OIV-MA-AS313-01 Total acidity

Type I method

 

1. Definition

The total acidity of the wine is the sum of its titratable acidities when it is titrated to pH 7 against a standard alkaline solution. Carbon dioxide is not included in the total acidity.

2. Principle

Potentiometric titration or titration with bromothymol blue as indicator and comparison with an end‑point color standard.

3. Apparatus

3.1.  Water vacuum pump.

3.2.  Vacuum flask, 500 mL.

3.3.  Potentiometer with scale graduated in pH values, and electrodes. The glass electrode must be kept in distilled water.  The calomel/saturated potassium chloride electrode must be kept in a saturated potassium chloride solution.

3.4.  Beakers of 12 cm diameter.

4. Reagents
   1.   Buffer solution pH 7.0:

• Potassium di-hydrogen phosphate, 107.3 g
• sodium hydroxide solution, NaOH, 1 mol/L 500 mL
• water to 1000 mL

Alternatively, ready-made buffer solutions are available commercially.

4.2.  Sodium hydroxide solution, NaOH, 0.1 mol/L.

4.3.  Bromothymol blue indicator solution, 4 g/L.

bromothymol blue 4 g

neutral ethanol, 96% (v/v) 200 mL

Dissolve and add:

water free of CO2 200 mL

sodium hydroxide solution, 1 mol/L, sufficient to produce

blue green color (pH 7) 7.5 mL

water to 1000 mL

5. Procedure

5.1.  Preparation of sample: elimination of carbon dioxide.

Place approximately 50 mL of wine in a vacuum flask; apply vacuum to the flask using a water pump for one to two min, while shaking continuously.

5.2.  Potentiometric titration

5.2.1.      Calibration of pH meter

The pH meter is calibrated for use at 20°C, according to the manufacturer's instructions, with the pH 7 buffer solution at 20°C.

5.2.2.      Method of measurement

Into a beaker, introduce a volume of the sample, prepared as described in 5.1, equal to 10 mL in the case of wine and 50 mL in the case of rectified concentrated must. Add about 10 mL of distilled water and then add sodium hydroxide solution, 0.1 mol/L, from a burette until the pH is equal to 7 at 20°C. The sodium hydroxide must be added slowly and the solution stirred continuously. Let n mL be the volume of sodium hydroxide, 0.1 mol/L, added.

5.3.  Titration with indicator (bromothymol blue)

5.3.1.      Preliminary test: end‑point color determination.

Into a beaker (3.4) place 25 mL of boiled distilled water, 1 mL of bromothymol blue solution and a volume prepared as in 5.1 equal to 10 mL in the case of wine and 50 mL in the case of rectified concentrated must. Add sodium hydroxide solution, 0.1 mol/L, until the color changes to blue-green. Then add 5 mL of the pH 7 buffer solution.

5.3.2.      Measurement

Into a beaker (3.4) place 30 mL of boiled distilled water, 1 mL of bromothymol blue solution and a volume of the sample, prepared as described in 5.1, equal to 10 mL in the case of wine and 50 mL in the case of rectified concentrated must. Add sodium hydroxide solution, 0.1 mol/L, until the same color is obtained as in the preliminary test above (5.3.1).  Let n mL be the volume of sodium hydroxide solution, 0.1 mol/L, added.

6. Expression of results
   1.   Method of calculation

The total acidity expressed in milliequivalents per liter is given by:

• A = 10 n.

It is recorded to one decimal place.

The total acidity expressed in grams of tartaric acid per liter is given by:

• A' = 0.075 x A

The result is quoted to two decimal places.

The total acidity expressed in grams of sulfuric acid per liter is given by:

• A' = 0.049 x A

The result is quoted to two decimal places.

6.2.  Repeatability (r) for titration with the indicator:(5.3):

• r = 0.9 meq/L
• r = 0.04 g sulfuric acid/L
• r = 0.07 g tartaric acid/L

6.3.  Reproducibility (R) for titration with the indicator (5.3):

For white and rosé wines:

• R = 3.6 meq/L
• R = 0.2 g sulfuric acid/L
• R = 0.3 g tartaric acid/L

For red wines:

• R = 5.1 meq/L
• R = 0.3 g sulfuric acid/L
• R = 0.4 g tartaric acid/L

Bibliography

• SEMICHON L., FLANZY M., Ann. Fals. Fraudes, 1930, 23,5.
• FÉRE L., Ibid., 1931, 24, 75.
• JAULMES P., Bull. O.I.V., 1953, 26, no 274, 42; Ann. Fals. Fraudes, 1955, 48, 157.

OIV-MA-AS313-02 Volatile acidity

Type I method

 

1. Definition

The volatile acidity is derived from the acids of the acetic series present in wine in the free state and combined as salts.

 

2. Principle

Carbon dioxide is first removed from the wine. Volatile acids are separated from the wine by steam distillation and titrated using standard sodium hydroxide.

The acidity of free and combined sulfur dioxide distilled under these conditions should be subtracted from the acidity of the distillate.

The acidity of any sorbic acid, which may have been added to the wine, must also be subtracted.

Note: Part of the salicylic acid used in some countries to stabilize the wines before analysis is present in the distillate. This must be determined and subtracted from the acidity.  The method of determination is given in the Annex of this Chapter.

3. Apparatus
   1.   Steam distillation apparatus consisting of:

• a steam generator; the steam must be free of carbon dioxide;
• a flask with steam pipe;
• a distillation column;
• a condenser.

This equipment must pass the following three tests:

(a) Place 20 mL of boiled water in the flask.  Collect 250 mL of the distillate and add to it 0.1 mL sodium hydroxide solution, 0.1 M, and two drops of phenolphthalein solution.  The pink coloration must be stable for at least 10 sec (i.e. steam to be free of carbon dioxide).

(b) Place 20 mL acetic acid solution, 0.1 M, in the flask.  Collect 250 mL of the distillate.  Titrate with the sodium hydroxide solution, 0.1 M: the volume of the titer must be at least 19.9 mL (i.e. at least 99.5% of the acetic acid entrained with the steam).

(c) Place 20 mL lactic acid solution, 1 M, in the flask. Collect 250 mL of the distillate and titrate the acid with the sodium hydroxide solution, 0.1 M.

The volume of sodium hydroxide solution added must be less than or equal to 1.0 mL (i.e. not more than 0.5% of lactic acid is distilled).

Any apparatus or procedure which passes these tests satisfactorily fulfils the requirements of official international apparatus or procedures.

3.2.   Water aspirator vacuum pump.

3.3.   Vacuum flask.

4. Reagents
   1.    Tartaric acid, crystalline.
   2.    Sodium hydroxide solution, 0.1 M.
   3.    Phenolphthalein solution, 1%, in neutral alcohol, 96% (m/v).
   4.    Hydrochloric acid (ρ20 = 1.18 to 1.19 g/mL) diluted 1/4 with distilled water.
   5.    Iodine solution, 0.005 M.
   6.    Potassium iodide, crystalline
   7.    Starch solution, 5 g/L.

Mix 5 g of starch with about 500 mL of water.  Bring to the boil, stirring continuously and boil for 10 min.  Add 200 g sodium chloride.  When cool, make up to one liter.

4.8.   Saturated solution of sodium tetraborate,, about 55 g/L at 20°C.

4.9.   Acetic acid, 0.1 M.

4.10.         Lactic acid solution, 0.1 M

100 mL of lactic acid is diluted in 400 mL of water. This solution is heated in an evaporating dish over a boiling water bath for four hours, topping up the volume occasionally with distilled water. After cooling, make up to a liter. Titrate the lactic acid in 10 mL of this solution with 1 M sodium hydroxide solution. Adjust the solution to 1 M lactic acid (90 g/L).

 

5. Procedure
   1.    Preparation of sample: elimination of carbon dioxide.  Place about 50 mL of wine in a vacuum flask; apply vacuum to the flask with the water pump for one to two min while shaking continuously. Other elimination systems may be used if the elimination is guaranteed.
   2.    Steam distillation

Place 20 mL of wine, freed from carbon dioxide as in 5.1, into the flask. Add about 0.5 g of tartaric acid. Collect at least 250 mL of the distillate.

5.3.   Titration

Titrate with the sodium hydroxide solution, (4.2), using two drops of phenolphthalein (4.3) as indicator. Let n mL be the volume of sodium hydroxide used.

Add four drops of the dilute hydrochloric acid (4.4), 2 mL starch solution (4.7) and a few crystals of potassium iodide (4.6).  Titrate the free sulfur dioxide with the iodine solution, 0.005 M (4.5).  Let n' mL be the volume used.

Add the saturated sodium tetraborate solution (4.8) until the pink coloration reappears.  Titrate the combined sulfur dioxide with the iodine solution, 0.005 M (4.5).  Let n" mL be the volume used.

6. Expression of results
   1.    Method of calculation

The volatile acidity, expressed in milliequivalents per liter to one decimal place, is given by:

• 5 (n ‑ 0.1 n' ‑ 0.05 n").

The volatile acidity, expressed in grams of sulfuric acid per liter to two decimal places, is given by:

• 0.245 (n ‑ 0.1 n' ‑ 0.05 n").

The volatile acidity, expressed in grams of acetic acid per liter to two decimal places, is given by:

• 0.300 (n - 0.1 n' ‑ 0.05 n").

6.2.   Repeatability (r)

• r = 0.7 meq/L
• r = 0.03 g sulfuric acid/L
• r = 0.04 g acetic acid/L.
  3.    Reproducibility (R) R = 1.3 meq/L

• R = 0.06 g sulfuric acid/L
• R = 0.08 g acetic acid/L.
  4.    Wine with sorbic acid present

Since 96% of sorbic acid is steam distilled with a distillate volume of 250 mL, its acidity must be subtracted from the volatile acidity, knowing that 100 mg of sorbic acid corresponds to an acidity of 0.89 milliequivalents or 0.053 g of acetic acid and knowing the concentration of sorbic acid in mg/L as determined by other methods.

