126, 2149 –2152.
• LUZZANA M., LARCHER R., MARCHITTI C. V. and BERTOLDI D. (2003): Quantificazione mediante pH-metria differenziale dell'urea negli spumanti metodo classico.in "Spumante tradizionale e classico nel terzo millennio" 27-28 giugno 2003, Istituti Agrario di San Mechele.
• MOSCA A., DOSSI g., LUZZANA M., ROSSI-BERNARDI L., FRIAUF W. S., BERGER R.L., HOPKINS H. P. and CAREY V (1981): Improved apparatus for the differential measurement of pH: application to the measurement of glucose. Anal. Biochem., 112, 287 – 294.
• MOIO L., GAMBUTI A., Di MARZIO L. and PIOMBINO P. (2001): Differential pHmeter determination of residual sugars in wine. Am. J. Enol. Vitic, 52(3), 271 – 274.
• TUSSEAU D., FENEUIL A., ROUCHAUSSE J.M. et VAN LAER S. (2004): Mesure différents paramètres d’intérêt œnologiques par pHmétrie différentielle. FV. OOIV 1199, 5 pages.

OIV-MA-AS311-08 Whole determination of glucose, fructose and saccharose content in wines by differential ph-metry

Type IV method

 

1. Scope

This method is applicable to the analysis of glucose and fructose in wines between 0 and 270 g/L.

This quantification is different from glucose and fructose quantification by its differential pH-metry which can not be substituted.

2. Principle

The determination by differential pH-metry of glucose, fructose and saccharose content consists in the preliminary hydroloysis of saccharose by invertase, followed by phosphorylation of the glucose and fructose by hexokinase. The H⁺ ions generated stoechiometrically in relation to the quantities of glucose and fructose are then quantified.

3. Reactions

Possible traces of saccharose are hydrolysed by invertase (EC 3.2.1.26)

The glucose and fructose initially or consecutively present to invertase action are phosphorylated by adenosine triphosphate (ATP) during an enzymatic reaction catalysed by hexokinase (HK) (EC. 2.7.1.1)

4. 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 ( ) 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.        Invertase (EC 3.2.1.26) 1 mg 500 U (ex Sigma ref I-4504)

4.11.        Hexokinase (EC. 2.7.1.1) 1 mg 145 U (e.g. Hofmann La Roche, Mannheim, Germany ref. Hexo-70-1351)

4.12.        Glycerol purity 98%

4.13.        Saccharose purity 99%

4.14.        Reagent buffer pH 8.0 commercial (ex. DIFFCHAMB GEN 644) or prepared according to the following method:

In a graduated 100-ml flask (5.2) pour roughly 70 ml (5.3) of water (4.1), and continuously stir (5.5). Add 0.242 g 0.001 g (5.4) of TRIS (4.2), 0.787 g 0.001 g (5.4) of ATP (4.3), 0.494 g  0.001 g (5.4) of sodium phosphate (4.4), 0.009 mg 0.001g (5.4) of sodium hydroxide (4.5), 0.203 g 0.001 g (5.4) of magnesium chloride (4.6), 2.000 0.001 g (5.4) of Triton X 100 (4.7), 0.820 g 0.001 g (5.4) of potassium chloride (4.8) and 0.010 0.001 g (4.9) of bronopol. Adjust to volume with water (4.1). The final pH must be 8.0 0.1 (5.6), otherwise adjust it with sodium hydroxide or hydrochloric acid. The buffer thus prepared is stable for two months at 4°C.

4.15.        Enzyme solution commercial or prepared according to the following method: Using a graduated pipette (5.7) place 5 ml of glycerol (4.11) into a graduated 10-ml flask, adjust to volume with water (4.1) and homogenize. Dissolve 300 mg 1 mg (5.4) of invertase (4.10) 10 mg 1 mg (5.4) of hexokinase (4.11) in 3 mL of glycerol solution. Enzyme solution activity must be 50 000 U 100 U per ml for intervase and 480 U 50 U for hexokinase. The enzyme solution is stable for 6 months at 4°C.

4.16.        Preparation of reference solution

Place 17,100 g 0.01 g (5.4) of saccharose (4.13) (desiccated 12 hours beforehand at 40 °C until constant weight), 0.745 g 0.001 g (5.4) of potassium chloride (4.8) and 0.010 g 0.001 g (5.4) of bronopol in a graduated 100-ml flask (5.2). Add water (4.1). Fully homogenize (5.5). Adjust to volume with water (4.1) after removing the magnetic bar. The final concentration is 171 g/L of saccharose. The solution is stable for 6 months at 4°C.

5. Apparatus

5.1.  Differential pH-metry apparatus (EUROCHEM CL 10 plus, Microlab EFA or equivalent) see appendix A

5.2.  Graduated 100-ml flask, class A

5.3.  Graduated 100-ml test-tube with foot

5.4.  Precision balance to weigh within 1 mg

5.5.  Magnetic stirrer and magnetic Teflon bar

5.6.  pH-meter

5.7.  Graduated 3-mL, 5-mL pipette, class A

5.8.  Graduated 10-ml flask, class A

5.9.  Automatic syringe pipettes, 25 and 50 μL

6. Preparation of samples

Samples must not contain excessive suspended matter. If this occurs, the solution centrifuge and filter. Sparkling wines must be degassed

7. Procedure

The operator must respect the instructions for use of the equipment (5.1). Before any use, the instrument must be stabilized in temperature. The circuits must be rinsed with the buffer solution (4.14) after cleaning, if required.

7.1.  Determination of the blank (determination of the enzyme signal)

Fill the electrode compartments (EL₁ and EL₂) of the differential pH-meter (5.1) with the buffer solution (4.14); the potential difference between the two electrodes (D₁) must range between 150 mpH;

Add 32 μL of enzyme solution (4.15) to the reaction vessel (using the micropipette 5.9 or the preparer) and fill electrode EL₂;

Measure the potential difference (D₂) between the two electrodes;

Calculate the difference in pH, for the blank using the following formula:

where

= the difference in pH between two measurements for the blank;

= the value of the difference in pH between the two electrodes filled with the buffer solution;

= the value of the difference in pH between the two electrodes, one of which is filled with the buffer solution and the other with the buffer solution and enzyme solution.

The value of is used to check the state of the electrodes during titration as well as their possible drift over time; it must lie between –30 and 0 mpH and 1.5 mpH between two consecutive readings. If not, check the quality of the buffer pH and the cleanliness of the hydraulic system and electrodes, clean if necessary and then repeat the blank.

7.2.  Calibration

Fill the electrode compartments (EL₁ and EL₂) with the buffer solution (4.14);

Add 10 μL (with the micropipette 5.9 or the preparer) of the standard saccharose solution (5) to the reaction vessel;

Fill the electrodes and with the buffer + standard solution;

Measure the potential difference (D₃) between the two electrodes;

Add 32 μL of enzyme solution (4.15) and fill electrode EL₂ with the buffer + standard solution + enzyme;

After the time necessary for the enzymatic reaction, measure the potential difference (D₄) between the two electrodes;

Calculate the difference in pH, for the calibration sample using the following formula:

where

= the difference between two measurements D₃ and D₄ for the calibration sample minus the difference obtained for the blank;

= the value of the difference in pH between the two electrodes filled with the reference buffer/solution mixture;

= the value of the difference in pH between the two electrodes, one of which is filled with the reference buffer/solution and the other with the buffer/ enzyme / reference solution.

Calculate the slope of the calibration line:

where

is the concentration of saccharose in the standard solution expressed in g/L.

Check the validity of the calibration by analysing 10 μL of standard solution (ML) of saccharose (5) according to the procedure (8.3). The result must range between 2% of the reference value. If not, repeat the calibration procedure.

7.3.  Quantification

Fill the electrode compartments ( and ) with the buffer solution (4.14)

Add 10 μL (with the micropipette 5.9 or the preparer) of the sample solution to the reaction vessel;

Fill electrodes and with the buffer + sample mixture;

Measure the potential difference (D₅) between the two electrodes;

Add 32 μL of the enzyme solution (4.15) and fill electrode with the buffer mixture + sample + enzyme;

Measure the potential difference (D6) between the two electrodes;

Calculate the quantity of aqueous solution in the sample using the following formula:

where

w = the quantity of aqueous solution in the sample (in g/L);

S is the slope determined by the calibration line;

= the difference in pH between two measurements for the blank;

= the value of the difference in pH between the two electrodes filled with the sample/ reference solution;

= the value of the difference in pH between the two electrodes, one of which is filled with the buffer/sample and the other with the buffer/ sample /enzyme.

8. Expression of results

The results are expressed in g/L of glucose with one significant figure after the decimal point.

9. Characteristics of the analysis

Due to the hydrolysis of saccharose in wines and musts, it is not possible to organise an inter-laboratory analysis according to the OIV protocol.

Inter-laboratory studies of this method demonstrate that for saccharose, the linearity between 0 and 250 g/l, a detection limit of 0.2 g/l, a quantification limit of 0.6 g/l, repeatability of 0.0837x -0.0249 g/l and reproducibility of 0.0935x -0.073 g/l (saccharose content).

10. Quality control

Quality controls can be carried out with certified reference materials, wines whose characteristics have been determined by consensus, or loaded wines regularly used in analytical series, and by following the related control charts.

Appendix A: Diagram of the differential pH-metry apparatus

A: differential amplifier; B: buffer solution; C: mixing chamber;  D: indicator; EL1 and EL2 capillary electrodes; EL: electronics; G: ground; K: keyboard; M: magnetic stirrer; P: printer; P1 to P3: peristaltic pumps; S: injection syringe for the sample and enzyme; W: waste.