Annex  Determination of Salicylic Acid entrained in the distillate from the volatile acidity

1. Principle

After the determination of the volatile acidity and the correction for sulfur dioxide, the presence of salicylic acid is indicated, after acidification, by the violet coloration that appears when an iron (III) salt is added.

The determination of the salicylic acid entrained in the distillate with the volatile acidity is carried out on a second distillate having the same volume as that on which the determination of volatile acidity was carried out.  In this distillate, the salicylic acid is determined by a comparative colorimetric method.  It is subtracted from the acidity of the volatile acidity distillate.

 

2. Reagents

• Hydrochloric acid, HCl, (ρ₂₀ = 1.18 to 1.19 g/L).
• Sodium thiosulfate solution,, 0.1 M.
• Iron (III) ammonium sulfate solution,, 10% (m/v)
• Sodium salicylate solution, 0.01 M: containing 1.60 g/L sodium salicylate, Na.

3. Procedure

 

3.1.   Identification of salicylic acid in the volatile acidity distillate

Immediately after the determination of the volatile acidity and the correction for free and combined sulfur dioxide, introduce into a conical flask 0.5 mL hydrochloric acid, 3 mL of the sodium thiosulfate solution, 0.1 M, and 1 mL of the iron (III) ammonium sulfate solution. If salicylic acid is present, a violet coloration appears.

3.2.   Determination of salicylic acid

On the above conical flask, indicate the volume of the distillate by a reference mark. Empty and rinse the flask. Subject a new test sample of 20 mL wine to steam distillation and collect the distillate in the conical flask up to the reference mark. Add 0.3 mL concentrated hydrochloric acid, and 1 mL of the iron (III) ammonium sulfate solution. The contents of the conical flask turn violet.

Into a conical flask identical to that carrying the reference mark, introduce distilled water up to the same level as that of the distillate. Add 0.3 mL concentrated hydrochloric acid and 1 mL of the iron (III) ammonium sulfate solution. From the burette run in the sodium salicylate solution, 0.01 M, until the violet coloration obtained has the same intensity as that of the conical flask containing the wine distillate.

Let n''' mL be the volume of solution added from the burette.

 

4. Correction to the volatile acidity

 

Subtract the volume 0.1 x n'''' mL from the volume n mL of sodium hydroxide solution, 0.1 M, used to titrate the acidity of the distillate during the determination of volatile acidity.

Bibliography

• Single method: JAULMES P., Recherches sur l'acidité volatile des vins, Thèse Diplom. Pharm. 1991, Montpellier, Nîmes.
• JAULMES P., Ann. Fals. Frauds, 1950, 43, 110.
• JAULMES P., Analyse des vins, 1951, 396, Montpellier.
• JAULMES P., Bull. O.I.V., 1953., 26, no 274, 48.
• JAULMES P., MESTRES R., MANDROU Mlle B., Ann. Fals. Exp. Chim., 1964, 57, 119.

OIV-MA-AS313-03 Fixed acidity

Type I method

 

1. Principle

The fixed acidity is calculated from the difference between total acidity and volatile acidity.

2. Expression of results

The fixed acidity is expressed in:

• milliequivalents per liter.
• grams of sulfuric acid per liter.
• grams of tartaric acid per liter.

OIV-MA-AS313-05A Tartaric acid

Type IV method

 

1. Principle

Tartaric acid is precipitated in the form of calcium ()tartrate and determined gravimetrically. This determination may be completed using a volumetric procedure for comparison. The conditions for precipitation (pH, total volume used, concentrations of precipitating ions) are such that precipitation of the calcium ()tartrate is complete whereas the calcium D(–) tartrate remains in solution.

When meta-tartaric acid has been added to the wine, which causes the precipitation of the calcium ()tartrate to be incomplete, it must first be hydrolyzed.

2. Method

2.1.  Gravimetric method

2.1.1.      Reagents

Calcium acetate solution containing 10 g of calcium per liter:

Calcium carbonate, Ca	25 g
Acetic acid, glacial, CCOOH (ρ= 1.05 g/mL)	40 mL
Water to	1000 mL

Calcium ()tartrate, crystallized: .

Place 20 mL of L(+) tartaric acid solution, 5 g/L, into a 400 mL beaker.

Add 20 mL of ammonium D(–) tartrate solution, 6.126 g/L, and 6 mL of calcium acetate solution containing 10 g of calcium per liter.

Allow to stand for two hours to precipitate. Collect the precipitate in a sintered glass crucible of porosity No 4, and wash it three times with about 30 mL of distilled water.  Dry to constant weight in the oven at 70°C. Using the quantities of reagent indicated above, about 340 mg of crystallized calcium () tartrate is obtained.  Store in a stoppered bottle.

- Precipitation solution (pH 4.75):

D(–) ammonium tartrate	150 mg
Calcium acetate solution, 10 g calcium/L	8.8 mL
Water to	1000 mL

Dissolve the D(‑) ammonium tartrate in 900 mL water; add 8.8 mL calcium acetate solution and make up to 1000 mL. Since calcium ()tartrate is slightly soluble in this solution, add 5 mg of calcium (±)tartrate per liter, stir for 12 hours and filter.

Note: The precipitation solution may also be prepared from D(-) tartaric acid.

D(–) tartaric acid	122 mg
Ammonium hydroxide solution (ρ= 0.97 g/mL), 25 % (v/v)	0.3 mL

Dissolve the D(–) tartaric acid, add the ammonium hydroxide solution and make up to about 900 mL; add 8.8 mL of calcium acetate solution, make up to a liter and adjust the pH to 4.75 with acetic acid.  Since calcium () tartrate is slightly soluble in this solution, add 5 mg of calcium ()tartrate per liter, stir for 12 hours and filter.

2.1.2.      Procedure

Wines with no added meta-tartaric acid

Place 500 mL of precipitation solution and 10 mL of wine into a 600 mL beaker.  Mix and initiate precipitation by rubbing the sides of the vessel with the tip of a glass rod.  Leave to precipitate for 12 hours (overnight).

Filter the liquid and precipitate through a weighed sintered glass crucible of porosity No. 4 fitted on a clean vacuum flask.  Rinse the vessel in which precipitation took place with the filtrate to ensure that all precipitate is transferred.

Dry to constant weight in an oven at 70°C.  Weigh.  Let ρ be the weight of crystallized calcium ()tartrate, Ca, obtained.

Wines to which meta-tartaric acid has been added.

When analyzing wines to which meta-tartaric acid has been or is suspected of having been added, proceed by first hydrolyzing this acid as follows:

Place 10 mL of wine and 0.4 mL of glacial acetic acid, COOH, (ρ_= 1.05 g/mL) into a 50 mL conical flask. Place a reflux condenser on top of the flask and boil for 30 min. Allow to cool and then transfer the solution in the conical flask to a 600 mL beaker. Rinse the flask twice using 5 mL of water each time and then continue as described above.

Meta-Tartaric acid is calculated and included as tartaric acid in the final result.

2.1.3.      Expression of results

One molecule of calcium ()tartrate corresponds to half a molecule of L(+) tartaric acid in the wine.

The quantity of tartaric acid per liter of wine, expressed in milliequivalents, is equal to: 384.5 p.

It is quoted to one decimal place.

The quantity of tartaric acid per liter of wine, expressed in grams of tartaric acid, is equal to 28.84 p.

It is quoted to one decimal place.

The quantity of tartaric acid per liter of wine, expressed in grams of potassium tartrate, is equal to: 36.15 p.

It is quoted to one decimal place.

2.2.  Comparative volumetric analysis

2.2.1.      Reagents

Hydrochloric acid (ρ20 = 1.18 to 1.19 g/mL) diluted 1:5 with distilled water

EDTA solution, 0.05 M:

EDTA (ethylenediaminetetraacetic acid disodium salt)	18.61 g
Water to	1000 mL

Sodium hydroxide solution, 40% (m/v):

Sodium hydroxide, NaOH	40 g
Water to	100 mL

Complexometric indicator: 1% (m/m) 2‑hydroxy‑1‑(2‑hydroxy‑4‑sulpho‑1‑naphthylazo)

3‑naphthoic acid

1 g

2.2.2.      Procedure

After weighing, replace the sintered glass crucible containing the precipitate of calcium ()tartrate on the vacuum flask and dissolve the precipitate with 10 mL of dilute hydrochloric acid.  Wash the sintered glass crucible with 50 mL of distilled water.

Add 5 mL 40% sodium hydroxide solution and about 30 mg of indicator. Titrate with EDTA solution, 0.05 M.  Let the number of mL used be n.

2.2.3.      Expression of results

The quantity of tartaric acid per liter of wine, expressed in milliequivalents, is equal to: 5 n.

It is quoted to one decimal place.

The quantity of tartaric acid per liter of wine, expressed in grams of tartaric acid, is equal to: 0.375 n.

It is quoted to one decimal place.

The quantity of tartaric acid per liter of wine, expressed in grams of potassium acid tartrate, is equal to: 0.470 n.

It is quoted to one decimal place.

Bibliography

• KLING A., Bull. Soc. Chim., 1910, 7, 567.
• KLING A., FLORENTIN D., Ibid, 1912, 11, 886.
• SEMICHON L., FLANZY M., Ann. Fals. Fraudes, 1933, 26, 404.
• PEYNAUD E., Ibid, 1936, 29, 260.
• PATO M., Bull. O.I.V., 1944, 17, no, 161, 59, no, 162, 64.
• POUX C., Ann. Fals. Fraudes, 1949, 42, 439.
• PEYNAUD E., Bull. Soc. Chim. Biol., 1951, 18, 911; Ref. Z. Lebensmit. Forsch. , 1953, 97, 142.
• JAULMES P., BRUN Mme S., VASSAL Mlle M., Trav. Soc, Pharm., Montpellier, 1961, 21, 46‑51.
• JAULMES P., BRUN Mme S., CABANIS J.C., Bull. O.I.V., 1969, nos 462‑463, 932.