Appendix B Bibliography

• LUZZANA M., PERELLA M. et ROSSI-BERNARDI L (1971) : Electrometric method for measurement of small pH changes in biological systems. Anal. Biochem, 43, 556-563.
• LUZZANA M., AGNELLINI D., CREMONESI P. et CARAMENTI G. (2001) : Enzymatic reactions for the determination of sugars in food samples using the differential pH technique. Analyst, 126, 2149 –2152.
• LUZZANA M., LARCHER R., MARCHITTI C. V. et BERTOLDI D. (2003) : Quantificazione mediante pH-metria differenziale dell'urea negli spumanti metodo classico.in "Spumante tradizionale e classico nel terzo millennio" 27-28 giugno 2003, Instituti Agrario di San Mechele.
• MOIO L., GAMBUTI A., Di MARZIO L. et PIOMBINO P. (2001) : Differential pHmeter determination of residual sugars in wine. Am. J. Enol. Vitic, 52(3), 271 – 274.
• MOSCA A., DOSSI G., LUZZANA M., ROSSI-BERNARDI L., FRIAUF W. S., BERGER R.L., HOPKINS H. P. et CAREY V (1981) : Improved apparatus for the differential measurement of pH : application to the measurment of glucose. Anal. Biochem., 112, 287 – 294.
• TUSSEAU D., FENEUIL A., ROUCHAUSSE J.-M. et VAN LAER S. (2004) : Mesure de différents paramètres d'interêt oenologique par pHmétrie différentielle. F.V. O.I.V. n° 1199, 5 pages.

OIV-MA-AS311-09 Determination of the isotope ratios of glucose, fructose, glycerol, ethanol in production of vitivinicultural origin by high-performance liquid chromatography coupled to isotope ratio mass spectrometry

Type II and III method

1. Scope of application

This method applies to products of vitivinicultural origin.

This method is:

• type II for glucose, fructose and glycerol,
• type III for ethanol.

2. Principle

The samples are injected into the HPLC instrument after any necessary dilution and filtration. After oxidation in a liquid interface, the ¹³C/¹²C isotope ratio of the compounds is determined using isotope ratio mass spectrometry. This liquid interface, symbolised by the acronym “co”, permits the chemical oxidation of the organic matter into CO2. HPLC-co-IRMS coupling can therefore be used to determine the isotope ratio of the following compounds simultaneously: glucose, fructose, glycerol and ethanol.

3. Reagents

 

3.1.  Pure water - resistivity > 18 M cm, HPLC quality

3.2.  Ammonium persulfate - analytical purity – [CAS No.: 7727-54-0]

3.3.  Orthophosphoric acid (concentration 85%) – analytical purity - [CAS No.: 7664-38-2]

3.4.  Analytical-grade helium, used as a carrier gas [CAS No.: 07440-59-7]

3.5.  Reference gas: analytical-grade (carbon dioxide), used as a secondary reference gas [CAS No.: 00124-38-9]

3.6.  International standards

4. Equipment

 

4.1.  Everyday laboratory equipment

4.2.  High-performance liquid chromatography instrument

4.3.  Liquid interface for the oxidation of eluted compounds

4.4.  Isotope ratio mass spectrometer

5. Analysis of the samples

5.1.  Preparation of the samples

Depending on the sugar, glycerol and ethanol contents, the samples should be diluted with the water (3.1) beforehand in order to obtain a concentration which is observable under the experimental conditions. Depending on the concentrations of the compounds, two measurements are needed with different dilutions.

5.2.  Example of analytical conditions

Total analysis duration: 20 minutes

As an indication, the dilution of grape juices and wines is around 1:200, while that of concentrated musts is approximately 1:500.

HPLC:

Column: carbohydrate-type column (e.g. 700-CH Carbohydrate column, HyperRez XP Carbohydrate H⁺)

Injection volume: 25 μl

Mobile phase: water (3.1)

Flowrate: 0.4 mL/min

Column T°: 80 °C

Liquid Interface:

Solution of ammonium persulfate (3.2) (15% in mass) and orthophosphoric acid (2.5% in volume)

Peristaltic pump flow: 0.6 mL/min

Heater temperature: 93 °C

Flow of the helium carrier gas: 15 mL/min

Helium flow for drying: 50 mL/min

IRMS:

Trap current: 300 μA

5.3.  Example chromatogram

Chromatogram of a sweet wine analysed using HPLC-co-IRMS

6. Determination of isotope ratios

 

 

The reference gas, CO₂, is calibrated from international commercial standards. The isotope ratios are expressed in  ‰ in relation to the Pee Dee Belemnite (PDB) and are defined as:

*

Where: Sam = sample; St = standard; R = ¹³C/¹²C isotope ratio

7. Method characteristics

The characteristics of the method for the measurement of the C isotope ratios of glucose, fructose, glycerol and ethanol by HPLC-co-IRMS have been determined from the results obtained from an inter-laboratory analysis of four samples (dry wine, sweet wine, grape juice and rectified concentrated must). The results obtained for each compound analysed and each type of matrix are annexed.

8. Bibliography

• Cabanero, AI.; Recio, JL.; Rupérez, M. (2008) Isotope ratio mass spectrometry coupled to liquid and gas chromatography for wine ethanol characterization. Rapid Commun. Mass Spectrom. 22: 3111-3118.
• Cabanero, AI.; Recio, JL.; Rupérez, M. (2010) Simultaneous stable carbon isotopic analysis of wine glycerol and ethanol by liquid chromatography coupled to isotope ratio mass spectrometry. J. Agric. Food Chem. 58: 722-728.
• Guyon, F.; Gaillard, L.; Salagoïty, MH.; Médina, B. (2011) Intrinsic Ratios of Glucose, Fructose, Glycerol and Ethanol ¹³C/¹²C Isotopic Ratio Determined by HPLC-co-IRMS: Toward Determining Constants for Wine Authentication. Anal. Bioanal. Chem. 401:1551-1558

Annex Statistical treatment of the HPLC-co-IRMS inter-laboratory analysis  for the determination of the precision of the method (repeatability and reproducibility)

List of laboratories in alphabetical order of country of origin.

Country	Laboratory
Belgium	IRMM
China	CNRIFFI
Czech Republic	SZPI
France	SCL-33
Germany	INTERTEK
Germany	UNI DUE
Germany	ELEMENTAR
Germany	QSI
Germany	LVI
Italy	FLORAMO
Japan	AKITA Univ.
Spain	MAGRAMA

Responses:

12 laboratories / 14 responses

Treatment of the results of inter-laboratory analyses according to ISO 5725-2

Samples:

• 1 dry wine (Wine A)
• 1 sweet wine (Wine B)
• 1 rectified concentrated must (RCM)
• 1 grape juice

Analytical conditions:

Each sample was analysed in duplicate (repeatability) and double blind (reproducibility)

Expression of results in % vs. PDB

Precision of the glucose measurement

Repeatability and reproducibility

	Wine B	RCM	Grape juice
Number of laboratories	12	12	12
Number of responses	14	13	14
Number of responses retained (elimination of outliers)	13	13	12
Minimum value	-26.33	-25.04	-25.78
Maximum value	-23.72	-23.74	-24.62
Mean value	-25.10	-24.24	-25.19
Repeatability variance	0.02	0.01	0.01
Repeatability standard deviation (Sr)	0.14	0.10	0.09
Repeatability limit (r ‰)	0.40	0.29	0.24
Reproducibility variance	0.39	0.14	0.11
Reproducibility standard deviation (SR)	0.62	0.38	0.33
Reproducibility limit (R ‰)	1.77	1.06	0.94

Precision of the fructose measurement

Repeatability and reproducibility

	Wine B	RCM	Grape juice
Number of laboratories	12	11	12
Number of responses	14	13	14
Number of responses retained (elimination of outliers)	13	13	13
Minimum value	-25.56	-24.19	-25.33
Maximum value	-24.12	-23.19	-23.98
Mean value	-24.87	-23.65	-24.56
Repeatability variance	0.02	0.03	0.02
Repeatability standard deviation (Sr)	0.14	0.16	0.14
Repeatability limit (r ‰)	0.40	0.46	0.39
Reproducibility variance	0.15	0.10	0.18
Reproducibility standard deviation (SR)	0.39	0.32	0.42
Reproducibility limit (R ‰)	1.10	0.90	1.19

Precision of the glycerol measurement

Repeatability and reproducibility

	Wine A	Wine B
Number of laboratories	12	12
Number of responses	12	12
Number of responses retained (elimination of outliers)	11	11
Minimum value	-32.91	-30.74
Maximum value	-30.17	-28.27
Mean value	-31.75	-29.54
Repeatability variance	0.13	0.04
Repeatability standard deviation (Sr)	0.36	0.19
Repeatability limit (r ‰)	1.03	0.55
Reproducibility variance	0.57	0.37
Reproducibility standard deviation (SR)	0.76	0.61
Reproducibility limit (R ‰)	2.14	1.72

Precision of the ethanol measurement

Repeatability and reproducibility

	Wine A	Wine B
Number of laboratories	12	12
Number of responses	11	12
Number of responses retained (elimination of outliers)	10	12
Minimum value	-27.85	-27.60
Maximum value	-26.50	-26.06
Mean value	-27.21	-26.82
Repeatability variance	0.03	0.03
Repeatability standard deviation (Sr)	0.16	0.17
Repeatability limit (r ‰)	0.47	0.47
Reproducibility variance	0.16	0.23
Reproducibility standard deviation (SR)	0.40	0.47
Reproducibility limit (R ‰)	1.14	1.34

OIV-MA-AS311-10 Determination of D-glucose and D-fructose in wines by automated enzymatic method

Type III method

 

1. Scope of application

This method makes it possible to determine the sum of D-glucose and D-fructose in wine by specific enzyme analysis using an automatic sequential analyser.

In this document a collaborative study is reported which demonstrates application of the method for measurement of D-glucose and D-fructose from 0.1 to 96.31 g/L, taking into account the introduction of a dilution of the sample above 5 g/L.

Note: Where necessary, each laboratory using this method may refine, and potentially widen, this range through a validation study.

2. Standard references

OIV Compendium of International Methods of Analysis: Glucose and fructose – enzymatic method, OIV-MA-AS311-02,

ISO 78-2: Chemistry – Layouts for standards.