OIV-MA-AS313-07 Lactic acid

Type II method

1. Principle

Total lactic acid (L‑lactate and D‑lactate) is oxidized by nicotinamide adenine dinucleotide (NAD) to pyruvate in a reaction catalyzed by L‑lactate dehydrogenase (L‑LDH) and D‑lactate dehydrogenase (D‑LDH).

The equilibrium of the reaction normally lies more strongly in favor of the lactate. Removal of the pyruvate from the reaction mixture displaces the equilibrium towards the formation of pyruvate.

In the presence of L‑glutamate, the pyruvate is transformed into L‑alanine in a reaction catalyzed by glutamate pyruvate transaminase (GPT):

(1) L-lactate + pyruvate +NADH+

(2) D-lactate + pyruvate +NADH+

(3) Pyruvate + L-glutamate L-alanine+ α-ketoglurarate

The amount of NADH formed, measured by the increase in absorbance at the wavelength of 340 nm, is proportional to the quantity of lactate originally present.

Note: L‑lactic acid may be determined independently by using reactions (1) and (3), while D‑lactic acid may be similarly determined by using reactions (2) and (3).

2. Apparatus

2.1.  A spectrophotometer permitting measurements to be made at 340 nm, the wavelength at which the absorbance of NADH is a maximum.

Failing that, a spectrophotometer with a discontinuous spectrum source permitting measurements to be made at 334 or 365 nm may be used.

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

2.3.   Micropipettes for pipetting sample volumes in the range 0.02 to 2 mL.

3. Reagents

Double‑distilled water

3.1.  Buffer solution, pH 10 (glycylglycine, 0.6 M; L‑glutamate, 0.1 M):

Dissolve 4.75 g of glycylglycine and 0.88 g of L‑glutamic acid in approximately 50 mL of double distilled water; adjust the pH to 10 with a few milliliters sodium hydroxide, 10 M, and make up to 60 mL with double distilled water.

This solution will remain stable for at least 12 weeks at 4°C.

3.2.   Nicotinamide adenine dinucleotide (NAD) solution, approximately 40 x 10‑3 M: dissolve 900 mg of NAD in 30 mL of double distilled water.  This solution will remain stable for at least four weeks at 4°C.

3.3.   Glutamate pyruvate transaminase (GPT) suspension, 20 mg/mL.

The suspension remains stable for at least a year at 4°C.

3.4.   L‑lactate dehydrogenase (L‑LDH) suspension, 5 mg/mL.

This suspension remains stable for at least a year at 4°C.

3.5.   D‑lactate dehydrogenase (D‑LDH) suspension, 5 mg/mL.

This suspension remains stable for at least a year at 4°C.

It is recommended that, prior to the determination, the enzyme activity should be checked.

Note: All the reagents are available commercially.

4. Preparation of the sample

 

Lactate determination is normally carried out directly on the wine, without prior removal of pigmentation (coloration) and without dilution provided that the lactic acid concentration is less than 100 mg/L. However, if the lactic acid concentration lies between:

100 mg/L and 1 g/L, dilute 1/10 with double distilled water, 1 g/L and 2.5 g/L, dilute 1/25 with double distilled water, 2.5 g/L and 5 g/L, dilute 1/50 with double distilled water.

5. Procedure

 

Preliminary note:

No part of the glassware that comes into contact with the reaction mixture should be touched with the fingers, since this could introduce L‑lactic acid and thus give erroneous results.

The buffer solution must be at a temperature between 20 and 25°C before proceeding to the measurement.

5.1.   Determination of total lactic acid

With the spectrophotometer adjusted to a wavelength of 340 nm, determine the absorbance using 1 cm cells, with air as the zero absorbance (reference) standard; (no cell in the optical path) or with water as the standard.

Place the following in the 1 cm cells:

	Reference cell		Sample cell
	(mL)		(mL)
Solution 3.1.	1.00	1.00
Solution 3.2.	0.20	0.20
Double distilled water	1.00	0.80
Suspension 3.3.	0.02	0.02
Sample to be measured	-	0.20

Mix using a glass stirrer or a rod of synthetic material with a flattened end; after about five min, measure the absorbencies of the solutions in the reference and sample cells ().

Add 0.02 mL of solution 3.4 and 0.05 mL of solution 3.5, homogenize, wait for the reaction to be completed (about 30 min) and measure the absorbencies of the solutions in the reference and sample cells ().

Calculate the differences (  – ) in the absorbencies of the solutions in the reference and sample cells,  and .

Finally, calculate the difference between those differences:

5.2.   Determination of L‑lactic acid and D‑lactic acid

Determination of the L‑lactic acid or D‑lactic acid can be carried out independently by applying the procedure for total lactic acid up to the determination of and then continuing as follows:

Add 0.02 mL of solution 3.4, homogenize, wait until the reaction is complete (about 20 min) and measure the absorbencies of the solutions in the reference and sample cells ().

Add 0.05 mL of solution 3.5, homogenize, wait until the reaction is complete (about 30 min) and measure the absorbencies of the solutions in the reference and sample cells ().

Calculate the differences ( – ) for L-lactic acid and ( – ) for D‑lactic acid between the absorbencies of the solutions in the reference and sample cells, and .

Finally, calculate the difference between those differences:

Note: The time needed for the completion of enzyme activity can 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. When determining the L‑lactic acid alone, the incubation time may be reduced to 10 min.

6. Expression of results

Lactic acid concentration is given in grams per liter (g/L) to one decimal place.

6.1.   Method of calculation

The general formula for calculating the concentration in g/L is:

where

V= volume of test solution in mL (V = 2.24 mL for L-lactic acid, V = 2.29 mL for D‑lactic acid and total lactic acid)

ν = volume of the sample in mL (0.2 mL)

M = molecular mass of the substance to be determined (for DL‑lactic acid, M = 90.08)

δ = optical path in the cell in cm (1 cm)

ε = absorption coefficient of NADH, at 340 nm

(ε= 6.3 mmol-1 x l x cm-1).

6.1.1. Total lactic acid and D‑lactic acid

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

Note:

• Measurement at 334 nm: C = 0.167 x , ( ε= 6.2 mmol^‑1 x 1 x cm^‑1).
• Measurement at 365 nm: C = 0.303 x A, (ε = 3.4 mmol^‑1 x 1 x cm^‑1).

6.1.2. L‑lactic acid

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

Note:

• Measurement at 334 nm: C = 0.163 x , (ε = 6.2 mmol^‑1 x 1 x cm^‑1).

Measurement at 365 nm: C = 0.297 x , (ε = 3.4 mmol^‑1 x 1 x cm^‑1).

6.2.   Repeatability (r)

 r = 0.02 + 0.07

xi is the lactic acid concentration in the sample in g/L.

6.3.   Reproducibility (R)

R = 0.05 + 0.125

xi is the lactic acid concentration in the sample in g/L.

Bibliography

• HOHORST H.J., in Méthodes d'analyse enzymatique, par BERGMEYER H.U., 2e éd., p. 1425, Verlag‑Chemie Weinheim/Bergstraße, 1970.
• GAWEHN K. et BERGMEYER H.U., ibid., p. 1450.
• BOEHRINGER, Mannheim, Méthodes d'analyse enzymatique en chimie alimentaire, documentation technique.
• JUNGE Ch., F.V., O.I.V., 1974, no 479.
• VAN DEN DRIESSCHE S. et THYS L., F.V., O.I.V., 1982, no 755.

---

OIV-MA-AS313-09 Citric acid

Type II method

 

1. Principle

 

Citric acid is converted into oxaloacetate and acetate in a reaction catalyzed by citratelyase (CL):

In the presence of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), the oxaloacetate and its decarboxylation derivative, pyruvate, are reduced to L‑malate and L‑lactate by reduced nicotinamide adenine dinucelotide (NADH):

The amount of NADH oxidized to NAD+ in these reactions is proportional to the amount of citrate present.  The oxidation of NADH is measured by the resultant decrease in absorbance at a wavelength of 340 nm.

 

2. Apparatus

2.1.  A spectrophotometer permitting measurement to be made at 340 nm, the wavelength at which absorbance of NADH is a maximum.

Alternatively, a spectrophotometer, with a discontinuous spectrum source permitting measurements to be made at 334 nm or 365 nm, may be used.

Since absolute absorbance measurements are involved (i.e. calibration curves are not used but standardization is made by consideration of the extinction coefficient of NADH), the wavelength scales and spectral absorbance of the apparatus must be checked.

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

2.3.   Micropipettes for pipetting volumes in the range 0.02 to 2 mL.

3. Reagents
   1.   Buffer solution pH 7.8 (glycylglycine, 0.51 M; pH 7.8; Zn⁺(0.6 x 10^‑3 M):

dissolve 7.13 g of glycylglycine in approximately 70 mL of double distilled water.  Adjust the pH to 7.8 with approximately 13 mL sodium hydroxide solution, 5 M, add 10 mL of zinc chloride, Zn, (80 mg in 100 mL double distilled water) solution and make up to 100 mL with double distilled water.

3.2.   Reduced nicotinamide adenine dinucleotide, NADH, solution (approximately 6 x 10^‑3M): dissolve 30 mg NADH and 60 mg NaH in 6 mL of double distilled water.

3.3.   Malate dehydrogenase/lactate dehydrogenase solution (MDH/LDH) (0.5 mg MDH/mL; 2.5 mg LDH/mL): mix together 0.1 mL MDH (5 mg MDH/mL), 0.4 mL ammonium sulfate solution, 3.2 M, and 0.5 mL LDH (5 mg/mL).

This suspension remains stable for at least a year at 4°C.

3.4.   Citrate‑lyase (CL, 5 mg protein/mL): dissolve 168 mg lyophilisate in 1 mL ice‑cold water. This solution remains stable for at least a week at 4°C and for at least four weeks if frozen.

It is recommended that, prior to the determination, the enzyme activity should be checked.

3.5.   Polyvinylpolypyrrolidone (PVPP).

Note: All the reagents above are available commercially.