3. Reaction principles

D-glucose and D-fructose are phosphorylated by adenosine triphosphate (ATP) during an enzymatic reaction catalysed by hexokinase (HK) to produce glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P).

Glucose-6-phosphate is first oxidised 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) is directly correlated with that of glucose-6-phosphate and thus with that of D-glucose.

Fructose-6-phosphate (F6P) is converted into glucose-6-phosphate (G6P) in the presence of phosphoglucose isomerase (PGI):

The glucose-6-phosphate thus formed reacts as shown in the above formula.

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) produced is measured based on its absorption at 340 nm.

4. Reagents and working solutions

During the analysis – unless stated otherwise – only use reagents of recognised analytical grade and water that is distilled, demineralised or of equivalent purity.

4.1.  Reagents

4.1.1.     Quality I or II water for analytical usage (ISO 3696 standard)

4.1.2.     Triethanolamine hydrochloride (CAS no. 637-39-8)

4.1.3.     NADP (nicotinamide adenine dinucleotide phosphate) (CAS no. 24292-60-2)

4.1.4.     ATP (adenosine-5'-triphosphate) (CAS no. 34369-07-8)

4.1.5.     MgSO4 (anhydrous magnesium sulphate) (CAS no. 7487-88-9

4.1.6.     Sodium hydroxide (CAS no. 1310-73-2)

4.1.7.     Hexokinase (HK) (CAS no. 9001-51-8)

4.1.8.     Glucose-6-phosphate dehydrogenase (G6PDH) (CAS no. 9001-40-5)

4.1.9.     Phosphoglucose isomerase (PGI): lyophilised powder, 400-600 units/mg protein (CAS no. 9001-41-6)

Note: One unit ensures the conversion of 1.0 μmole of D-fructose-6-phosphate into D-glucose-6-phosphate per minute at pH 7.4 and 25 °C

4.1.10. Polyvinylpyrrolidone (PVP) (CAS no. 9003-39-8

4.1.11. D-glucose: purity 99.5% (CAS no. 50-99-7)

4.1.12. D-fructose: purity 99% (CAS no. 57-48-7)

Note 1: There are commercial kits for the determination of D-glucose and D-fructose. The user needs to check the composition to ensure it contains the above-indicated reagents.

Note 2: The use of PVP is recommended to eliminate any possible negative effect of tannins in wine on the enzyme protein molecules. This is the case particularly in red wines. Should the use of PVP not prove effective, the laboratory should ensure that the wine tannins do not interfere with the enzymes.

4.2.  Working solutions

4.2.1.     Triethanolamine hydrochloride buffer and magnesium sulphate adjusted to pH 7.6. The preparation may be as follows:

• triethanolamine hydrochloride (4.1.2): 11.2 g,
• magnesium sulphate (4.1.5): 0.2 g,
• PVP (4.1.10): 2 g,
• water for analytical usage (4.1.1): 150 mL.

The mixture is adjusted to pH 7.6 using a 5 M sodium hydroxide solution, then made up to 200 mL with water for analytical usage. The solution is stable for at least 4 weeks at 2-8 °C.

4.2.2.     R1 working solution (example):

• triethanolamine buffer (4.2.1): 50 mL,
• NADP (4.1.3): 117 mg,
• ATP (4.1.4): 150 mg.
  3.      R2 working solution (example):

• triethanolamine buffer (4.2.1): 2 mL,
• HK (4.1.7): 270 U,
• G6PDH (4.1.8): 340 U,
• PGI (4.1.9): 640 U.

Note: Commercial preparations of a HK/6GPDH mixture may be used.

Note: When preparing these solutions, they should be mixed gently to prevent foam from forming. The life cycle of the working solutions is limited and should be evaluated and respected by the laboratory.

Calibration solutions

To ensure the closest possible connection to the International System of Units (SI), the calibration range should be created using pure solutions of D-glucose and D-fructose prepared by weighing and covering the measurement range.

5. Apparatus

5.1.  Analyser

5.1.1.     Equipment type

Automatic sequential analyser equipped with a spectrophotometer with UV detector. The reaction temperature should be stable (around 37 °C). The reaction cuvettes are glass, methacrylate or quartz. The equipment is controlled by software ensuring its operation, data acquisition and useful calculations.

5.1.2.     Absorbance reading

The concentration of the analytes directly relates to the absorbance difference read by the spectrophotometer. The precision of the absorbance reading should be a minimum of 0.1 absorbance unit (AU). It is preferable not to use absorbance values higher than 2.0.

5.1.3.     Precision of volumes collected

The precision of the volumes of reagents and samples collected by the pipettes of the analyser influences the measurement result. Quality control of the results using appropriate strategies (e.g. according to the guides published by the OIV) is recommended.

5.1.4.     Reaction duration and temperature

In general, the reaction time is 10 minutes and the temperature is 37 °C. Certain pieces of apparatus may use slightly different values.

5.1.5.     Wavelength

The wavelength of maximum absorption of the NADPH formed by the reaction is 340 nm. This wavelength will be selected for the spectrophotometers commonly used. Some analysers are equipped with photometers that use a mercury-vapor lamp. In this case, a wavelength with a reading of 365 or 334 nm is to be selected.

5.2.  Balance

This should be calibrated to the International System of Units and have 1 mg precision.

5.3.  pH meter

5.4.  Measuring glassware

The measuring glassware for the preparation of reagents and calibration solutions is class A.

6. Sampling

6.1.  Preparation of samples of musts and wines

The majority of wine and must samples may be analysed without preparation. In some cases, a preparation may be introduced:

filtration should be used for highly turbid samples,

sample dilution (manual or automatic) with water for analytical usage (4.1.1) should be used for values exceeding the measurement range. By way of example, factors of 10x, 20x or 40x are used for musts. Given their impact on the uncertainty budget, these dilutions should be conducted with the utmost care.

 

6.2.  Preparation of samples of wines containing CO₂

Wine samples containing CO₂ may produce bubbling effects. They must be degassed beforehand by stirring under vacuum, ultrasonic processing or any method enabling the required degassing.

7. Procedure

Given that different analysers may be used, it is recommended that the conditions of use provided by the manufacturer are strictly observed. This also applies to the different enzymatic kits available on the market.

The procedure takes place as follows:

1.The sample (S) is placed in a reaction cuvette.

2.Working solution R1 (4.2.2) is then added to the cuvette.

3.The two are mixed together. Time is then allowed for a lag period, in order to guarantee absorbance stability. This lag period may last from 1-5 min, and is defined by the laboratory, according to the characteristics of the equipment used.

Working solution R2 (4.2.3) is added and the reaction takes place.

By way of example, the quantities of different elements may be as follows:

• sample: 2.0 μL,
• R1: 40 μL,
• R2: 40 μL.

The equipment takes regular measurements (every 12 seconds, for example) that make it possible to obtain a reaction curve, an example of which is given in Figure 1.

Figure 1

The equipment makes it possible to choose the reading points for the difference in absorbance sought, for example A and B in Figure 1.

8.      Calculation of results

The measurement used for the determination of the result is as follows:

In order to correlate this value with the desired concentration of D-glucose and D-fructose, calibration of the equipment is carried out using the calibration solutions at a minimum of 3 points (§4.3) covering the measurement range. In addition, a reagent blank is used comprising all of the reagents but no sample (point 0 of the calibration).

Figure 2: Calibration curve

The calibration curve can be order 1 (Concentration = a. + b) or even order 2 (Concentration = a. .+ b. . + c). If using a calibration curve of order 2, the laboratory should take care to limit the calibration domain in order to maintain sufficient sensitivity of the method (risk of crushing the curve).

The final value obtained should be multiplied by any coefficient of dilution used.

9.      Expression of results

The D-glucose + D-fructose results are expressed in g/L to 2 d.p.

10    Precision

 

Interlaboratory reproducibility

RSD_R = 5% (from 1 g/L)

CV_R% (k=2) = 2·RSD_R= 10%, (from 1 g/L)

Repeatability

= 1.5% (from 1 g/L)

% (k=2) = 2 = 3% (from 1 g/L)

Limit of quantification

Validated LOQ = 0, 10 g/L

(Concentration where % (k=2) = 60%)

 

Annex Results of the interlaboratory tests

 

Collaborative study

A total of 17 laboratories from different countries participated in the collaborative study, organised in 2016.

Labo	Country
Miguel Torres S.A.- Finca Mas La Plana	SPAIN
Estación Enológica de Castilla y León	SPAIN
INGACAL -Consellería do Medio Rural Estación de Viticultura e Enoloxía de Galicia	SPAIN
Estación Enológica de Haro	SPAIN
Instituto dos Vinhos do Douro e do Porto, IP	Portugal
Comissão de Viticultura da Região dos Vinhos Verdes	Portugal
Laboratoires Dubernet	France
Laboratoire Diœnos Rhône	France
Laboratoire Natoli	France
SCL Montpellier	France
Agricultural institute of Slovenia	Slovenia
Fachbereich: Wein, Weinüberwachung - Chemisches und Veterinärunterchungsamt Karlsruhe	Germany
HBLAuBA Wein - und Obstbau	AUSTRIA
Landesuntersuchungsamt Mainz	Germany
Hochschule GEISENHEIM University Institut Weinanalytik und Getränkeforschung	Germany
Unità Chimica Vitienologica e Agroalimentare - Centro Trasferimento Tecnologico - Fondazione Edmund Mach	ItalY
Unione Italiana Vini soc. Coop.	ItalY

For analysis, 2 x 10 blind duplicate samples were used, with 1 repetition. The wines analysed are wines originating from France and Portugal, dry wines and liqueur

 