 

4. Preparation of the sample

Citrate determination is normally carried out directly on wine, without preliminary removal of pigmentation (coloration) and without dilution, provided that the citric acid content is less than 400 mg/L. If not, dilute the wine until the citrate concentration lies between 20 and 400 mg/L (i.e. between 5 and 80 μg of citrate in the test sample).

With red wines that are rich in phenolic compounds, preliminary treatment with PVPP is recommended:

Form a suspension of about 0.2 g of PVPP in water and allow to stand for 15 min.  Filter using a fluted filter.

Place 10 mL of wine in a 50 mL conical flask, add the moist PVPP removed from the filter with a spatula.  Shake for two to three minutes.  Filter.

 

5. Procedure

With the spectrophotometer adjusted to a wavelength of 340 nm, determine the absorbance using the 1 cm cells, with air as the zero absorbance (reference) standard (no cell in the optical path).  Place the following in the 1 cm cells:

	Reference cell	Sample cell
Solution 3.1	1.00	1.00
Solution 3.2	0.10	0.10
Sample to be measured	-	0.20
Double distilled water	2.00	1.80
Solution 3.3	0.02	0.02
Mix, and after about five min read the absorbance of the solutions in the reference and sample cells (A1).
Solution 3.4	0.02 mL	0.02 mL

Mix; wait until the reaction is completed (about five min) and read the absorbance of the solutions in the reference and sample cells ().

Calculate the absorbance difference () for the reference and sample cells, and .

Finally, calculate the difference between those differences:

Note: The time needed for the completion of enzyme activity can 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.

6. Expression of results

Citric acid concentration is given in milligrams per liter to the nearest whole number.

6.1.   Method of calculation

The general formula for calculating the concentration in mg/L is:

where:

V = volume of test solution in mL (3.14 mL)

v = volume of the sample in mL (0.2 mL)

M = molecular mass of the substance to be determined (for anhydrous citric acid, M = 192.1)

d = optical path in the cell in cm (1 cm)

ε = absorption coefficient of NADH, (at 340 nm, ε = 6.3 mmol-1 x l x cm-1).

so that:

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

Note:

  At 334 nm: C = 488 x (ε=  6.3 mmol‑1 x l x cm‑1).

  At 365 nm: C = 887 x (ε=  3.4 mmol‑1 x l x cm‑1).

6.2.   Repeatability (r)

Citric acid concentration less than 400 mg/L: r = 14 mg/L.

Citric acid concentration greater than 400 mg/L: r = 28 mg/L.

6.3.   Reproducibility (R)

Citric acid concentration less than 400 mg/L: R = 39 mg/L.

Citric acid concentration greater than 400 mg/L: R = 65 mg/L.

Bibliography

• Mayer K. et Pause G., Lebensm. Wiss. u. Technol., 1969. 2, 143
• Junge Ch., F.V., O.I.V., 1970, no 364
• Boehhringer, Mannheim, Méthodes d'analyse enzymatique en chimie alimentaire, documentation technique.
• Van den Dreische S. et Thys L., F.V., O.I.V., 1982, no 755.

OIV-MA-AS313-10 Total malic acid

Type IV method

1. Principle

Malic acid, separated by means of an anion exchange column, is determined colorimetrically in the eluent by measuring the yellow coloration it forms with chromotropic acid in the presence of concentrated sulfuric acid.  A correction for interfering substances is made by subtracting the absorbance, obtained using 86% sulfuric and chromotropic acid respectively (malic acid does not react at these acid concentrations), from the absorbance obtained from using 96% strength acids.

2. Apparatus

2.1.  Glass column 250 mm approximately in length and 35 mm internal diameter, fitted with drain tap.

2.2.  Glass column approximately 300 mm in length and 10 to 11 mm internal diameter, fitted with drain tap.

2.3.  Thermostatically controlled water bath at 100°C.

2.4.  Spectrophotometer set to measure absorbance at 420 nm using cells of 1 cm optical path.

3. Reagents
   1.   A strongly basic anion exchanger (e.g. Merck III)
   2.   Sodium hydroxide, 5% (m/v).
   3.   Acetic acid, 30% (m/v).
   4.   Acetic acid, 0.5% (m/v).
   5.   Sodium sulfate solution, 10% (m/v).
   6.   Concentrated sulfuric acid, 95‑97% (m/m).
   7.   Sulfuric acid, 86% (m/m).
   8.   Chromotropic acid, 5% (m/v).

Prepare fresh solution before each determination by dissolving 500 mg sodium chromotropate, , in 10 mL distilled water

3.9.  0.5 g DL‑malic acid per liter solution

Dissolve 250 g malic acid () in sodium sulfate solution, 10%, to obtain 500 mL.

4. Procedure

4.1.  Preparation of ion exchanger

Place a plug of cotton impregnated with distilled water in a 35 x 250 mm glass column.  Pour a suspension of the anion exchange resin into the glass column. The level of the liquid should be 50 mm above the top of the resin.  Rinse with 1000 mL of distilled water. Wash the column with sodium hydroxide solution, 5%, allow to drain to approximately 2 to 3 mm of the top of the resin and repeat with two further washings of sodium hydroxide, 5%, and leave for one hour. Wash the column with 1000 mL of distilled water. Refill the column with acetic acid, 30%, allow to drain to approximately 2 to 3 mm above the top of the resin and repeat with two further washings of acetic acid, 30%. Leave for at least 24 hours before use.  Keep the ion exchange resin in acetic acid, 30%, for the subsequent analysis.

4.2.  Preparation of ion exchange column.

Place a plug of cotton wool at the bottom of the column measuring 11 x 300 mm above the tap.  Pour in the ion exchanger prepared as described above in 4.1 to a height of 10 cm.  Open the tap and allow the acetic acid solution, 30%, to drain to approximately 2 to 3 mm above the surface of the exchanger.  Wash the exchanger with a 50 mL acetic acid solution, 0.5%.

4.3.  Separation of DL‑Malic acid

Pour onto the column (4.2) 10 mL of wine or must.  Allow to drain drop by drop (average rate of one drop per second) and stop the flow 2 to 3 mm from the top of the resin.  Wash the column with 50 mL acetic acid, 0.5% (m/v), then with 50 mL of distilled water and allow to drain at the same rate as previously, stopping the flow 2 to 3 mm from the top of the resin.

Elute the acids absorbed on the exchange resin with sodium sulfate solution, 10%, at the same rate as in the previous steps (1 drop/sec).  Collect the eluate in a 100 mL volumetric flask. The ion exchange column can be regenerated using the procedure described in 4.1

4.4.  Determination of malic acid

Take two wide necked 30 mL tubes fitted with ground glass stoppers, A and B.  In each tube add 1.0 mL of the eluate and 1.0 mL chromotropic acid solution, 5%.  Add to tube A 10.0 mL sulfuric acid, 86% (m/m), (reference) and to the tube B 10.0 mL sulfuric acid, 96% (m/m), (sample).  Stopper and shake to homogenize carefully, without wetting the glass stopper.  Immerse the tubes in a boiling water bath for exactly 10 min.  Cool the tubes in darkness at 20 C for exactly 90 min.  Immediately measure the absorbance of tube B relative to the sample tube A at 420 nm in 1 cm cells.

4.5 Plotting the calibration curve

Pipette 5, 10, 15 and 20 mL of the DL‑malic acid solution (0.5g/L) into separate 50 mL volumetric flasks.  Make up to the mark with sodium sulfate solution, 10%.

These solutions correspond to eluates obtained from wines containing 0.5, 1.0, 1.5 and 2.0 g DL‑malic acid per liter.

Continue as indicated in 4.4.  The graph of the absorbencies of these solutions verses their malic acid concentration should appear as a straight line passing through the origin.

The intensity of the coloration depends to a large extent on the strength of the sulfuric acid used.  It is necessary to check the calibration curve to see if the concentration of the sulfuric acid has changed.

5. Expression of results

Plot the absorbance on calibration graph to obtain the content of DL‑malic acid in grams per liter. This content is expressed with 1 decimal.

Bibliography

• REINHARD C., KOEDING, G., Zur Bestimmung der Apfelsäure in Fruchtsäften, Flüssiges Obst., 1989, 45, S, 373 ff.

OIV-MA-AS313-11 L-Malic acid

Type II method

1. Principle of the method

L-malic acid (L-malate) is oxidized by nicotinamide adenine dinucleotide (NAD) to oxaloacetate in a reaction catalysed by L-malate dehydrogenase (L-MDH).

The equilibrium of the reaction normally lies more strongly in favour of the malate. Removal of the oxaloacetate from the reaction mixture displaces the equilibrium towards the formation of oxaloacetate. In the presence of L-glutamate, the oxaloacetate is transformed into L-aspartate in a reaction catalysed by glutamate oxaloacetate transaminase (GOT):

The amount of NADH formed, measured by the increase in absorbance at the wavelength of 340 nm, is proportional to the quantity of L-malate originally present.

2. Apparatus

 

2.1.  A spectrophotometer permitting measurement to be made at 340 nm, the wavelength at which absorption by NADH is at a maximum. Failing that, a spectrophotometer, with a discontinuous spectrum source permitting measurements to be made at 334 or 365 nm, may be used.

Since absolute measurements of absorbance are involved (i.e. calibration curves are not used, but standardization is made by consideration of the extinction coefficient of NADH), the wavelength scales and spectral absorbance of the apparatus must be checked.

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

2.3.  Micropipettes for pipetting sample volumes in the range 0,01 to 2 ml.

3. Reagents

Doubly distilled water

3.1.  Buffer solution, pH 10

(glycylglycine 0,6 M; L-glutamate 0,1 M):

dissolve 4,75 g of glycylglycine and 0,88 g of L-glutamic acid in approximately 50 ml of doubly distilled water; adjust the pH to 10 with about 4,6 ml of 10 M sodium hydroxide and make up to 60 ml with doubly distilled water. This solution will remain stable for at least 12 weeks at 4 °C.

3.2.  Nicotinamide adenine dinucleotide (NAD) solution, approximately 47 × 10 3 M: dissolve 420 mg of NAD in 12 ml of doubly distilled water. This solution will remain stable for at least four weeks at 4 °C.