Sample		A		B		C		D		E		F		G		H		I		J
Position		1	9	2	13	3	4	5	15	6	10	16	20	7	11	12	17	8	19	14	18
Labo3	rep#1	94.00	96.00	3.40	3.50	0.40	0.40	0.90	1.10	2.10	2.50	0.10	0.10	1.40	1.40	5.60	5.90	4.70	4.20	17.50	17.00
	rep#2	96.00	98.00	3.50	3.60	0.40	0.30	1.00	1.10	2.20	2.40	0.10	0.10	1.40	1.40	5.70	6.00	4.30	4.50	17.50	17.00
Labo6	rep#1	97.50	95.00	3.42	3.25	0.35	0.48	1.05	0.98	3.24	2.65	0.08	0.05	1.42	1.40	5.49	5.57	4.04	4.11	13.63	19.00
	rep#2	97.00	94.50	3.39	3.29	0.37	0.57	1.08	1.01	3.34	2.66	0.08	0.08	1.52	1.45	5.42	5.52	3.95	4.13	13.70	20.50
Labo7	rep#1	99.22	99.53	3.46	3.56	0.31	0.34	1.00	0.98	2.50	2.58	0.04	0.04	1.49	1.39	5.77	5.75	4.26	4.35	17.66	17.35
	rep#2	100.30	98.90	3.53	3.53	0.31	0.32	1.02	1.02	2.48	2.50	0.04	0.02	1.48	1.34	5.89	5.79	4.23	4.40	17.21	17.94
Labo9	rep#1	92.00	94.20	3.05	3.03	0.29	0.30	0.93	0.97	2.30	2.16	0.04	0.04	1.25	1.25	5.02	5.01	3.98	3.76	15.60	15.76
	rep#2	95.00	97.25	3.03	3.23	0.32	0.31	0.94	0.90	2.20	2.29	0.03	0.04	1.27	1.25	5.14	5.39	3.80	4.06	16.64	16.40
Labo10	rep#1	90.79	92.31	3.27	3.36	0.34	0.34	0.97	1.01	2.28	2.30	0.09	0.07	1.28	1.26	5.46	5.42	3.27	3.36	17.92	17.99
	rep#2	92.13	91.65	3.34	3.24	0.32	0.35	0.97	1.04	2.28	2.33	0.08	0.08	1.32	1.28	5.18	5.37	3.34	3.24	17.58	17.68
Labo11	rep#1	91.40	91.28	3.06	3.12	0.57	0.30	0.95	0.93	2.15	2.18	0.07	0.05	1.16	1.22	5.19	5.34	3.70	3.86	16.22	16.47
	rep#2	90.13	89.94	3.10	3.14	0.56	0.30	0.93	0.93	2.14	2.18	0.07	0.06	1.16	1.20	5.28	5.18	3.76	3.86	16.13	16.33
Labo12	rep#1	100.00	100.00	3.25	3.27	0.34	0.33	1.03	1.10	2.35	2.75	0.08	0.10	1.30	1.39	5.66	5.64	4.07	4.13	17.30	17.44
	rep#2	101.00	97.00	3.22	3.25	0.34	0.33	1.03	1.11	2.36	2.75	0.08	0.10	1.30	1.39	5.62	5.68	4.07	4.15	17.50	17.80
Labo13	rep#1	96.60	96.00	3.04	3.07	0.34	0.31	0.97	0.94	2.26	2.50	0.05	0.04	1.25	1.25	5.21	5.29	3.84	3.99	16.08	16.03
	rep#2	96.00	95.10	3.07	3.12	0.32	0.32	0.97	1.04	2.25	2.25	0.04	0.04	1.25	1.28	5.24	5.31	3.90	3.97	15.95	16.18
Labo14	rep#1	104.00	98.00	3.19	3.16	0.33	0.33	0.97	0.96	2.47	2.44	0.05	0.05	1.34	1.32	5.77	5.81	4.20	4.21	17.76	17.04
	rep#2	103.00	96.00	3.18	3.17	0.33	0.33	0.97	0.97	2.48	2.44	0.05	0.05	1.34	1.32	5.77	5.78	4.20	4.14	17.44	17.24
Labo15	rep#1	110.03	99.25	3.63	3.60	0.20	0.19	0.94	0.97	2.54	2.36			1.30	1.20	5.65	6.14	4.56	4.43	17.16	19.33
	rep#2	104.39	99.34	3.59	3.72	0.20	0.20	0.94	0.95	2.52	2.32			1.32	1.20	5.62	6.19	4.39	4.54	17.41	19.29
Labo16	rep#1	95.20	94.08	3.20	3.22	0.32	0.32	0.96	0.96	2.24	2.26	0.06	0.06	1.23	1.23	5.19	5.19	3.89	3.84	17.82	17.38
	rep#2	96.00	94.41	3.17	3.18	0.31	0.33	0.95	0.94	2.25	2.22	0.06	0.06	1.24	1.22	5.13	5.15	3.85	3.86	17.84	17.24
Labo17	rep#1	96.68	97.10	3.28	3.38	0.47	0.43	1.03	1.03	2.41	2.46	0.10	0.20	1.36	1.36	5.52	5.53	4.09	4.00	16.42	17.30
	rep#2	97.08	99.40	3.24	3.33	0.39	0.38	0.95	0.96	2.30	2.36	0.20	0.15	1.32	1.24	5.38	5.40	3.95	4.10	16.50	16.60
Labo18	rep#1	90.23	91.39	3.14	3.26	0.46	0.47	1.12	1.10	2.30	2.44	0.23	0.24	1.38	1.30	5.19	5.49	3.91	4.10	14.83	14.89
	rep#2	90.02	91.74	3.18	3.31	0.47	0.47	1.07	1.07	2.31	2.40	0.23	0.24	1.38	1.32	5.23	5.45	3.94	4.04	14.82	14.85
Labo19	rep#1	99.63	103.55	3.34	3.41	0.32	0.32	0.98	0.97	2.38	2.41	0.04	0.05	1.29	1.30	5.68	5.56	4.10	4.11	17.61	17.49
	rep#2	100.57	103.28	3.36	3.42	0.32	0.32	0.98	0.97	2.36	2.42	0.05	0.05	1.29	1.31	5.61	5.59	4.10	4.11	17.53	17.51
Labo20	rep#1	96.41	96.18	3.20	3.23	0.32	0.32	0.96	0.95	2.26	2.32	0.07	0.08	1.24	1.24	5.35	5.40	3.92	4.03	16.36	16.51
	rep#2	96.32	95.89	3.18	3.23	0.32	0.32	0.96	0.95	2.26	2.32	0.07	0.08	1.24	1.24	5.35	5.38	3.92	4.03	16.38	16.49
Labo21	rep#1	103.60	102.02	3.37	3.60	0.23	0.25	0.95	0.98	2.41	2.49	0.05	0.05	1.27	1.33	5.95	6.12	4.02	4.53	18.41	19.70
	rep#2	102.50	103.02	3.34	3.51	0.23	0.26	0.92	0.98	2.45	2.45	0.03	0.05	1.26	1.27	6.02	5.99	4.09	4.42	18.96	19.90
Labo22	rep#1	96.73	96.59	3.25	3.28	0.28	0.28	0.92	0.93	2.25	2.31	0.06	0.05	1.23	1.28	5.51	5.47	4.02	3.98	17.09	17.10
	rep#2	97.06	96.34	3.24	3.21	0.30	0.30	0.93	0.93	2.26	2.30	0.04	0.05	1.21	1.24	5.40	5.39	4.03	4.04	17.05	17.01

 

Table of the data obtained. The values in bold correspond with the values rejected in accordance with the Cochran (variance outliers) test with a 2.5% significance level (one-tailed test), and the Grubbs (outliers from the mean) test with significance levels of 2.5% (two-tailed test).

Note: The absent values have not been provided by the laboratory in question.

 

Sample	A	B	C	D	E	F	G	H	I	J
No. of laboratories selected	15	17	14	17	14	14	17	16	15	14
No. of repetitions	4	4	4	4	4	4	4	4	4	4
Min.	90.69	3.08	0.20	0.93	2.16	0.04	1.19	5.14	3.80	14.85
Max.	102.79	3.64	0.47	1.09	2.52	0.10	1.45	6.02	4.48	17.79
Overall average	96.31	3.29	0.32	0.98	2.34	0.06	1.30	5.50	4.05	16.86
Repeatability variance	1.449	0.004	0.000	0.001	0.004	0.000	0.001	0.009	0.005	0.065
Inter-laboratory stand. dev.	3.60	0.16	0.06	0.05	0.10	0.02	0.07	0.26	0.17	0.83
Reproducibility variance	14.037	0.029	0.004	0.003	0.013	0.000	0.006	0.073	0.034	0.739
Repeatability variance	1.20	0.06	0.01	0.04	0.06	0.01	0.04	0.09	0.07	0.26
r limit	3.40	0.17	0.04	0.10	0.17	0.02	0.11	0.26	0.21	0.72
Repeatability RSDr	1.2%	1.8%	4.4%	3.6%	2.5%	13.2%	2.9%	1.7%	1.8%	1.5%
Reproducibility stand. dev.	3.75	0.17	0.07	0.06	0.11	0.02	0.08	0.27	0.19	0.86
R limit	10.60	0.48	0.19	0.16	0.32	0.06	0.22	0.76	0.52	2.43
Reproducibility RSDR	3.9%	5.1%	20.4%	5.7%	4.8%	35.3%	6.1%	4.9%	4.6%	5.1%
Horwitz RSDr	1.877	3.120	4.425	3.742	3.284	5.694	3.588	2.889	3.025	2.440
Horratr	0.666	0.587	1.001	0.952	0.773	2.315	0.804	0.585	0.593	0.621
Horwitz RSDR	2.84	4.73	6.70	5.67	4.98	8.63	5.44	4.38	4.58	3.70
HorratR	1.368	1.086	3.036	0.997	0.965	4.087	1.123	1.122	1.000	1.378

Table of the results obtained

Note: The results from sample F should be taken with caution due to the very low concentration levels, which are below to the laboratories’ limit of quantification.

 

Figure 3: R limit according to concentration

 

Figure 4: Interlaboratory RSDR % according to concentration.

 

Modelling: % = 1·+5