3.3.  Glutamate oxaloacetate transaminase (GOT) suspension, 2 mg/ml. The suspension remains stable for at least a year at 4 °C.

3.4.  L-malate dehydrogenase (L-MDH) solution, 5 mg/ml. This solution remains stable for at least a year at 4 °C.

Note: All the reagents above are available commercially

4. Preparation of the sample

L-malate determination is normally carried out directly on the wine, without prior removal of pigmentation (colouration) and without dilution provided that the Lmalic acid concentration is less than 350 mg/l (measured at 365 mg/l). If this is not so, dilute the wine with doubly distilled water until the L-malate concentration lies between 30 and 350 mg/l (i.e. amount of L-malate in the test sample lies between 3 and 35 μg).

If the malate concentration in the wine is less than 30 mg/l, the volume of the test sample may be increased up to 1 ml. In this case, the volume of water to be added is reduced in such a way that the total volumes in the two cells are equal.

5. Procedure

With the spectrophotometer adjusted to a wavelength of 340 nm, determine the absorbance using the cells having optical paths of 1 cm, with air as the zero absorbance (reference) standard (no cell in the optical path) or with water as the standard.

Place the following in the cells having 1 cm optical paths:

	Reference cell (ml)	Sample cell (ml)
Solution 3.1	1,00	1,00
Solution 3.2	0,20	0,20
Doubly distilled water	1,00	0,90
Suspension 3.3	0,01	0,01
Sample to be measured	)	0,10
Mix; after about three minutes, measure the absorbances of the solutions in the reference and sample cells (A1).
Add:
Solution 3.4	0.01 ml	0.01 ml

Mix; wait for the reaction to be completed (about 5 to 10 minutes) and measure the absorbances of the solutions in the reference and sample cells (A₂).

Calculate the differences () in the absorbances of the solutions in the reference and sample cells,.

Finally, calculate the difference between those differences:

Note: The time needed for the completion of enzyme activity can 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.

6. Expression of results

L-malic acid concentration is given in grams per litre to one decimal place.

6.1.  Method of calculation

The general formula for calculating the concentration in g/l is:

where:

V = volume of test solution in ml (here 2,22 ml)

V = volume of the sample in ml (here 0,1 ml)

M = molecular mass of the substance to be determined (here, for L-malic acid, M=134,09)

δ= optical path in the cell in cm (here, 1 cm)

ε= absorption coefficient of NADH, (at 340 nm

ε= 6,3 m mol 1 × l × cm 1),

so that for L-malate:

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

Note:

Measurement at 334 nm, ε = 6,2 ( x 1 x cm²)

C = 0,482 ×

Measurement at 365 nm, ε = 6,2 (x  l x  cm²)

C = 0,876 ×

6.2.  Repeatability (r)

r = 0,03 + 0,034 x_i

is the malic acid concentration in the sample in g/l.

6.3.  Reproducibility (R)

R = 0,05 + 0,071 x_i

is the malic acid concentration in the sample in g/l.

Bibliography

• BERGMEYER H.U., Méthodes d’analyse enzymatique, 2^e éd., Verlag-Chemie Weinheim/Bergstrasse, 1970
• BOERHINGER, Mannheim, Méthodes d’analyse enzymatique en chimi alimentaire, documentation technique.
• VAN DEN DRIESSCHE S. et THYS L., F.C. O.I.V., 1982, n° 755

OIV-MA-AS313-12A D-Malic acid (Enzymatic method)

Type II method

1. Principle

In the presence of D-malate-dehydrogenase (D-MDH), D-malic acid (D-malate) is oxidized to oxalo-acetate by nicotinamide-adenine-dinucleotide (NAD). The formed oxalo-acetate is transformed into pyruvate and carbon dioxide.

(1)

The formation of NADH, measured by the increase of absorbance for 334, 340 or 365 nm wave lengths, is proportional to the quantity of D-malate present.

2. Reagents

Reagents that allow 30 determinations to be made are marketed in a set which includes:

• 1/ Flask 1 containing about 30 ml of solution of Hepes buffer acid [N-(2-hydroxyethyl)piperazine-N’-2-ethane sulfonic] pH = 9.0 and stabilizers;
• 2/ Flask 2 containing about 210 mg of NAD lyophilizate;
• 3/ Flask 3 (three flasks), containing D-MDH lyophilizate, with a titer of about 8 units.

 

Preparation of the solutions

• 1/ Use the content of flask 1 without dilution. Bring the solution to a temperature of 20-25°C before using it.
• 2/ Dissolve the content of flask 2 in 4 ml of double-distilled water.
• 3/Dissolve the content of one the flasks 3 in 0,6 ml of double-distilled water.

Bring the solution to a temperature of 20-25 °C before using it.

Stability of the solutions

The contents of flask 1 can be kept for at least one year at + 4°C; solution 2 can be kept about 3 weeks at + 4 °C and 2 months at - 20 °C; solution 3 can be kept 5 days at + 4 °C.

 

3. Apparatus

3.1.   Spectrophotometer which is able to measure at the NADH absorption maximum of 340 nm. If this is not available, a spectrophotometer with a discontinuous spectrum source permitting measurements to be made at 334 or 365 nm may be used.  Since absolute absorbance measurements are involved (i.e. calibration curves are not used, but standardization is made by consideration of the extinction coefficient of NADH), the wavelength scales and spectral absorbance of the apparatus must be checked.

3.2.   Cells with a 1 cm path of glass or single-use cells.

3.3.   Micropipettes capable of pipetting volumes between 0.01 and 2 ml.

4. Preparation of the sample

The analysis of D-malate is generally carried out directly on the wine without preliminary decoloration.

The quantity of D-malate in the cell must be between 2 µg and 50 µg; wine should be diluted so the malate concentration will be between 0.02 and 0.5 g/L or 0.02 and 0.3 g/L depending on the apparatus used.

Dilution table:

Estimated quantity of D-malate/liter	Dilution with water	Dilution factor F
Measured at: 340 or 334 nm 365 nm
< 0.3 g  < 0.5 g	-	1
0.3-3.0 g 0.5-5.0 g	1 + 9	10

 

5. Procedure

 

With the spectrophotometer adjusted to a wavelength of 340 nm, determine the absorbance using 1 cm cells, with air as the zero absorbance (reference) standard (no cell in the optical path) or with water as the standard.

Place the following in the 1 cm cells:

	Reference cell (mL)	Sample cell (mL)
Solution 1	1.00 mL	1.00 mL
Solution 2	0.10 mL	0.10 mL
Double-distilled Water	1.80 mL	1.70 mL
Sample	-	0.10 mL

Mix: after approximately 6 minutes, measure the absorbance of the reference and sample solutions ().

Add

	Reference	Sample
Solution 3	0.05 mL	0.05 mL

Mix: wait for the end of the reaction (about 20 min.) and measure the absorbance of the reference and sample solutions ().

Determine the absorbance differences () of the control () and trial ().

Deduct the control absorbance difference from the trial absorbance difference:

Comment: the time required for the enzymes’ action can vary from one batch to the other. It is given here only as an indication. It is recommended it be determined for each batch.

D-malic acid reacts rapidly. An additional activity of the enzyme also transforms L-tartaric acid even though it is not as rapid. This is the reason why there is a small side reaction which may be corrected by means of extrapolation (see annex 1).

 

6. Expression of the results

 

The concentration in milligrams per liter is calculated with the general formula:

V = volume of the test in ml (here 2.95 mL)

 = volume of the sample in ml (here 0.1 mL)

PM = molecular mass of the substance to be measured

 (here, D-malic acid = 134.09)

d= cell path length in cm (here 1 cm)

ε= absorption coefficient of NADH:

• at 340 nm = 6.3 (l mmol^-1 cm^-1)
• at 365 nm = 3.4 (l mmol^-1 cm^-1)
• at 334 nm = 6.18(l mmol^-1 cm^-1).

If a dilution was made during the preparation of the sample, multiply the result by the dilution factor. The concentration in D-malic acid is given in milligrams per liter (mg/L) without decimal.

 

7. Accuracy

The details of the interlaboratory trial on the accuracy of the method are summarized in annex 2. The derived values of the interlaboratory study may not be applicable to ranges of concentration of the analyte and to other matrices other than those given in annex 2.

7.1.   Repeatability

The absolute difference between individual results obtained on an identical matter submitted to a trial by an operator using the same apparatus, within the shortest time interval, will not exceed the value of repeatability r in more than 5% of the cases. The value is: r = 11 mg/L.

7.2.   Reproducibility

The absolute difference between individual results obtained on an identical material submitted to a test in two laboratories will not exceed the value of reproducibility R in more than 5% of the cases. The value is: R = 20 mg/L.

8. Comments

Taking into account the method's accuracy, the values of D-malic acid less than 50 mg/L must be confirmed by another analytical method using another measuring principle such as that of PRZYBORSKI et al, (1993). Values of D-malic acid less than 100 mg/L must not be interpreted as an addition of D, L-malic acid to wine.

The wine content in the cuvette must not exceed 0.1mL to avoid a possible inhibition of enzymatic activity by polyphenols.

Bibliogaphy

 

PRZYBORSKI et al. Mitteilungen Klosterneuburg 43, 1993; 215-218.

Annex 1 :How to treat side reactions

Side reactions are generally due to secondary reactions of the enzyme, in the presence of other enzymes in the sample’s matrix, or the interaction of one or several elements of the matrix with a co-factor of the enzymatic reaction.

With a normal reaction, absorbance reaches a constant value after a certain time, generally between 10 min and 20 min, according to the speed of the specific enzymatic reaction. However, when secondary reactions occur, the absorbance does not reach a constant value, but increases regularly with time; this type of process is commonly called a « side reaction ».

When this problem arises, one should measure the solution’s absorbance at regular intervals (2 min to 5 min), after the required time for the standard solution to reach its final absorbance. When the absorbance increases regularly, carry out 5 or 6 measurements, than establish a graphic or calculated extrapolation, in order to obtain what the solution’s absorbance would have been when the final enzyme was added (T0). The difference in extrapolated absorbance at this time (Af-Ai) is used for the calculation of the substrate concentration.

Figure 1: Side reaction

Annex 2

Interlaboratory trials statistical results

 

Year of the interlaboratory trial	1995
Number of laboratories	8
Number of samples	5 with addition of D-malic acid

Sample	A	B	C	D	E
Number of laboratories retained after elimination of laboratories presenting aberrant results Number of laboratories presenting aberrant results Number of accepted results	7 1 35	8 - 41	7 1 35	8 - 41	7 1 36
Average value(x (mg/L)	161.7	65.9	33.1	106.9	111.0
Standard deviation of repeatability (sr) (mg/L) Relative standard deviation of repeatability (RSDr) (%)	4.53 2.8	4.24 6.4	1.93 5.8	4.36 4.1	4.47 4.00
Limit of repeatability (r) (mg/L)	12.7	11.9	5.4	12.2	12.5
Standard deviation of reproducibility (sR) (mg/L) Relative standard deviation of reproducibility (RSDR) (%)	9.26 5.7	7.24 11	5.89 17.8	6.36 5.9	6.08 5.5
Limit of reproducibility (R) (mg/L)	25.9	20.3	16.5	17.8	17.0

Types of samples:

A	Red wine
B	Red wine
C	White wine
D	White wine
E	White wine

OIV-MA-AS313-12B Determination of d-malic acid in wines at low concentrations using the enzymatic method

Type IV method

 

1. Field of application

The method described is applied to dosage, by the enzymatic means, of malic acid D of wines with contents under 50 mg/l.

2. Principle

The principle of the method is based on  malic acid D(+) oxidation (D-malate) by nicotinamide-adenine-dinucleotide (NAD) in oxaloacetate that is transformed into pyruvate and carbon dioxide; the formation of NADH, measured by the increase of absorbance in wave length at 340 nm, is proportional to the quantity of D-malate present (principle of the method described for malic acid D determination for concentrations above 50 mg/l), after introducing a quantity of malic acid D of 50 mg/l in a cuvette.

3. Reagents

Malic acid D solution of 0.199 g/l, above reagents indicated in the methods described for contents above 50 mg/l.

4. Apparatus

Apparatus indicated in the method described for concentration above 50 mg/l.

5. Sample preparation

Sample preparation is indicated in the method described for concentrations above 50 mg/l.

6. Procedure

The procedure is indicated in the method described for concentrations above 50 mg/l. (Resolution Oeno 6/98), but with the introduction in the tank of a quantity of malic acid D equivalent to 50 mg/l. (Introduction of 0.025 mL of malic acid D at 0.199 g/l, substituting the equivalent volume of water); the values obtained are decreased by 50 mg/l.

7. Internal validation

Summary of the internal validation file on the dosage of malic acid D(+)-after the addition of 50 mg/l of this isomer

Work level	0 mg of 70 mg of malic acid D(+)-per liter. Within these limits, the method is linear with a correlation coeffiency between 0.990 and 0.994
Setting limit	24.4 mg/l
Detection limit	8.3 mg/l
Sensitivity	0.0015 abs / mg/l
Recovery percent range	87.5 to 115.0% for white wines and 75 to 105% for red wines
Repeatability	=12.4 mg/l for white wines (according to the OIV method =12,5 mg/l) =12.6 mg/l for red wines (according to OIV method=12,7 mg/l)
Percentage standard deviation	4.2% to 7.6% (white wines and red wines)
Intralaboratory variability	CV=7.4% (s=4.4mg/l; X average=59.3 mg/l)

8. Bibliography

• Chretien D., Sudraud P., 1993. Présence naturelle d'acide D(+)-malique dans les moûts et les vins, Journal International des Sciences de la Vigne et du Vin, 27: 147-149.
• Chretien D., Sudraud P., 1994. Présence naturelle d'acide D(+)-malique dans les moûts et les vins, Feuillet Vert de l'OIV, 966.
• Delfini C., Gaetano G., Gaia P., Piangerelli M.G., Cocito C., 1995. Production of D(+)-malic acid by wine yeasts, Rivista de Viticoltura e di Enologia, 48: 75-76.
• OIV, 1998. Recueil des méthodes internationales d'analyse des vins et des moûts.Mise à jour Septembre 1998. OIV, Paris.
• Przyborski H., Wacha C., Bandion F., 1993. Zur bestimmung von D(+)Apfelsäure in wein, Mitteilung Klosterneuburg, 43: 215-218.
• Machado M. and Curvelo-Garcia A.S., 1999; FV.O.I.V. N° 1082, Ref. 2616/220199.

OIV-MA-AS313-14A Sorbic acid

Type IV method

1. Principle of Method

 

Determination using ultraviolet absorption spectrophotometry

Sorbic acid (trans, trans, 2,4-hexadienoic acid) extracted by steam distillation is determined in wine distillate by ultraviolet absorption spectrophotometry. Substances that interfere with the measure of absorption in ultraviolet are removed by evaporation to dryness using a slightly alkaline calcium hydroxide solution. Samples with less than 20 mg/L are confirmed using thin layer chromatography (sensitivity: 1 mg/L).

2. Determination by ultraviolet absorption spectrophotometry
   1.    Apparatus
      1. Steam distillation apparatus (see chapter "Volatile Acidity")
      2. Water bath 100 °C
      3. Spectrophotometer allowing absorbance measurements to be made at a wavelength of 256 nm and having a quartz cell with a 1 cm optical path
   2.    Reagents
      1. Crystalline tartaric acid
      2. Calcium hydroxide solution, approx. 0.02 M
      3. Sorbic acid standard solution, 20 mg/L:

Dissolve 20 mg sorbic acid in approximately 2 mL 0.1 M sodium hydroxide solution. Pour into a 1 L volumetric flask, and make up to volume with water. Alternatively dissolve 26.8 mg of potassium sorbate,, in water and make up to 1 L with water.

2.3.   Procedure

2.3.1. Distillation

Place 10 mL of wine in the bubbler of the steam distillation apparatus and add about 1 g tartaric acid. Collect 250 mL of distillate.

2.3.2. Preparation of the calibration curve

Prepare, by dilution of the standard solution (2.2.3) with water, four dilute standard solutions containing 0.5, 1.0, 2.5 and 5 mg of sorbic acid per liter.  Measure their absorbance with the spectrophotometer at 256 nm using distilled

water as a blank.  Plot a curve showing the variation of absorbance as a function of concentration. The relationship is linear.

2.3.3.      Determination

Place 5 mL of the distillate in an evaporating dish of 55 mm diameter, add 1 mL of calcium hydroxide solution (2.2.2).  Evaporate to dryness on a boiling water bath.  Dissolve the residue in several mL of distilled water, transfer completely to a 20 mL volumetric flask and bring to volume with the rinsing water.  Measure the absorbance at 256 nm using a solution obtained by diluting 1 mL of calcium hydroxide solution to 20 mL with water as the blank.  Plot the value of the absorbance on the calibration curve and from this interpolate the concentration C of sorbic acid in the solution.

Note: In this method the loss due to evaporation is negligible and the absorbance is measured on the treated distillate diluted 1/4 with distilled water.

2.4.   Expression of results

2.4.1. Calculation

The sorbic acid concentration in the wine expressed in mg/L is given by:

C = concentration of sorbic acid in the solution obtained in 2.3.3 expressed in mg/L.

Bibliography

• Jaulmes P., Mestres R. & Mandrou B., Ann. Fals. Exp. Chim., n° spécial, réunion de Marseille, 1961, 111-116.
• Mandrou, B., Brun, S. & Roux E., Ann. Fals. Exp. Chim., 1975, 725, 29-48.
• Chretien D., Perez L. & Sudraud P., F.V., O.I.V., 1980, n° 720

OIV-MA-AS313-14C Sorbic acid

Type IV method

 

1. Principle of Methods

 

Identification of traces by thin-layer chromatography

Sorbic acid extracted in ethyl ether is separated by thin layer chromatography and its concentration is evaluated semi-quantitatively.

2. Identification of traces of sorbic acid by thin layer chromatography

 

2.1.  Apparatus

2.1.1.      Precoated 20 x 20 cm plates for thin layer chromatography coated with polyamide gel (0.15 mm thick) with the addition of a fluorescence indicator

2.1.2.      Chamber for thin layer chromatography

2.1.3.      Micropipette or microsyringe for delivering volumes of 5 μL to within 0.1 μL

2.1.4.      Ultraviolet lamp (254 nm)

2.2.  Reagents

2.2.1.      Diethyl ether, (

2.2.2.      Aqueous sulfuric acid solution: sulfuric acid (ρ₂₀= 1.84 g/mL), diluted 1/3 (v/v)

2.2.3.      Standard solution of sorbic acid, approximately 20 mg/L, in a 10% (v/v) ethanol/water mixture.

Mobile phase: hexane + pentane + acetic acid (20:20:3).

2.3.  Procedure

2.3.1.      Preparation of sample to be analyzed

Into a glass test tube of approximately 25 mL capacity and fitted with a ground glass stopper, place 10 mL of wine; add 1 mL of dilute sulfuric acid (2.2.2) and 5 mL of diethyl ether (2.2.1).  Mix by repeatedly inverting the tube. Allow to settle.

2.3.2.      Preparation of dilute standard solutions

Prepare five dilute standard solutions from the solution in 2.2.3. containing 2, 4, 6, 8 and 10 mg sorbic acid per liter.

2.3.3.      Chromatography

Using a microsyringe or micropipette, deposit 5 μL of the ether-extracted phase obtained in 2.3.1 and 5 μL each of the dilute standard solutions (2.3.2) at points 2 cm from the lower edge of the plate and 2 cm apart from each other.

Place the mobile phase in the chromatograph tank to a height of about 0.5 cm and allow the atmosphere in the tank to become saturated with solvent vapor. Place the plate in the tank. Allow the chromatogram to develop over 12 to 15 cm (development time approximately 30 minutes). Dry the plate in a current of cool air. Examine the chromatogram under a 254 nm ultraviolet lamp.

The spots indicating the presence of sorbic acid will appear dark violet against the yellow fluorescent background of the plate.

2.4.  Expression of the results

A comparison of the intensities of the spots produced by the test sample and by the standard solutions will enable a semi-quantitative assessment of a sorbic acid concentration between 2 and 10 mg/L. A concentration equal to 1 mg/L may be determined by using a 10 μL sample size.

Concentrations above 10 mg/L may be determined using a sample volume of less than 5 μL (measured out using a microsyringe).

Bibliography

• Jaulmes P., Mestres R. & Mandrou B., Ann. Fals. Exp. Chim., n° spécial, réunion de Marseille, 1961, 111-116.
• Mandrou, B., Brun, S. & Roux E., Ann. Fals. Exp. Chim., 1975, 725, 29-48.
• Chretien D., Perez L. & Sudraud P., F.V., O.I.V., 1980, n° 720

OIV-MA-AS313-15 pH

Type I method

 

1. Principle

The difference in potential between two electrodes immersed in the liquid under test is measured. One of these two electrodes has a potential that is a function of the pH of the liquid, while the other has a fixed and known potential and constitutes the reference electrode.

2. Apparatus

2.1.  pH meter with a scale calibrated in pH units and enabling measurements to be made to at least 0.01 pH units.

2.2.  Electrodes:

• glass electrode, kept in distilled water;
• calomel-saturated potassium chloride reference electrode, kept in a saturated solution of potassium chloride; or,
• a combined electrode, kept in distilled water.

3. Reagents

Buffer solutions

• Saturated potassium hydrogen tartrate solution, containing 5.7 g/L potassium hydrogen tartrate ( at 20°C. (This solution may be kept for up to two months by adding 0.1 g of thymol per 200 mL.)

pH

 Potassium hydrogen phthalate solution, 0.05 M, containing 10.211 g/L potassium hydrogen phthalate,, at 20oC.  (This solution may be kept for up to two months.)

pH

Solution containing:

potassium di-hydrogen phosphate,: 3.402 g

di-potassium hydrogen phosphate,:4.354 g

water to: 1 litre

(This solution may be kept for up to two months)

pH

Note: commercial reference buffer solutions traceable to the SI may be used.

For example:

pH 1.679 0.01 at 25°C

pH 4.005 0.01 at 25°C

pH 7.000 0.01 at 25°C

4. Procedure

4.1.  Zeroing of the apparatus

Zeroing is carried out before any measurement is made, according to the instructions provided with the apparatus used.

4.2.  Calibration of the pH meter

The pH meter must be calibrated at 20°C using standard buffer solutions connected to the SI. The pH values selected must encompass the range of values that may be encountered in musts and wines. If the pH meter used is not compatible with calibration at sufficiently low values, a verification using a standard buffer solution linked to the SI and which has a pH value close to the values encountered in the musts and wines may be used.

4.3.  Determination

Dip the electrode into the sample to be analyzed, the temperature of which should be between 20 and 25°C and as close as possible to 20°C. Read the pH value directly off the scale.

Carry out at least two determinations on the same sample.

The final result is taken to be the arithmetic mean of two determinations.

5. Expression of results

The pH of the must or the wine is reported to two decimal places.

OIV-MA-AS313-16 Determination of organic acids and mineral anions in wines by ionic chromatography

Type IV method

Preamble

The development of high performance ionic chromatography in laboratories has enabled the study the determination of organic acids and mineral anions in alcoholic and non alcoholic beverages by this technique.

Particularly concerning the analysis of wines, the results of intercomparison test trials and the measurements of recovery rates have enabled us to validate an analytical methodology.

The major interest of this method is that the ion exchange columns allow the separation of most organic acids and anions, and the detection by conductimetry frees the analysis from interferences due to the presence of phenolic compounds. This type of interference is very notable in chromatographic methods that include detection in ultra-violet radiation at 210 nm.

1. Object and field of application

This method for mineral anions and organic acids by ionic chromatography is applicable to alcoholic beverages (wines, wine spirits and liqueurs). It enables the determination of organic acids in the ranges of concentration listed in table 1; these concentrations are obtained by diluting samples.

Table 1: range of concentration of anions for their analysis by ionic chromatography

Sulfate: 0.1 to 10 mg/l

Ortho-phosphate: 0.2 to 10 mg/l

Malic acid: 1 to 20 mg/l

Tartaric acid: 1 to 20 mg/l

Citric acid: 1 to 20 mg/l

Isocitric acid: 0.5 to 5 mg/l

The ranges of the above-mentioned work are given as an example. They include the methods of calibration commonly practiced and are therefore adaptable according to the type of apparatus used (nature of column, sensitivity of the detector, etc.) and procedure (volume of sample injected, dilution, etc.).

2. Principle

Separation of mineral and organic anions on an ion exchanger resin.

Detection by conductimetry.

Identification after the retention time and quantification using the calibration curve.

3. Reagents

All the reagents used during the analysis must be of analytical quality. The water used for the preparation of solutions must be distilled or deionised water of a conductivity lower than 0.06 µS, free from anions determined at thresholds compatible with the detection limits of the apparatus used.

3.1.   Eluant

The composition of the eluant depends on the nature of the separation column and the nature of the sample to be analysed. Nevertheless it is always prepared using aqueous solutions of sodium hydroxide.

The performances of the chromatographic analysis are alternated by carbonation of the sodium hydroxide solution; consequently, the mobile phase flasks are swept with helium before adding sodium hydroxide and all precautions should be taken in order to avoid contaminating them with room air.

Lastly, commercial concentrated sodium hydroxide solutions will be used.

Remark

The table in chapter 9 mentions the main interferents susceptible of being present in the samples.

It is therefore necessary to know beforehand if they coelute with the ions to be determined and if they are present at such a concentration that the analysis is disrupted.

Fermented drinks contain succinic acid which can interfere with the malic acid determination. To this effect, it is necessary to add methanol to the eluant in order to improve the resolution of the column for these two substances (20% of methanol).

3.2.   Calibration reference solutions

Prepare calibration reference solutions of precise concentrations close to those indicated in the following table. Dissolve in water, quantities of salts or corresponding acids in 1000 ml volumetric flasks. (Table 2)

Table 2: Concentration of anions determined in calibration reference solutions

Anions and acids	Compounds weighed	Concentration final (mg/l)	Quantity weighed (mg)
Sulphate	Na2SO4	500	739.5
Orthophosphate	KH2PO4	700	1003.1
Malic acid	Malic acid	1000	1000.0
Tartaric acid	Tartaric acid	1000	1000.0
Citric acid	Citric acid, H2O	1000	1093.8
Isocitric acid	Isocitrate 3Na, 2H2O	400	612.4

Remark

The laboratory must take the necessary precautions regarding the hygroscopic character of certain salts.

3.3.   Calibration solutions

The calibration solutions are obtained by diluting the reference solutions of each ion or acid in water.

These solutions should contain all the ions or acids determined in a range of concentrations covering those corresponding to the samples to be analysed. They must be prepared the day of their use.

At least two calibration solutions and a blank must be analysed so as to establish, for each substance, the calibration curves using three points (0, maximum semi-concentration, maximum concentration).

Remark

Table 1 gives indications on the maximum concentrations of anions and acids in calibration solutions but the performances of the chromatographic columns are better with very diluted solutions.

So the best adequation possible between the performances of the column and the level of dilution of the samples should be looked for.

In general, the sample is diluted between 50 and 200 times maximum except for particular cases.

For prolonging the life span of the dilution solutions, it is preferable to prepare them in a water/methanol solution (80/20).

4. Apparatus

 

4.1.   Instrument system for ionic chromatography including:

4.1.1. Eluant reservoir(s)

4.1.2. Constant-stroke pump, without pulsing action

4.1.3. Injector, either manual or automatic with a loop sampling valve (for example 25 or 50 μl).

4.1.4. Separation columns

System made up of an anion exchanger column of controlled performance, possibly a precolumn of the same type as the main column. For example, it is possible to use the AS11 columns and DIONEX AG11 precolumn.

4.1.5. Detection system

Circulation conductivity cell of very low volume connected to a conductivity meter with several ranges of sensitivity.

In order to lower the conductivity of the eluant, a chemical suppression mechanism, a cation exchanger is installed in front of the conductivity cell.

4.1.6. Recorder, integrator or other device for the treatment of signals.

4.2.   Precise balance to 1 mg

4.3.   Volumetric flasks from 10 to 1000 ml

4.4.   Calibrated pipettes from 1 to 50 ml

4.5.   Filtrating membranes with an average pore diameter of 0.45 μm.

5. Sampling

 

The samples are diluted while taking into account the mineral anions and organic acids that are to be determined.

If their concentration is very variable in the sample, two levels of dilution will be necessary in order to respect the ranges of concentration covered by the calibration solutions.

6. Procedure

Turn on the apparatus by following the manufacturer’s instructions.

Adjust the pumping (eluant flux) and detection conditions so as to obtain good separations of the peaks in the range of concentrations of ions to be analysed.

Allow the system to balance until a stable base line is obtained.

6.1.   Calibration

Prepare the calibration solutions as indicated in 3.3.

Inject the calibration solutions so that the volume injected is at least 5 times that of the sampling loop to allow the rinsing of the system.

Trace the calibration curves for each ion. These must normally be straight.

6.2.   Blank trial

Inject the water used for the preparation of the calibration solutions and samples.

Control the absence of parasite peaks and quantify the mineral anions present (chloride, sulphate, etc.).

 

6.3.   Analysis

Dilute the sample possibly at two different levels as indicated in 5, so that the anions and acids to be determined are present in the range of concentrations of the calibration solutions.

Filter the diluted sample on a filtrating membrane (4.5) before injection.

Then proceed as for the calibration (6.1).

7. Repetability, reproducibility

 

An interlaboratory circuit tested this method, but this does not constitute a formal validation according to The OIV protocol (Oeno 6/99).

A repeatability limit and a reproducibility limit for the determination of each ion in wine were calculated according to the ISO 5725 standard.

Each analysis was repeated 3 times.

Number of participating laboratories: 11; the results were as follows:

White wine

	No labs	Average (mg/l)	Repeatability (mg/l)	Reproducibility (mg/l)
Malic acid	11/11	2745	11à	559
Citric acid	9/11	124	13	37
Tartaric acid	10/11	2001	96	527
Sulphate	10/11	253	15	43
O.phosphate	9/11	57	5	18

Red wine

	No labs (mg/l)	Average	Repeatability (mg/l)	Reproducibility (mg/l)
Malic acid	8/11	128	16	99
Citric acid	8/10	117	8	44
Tartaric acid	9/11	2154	48	393
Sulphate	10/11	324	17	85
O.phosphate	10/11	269	38	46

8. Calculation of recovery rate

The supplemented sample is a white wine.

Determination	No labs	Concentration initial (mg/l)	Real addition (mg/l)	Measured addition (mg/l)	Recovery rate (%)
Citric acid	11/11	122	25.8	24.2	93.8
Malic acid	11/11	2746	600	577	96.2
Tartaric acid	11/11	2018	401	366	91.3

9. Risks of interferences

Any substance whose retention time coincides with that of one of the ions analysed can constitute an interference.

The most common interference include the following:

Anions	Or interferents acids
Nitrate	Bromide
Sulphate	Oxalate, maleate Ortophophatephtalate
Malic acid	Succinic acid, citramalic acid
Tartric acid	Malonic acid
Citric acid	-
Isocitric acid	-

Remark: The addition of methanol in the mobile phase can resolve certain analytical problems.

---

OIV-MA-AS313-18 Determination of sorbic acid in wines by capillary electrophoresis

Type IV method

 

1. Scope

The present method is used to determine the sorbic acid in wines in a range from 0 to 300 mg/l.

2. Principle

The negatively charged sorbate ion naturally enables easy separation by capillary electrophoresis. At the capillary outlet, detection is carried out in the ultraviolet spectrum at 254 Nm.

3. Reagents and products

3.1.  Reagents

3.1.1.      Sodium dihydrogenophosphate [10049-21-5] purity > 96%

3.1.2.      Sodium hydrogenophosphate [10028-24-7] purity > 99%

3.1.3.      Sodium hydroxide [1310-73-2] purity > 97%

3.1.4.      Hippuric sodium [532-94-5] purity > 99%

3.1.5.      Demineralised water (< 15 MOHMS) or double-distilled

3.2.  Migration buffer solution

The migration buffer is made up in the following way:

• Sodium dihydrogenophosphate (3.1.1): 5 mM
• Sodium hydrogenophosphate(3.1.2) 5 mM
  3.   Internal standard

Hippuric spdium (3.1.4) in an aqueous solution 0.5 g.L-1

3.4.  Rinse solutions

3.4.1.      Sodium hydroxide (3.1.3) N/10

3.4.2.      Sodium hydroxide (3.1.3) N

4. Sample preparation

The wine samples are prepared as follows, which involves a 1/20 dilution:

Wine to be analyzed:  0.5 ml

Sodium hydroxide (3.1.3):0.5 ml

Internal standard (3.1.4) with 0.5 g. : 0.5 ml

Qsp 10 ml with demineralized water (3.1.5)

5. Operating conditions

5.1.  Conditioning the capillary

Before its first use, and as soon as the migration times increase, the capillary must be conditioned according to the following process:

5.1.1.      Rinse with sodium hydroxide solution 1N (3.4.2) at 20 psi (140 kPA) for 8 min.

5.1.2.      Rinse with sodium hydroxide solution (3.4.1) 0.1 N at 20 psi (140 kPA) for 12 min.

5.1.3.      Rinse with water (3.1.5) at 20 psi (140 kPA) for 10 min.

5.1.4.      Rinse with the migration buffer (3.2) at 20 psi (140 kPA) for 30 min.

5.2.  Migration conditions

These conditions may be slightly changed depending on the equipment used.

 

5.2.1.      The molten silica capillary is 31 cm long, with a diameter of 50 microns.

5.2.2.      Migration temperature: 25°C

5.2.3.      Reading wavelength: 254 nm.

5.2.4.      Reading of the signal in direct mode (sorbic acid absorbs in the UV spectrum).

5.2.5.      First Pre-rinse under pressure 30 psi (210 kPA) with sodium hydroxide solution 0.1 N (3.4.1) for 30 seconds

5.2.6.      Second Pre-rinse under pressure 30 psi (210 kPA) with the migration buffer (3.2) for 30 seconds.

5.2.7.      The injection is done under a pressure of 0.3 psi (2.1 kPA) for 10 seconds.

5.2.8.      The migration lasts approximately 1.5 to 2 minutes under a potential difference of + 25 kV, in normal polarity (cathod at the exit).

5.2.9.      Certain capillary electrophoresis apparatus propose large-capacity vials for migration buffer solutions. This is preferable when several analyses are carried out in series, because the electrolytic properties are maintained longer.

5.3.  Reading the results

The absorption peaks for the internal standard and the sorbic acid are obtained on average 1 to 1.5 minutes after the start of the migration phase live. Migration time is fairly constant, but can slightly vary according to the state of the capillary. If the migration time degrades, reconditioning of the capillary is necessary, and if the nominal conditions are not restored, the capillary must be replaced.

6. Characteristics of the method

 

The different validation steps described were carried out according to the OIV resolution OENO 10/2005.

6.1.  Intralaboratory repeatability

Standard repeatability deviation Sr	1.6 mg/ L-1
Repeatability r	4.6 mg/ L-1

6.2.  Linearity

Regression line	Y = 0,99491 X + 2,52727
Correlation coefficient r	0,9997
Residual standard deviation Sxy	1,6 mg.L-1
Standard deviation slope Sb	0,008 mg.L-1

 

6.3.  Intralaboratory reproducibility

 

Standard reproductibility deviation Sr	2.1 mg/ L-1
Reproductibility R	5.8 mg/ L-1

6.4.  Detection and quantification limits

Detection limit Ld	1.8 mg/ L-1
Quantification limit Lq	4.8 mg/ L-1

6.5.  Robustness

6.5.1.      Determination

Since the method is relative, any slight variations in the analysis conditions will have no effect on the final result, but will primarily influence the migration time.

6.6.  Method specificity

Possible influence of principle oenological additives were tested. None of them modify the results obtained.

6.7.  Correlating the method with the OIV reference method

The OIV reference method is determination by ultraviolet absorption spectrometry. The sorbic acid, extracted by steam distillation, is determined in the wine distillate by ultraviolet absorption spectrometry at 256 Nm.

6.7.1.      Comparison of repeatabilities

	Capillary electrophoresis	OIV reference method
Standard deviation of repeatability Sr	1.6 mg/l	2.5 mg/ L-1
Repeatability r	4.6 mg/l	7.0 mg/ L-1

 

6.7.2.      Accuracy of the usual method in relation to the reference method

Coefficient of correlation r	0.999
Average bias Md	0.03 mg L-1
Average bias standard deviation Sd	3.1 mg L-1
Z-score (Md/Sd)	0.01

 

OIV-MA-AS313-19 Determination of the principal organic acids of wines and sulphates by capillary electrophoresis

Type II method (for organic acids)

Type III method (for sulphate)

1. Introduction

Tartaric, malic and lactic acids and sulphates are separated and assayed by capillary electrophoresis after simple dilution and addition of an internal standard.

2. Title

Determination of the principal organic acids of wines and sulphates by capillary electrophoresis

3. Scope

Capillary electrophoresis can be used to assay the tartaric and malic acid in musts, as well as the tartaric, malic and lactic acids and sulphates in wines that have been diluted, degassed and filtered beforehand if need be.

4. Definitions

4.1.  Capillary electrophoresis

Capillary electrophoresis: all the techniques that use a capillary tube of very small diameter with an appropriate buffer solution to effectively separate small and large electrically charged molecules in the presence a high-voltage electric current.

4.2.  Buffer for electrophoresis

Solution containing one or more solvents and aqueous solutions with suitable electrophoretic mobilities to buffer the pH of the solution.

4.3.  Electrophoretic mobility

Aptitude of an ion to move quickly under the effect of an electric field.

4.4.  Electroosmotic flow

Flow of solvent in the buffer solution along the internal wall of the capillary tube due to displacement of the solvated ions under the effects of the field and the electric charges of the silica.

5. Principle

Separations of the aqueous solutions of a mixture by capillary electrophoresis are obtained by differential migrations in a buffered electrolyte referred to as a buffer. The electrophoresis takes place in a silica tube with an inside diameter ranging between 25 and 75 µm. The aqueous solutions to be separated are simultaneously driven by 2 forces that can act in the same direction or in the opposite direction. These two forces are caused by the electric field and the electroosmotic flow.

The electric field is represented by the voltage in volts applied between the electrodes brought to within one centimetre of the capillary tube, and is expressed in V.cm^-1. Mobility is a characteristic of ions. The smaller the molecules, the greater their electrophoretic mobility. 

If the internal wall of the capillary tube is not coated, the negative electric charges of the silica fix part of the cations of the buffer. The solvation and displacement towards the cathode of part of the cations of the buffer create the electroosmotic flow. The pH of the buffer and additives can be chosen in order to control the direction and the intensity of the electroosmotic flow.

The addition of a chromophoric ion in the buffer can be used to obtain negative peaks that quantitatively represent the solutions to be separated which do not absorb at the used wavelength.

6. Reagents and products

6.1.  Chemically pure grade products for analysis at least at 99%

6.1.1.     Sodium sulphate or Potassium sulphate

6.1.2.     L-tartaric acid

6.1.3.     D,L- malic acid

6.1.4.     Monohydrated citric acid

6.1.5.     Succinic acid

6.1.6.     D,L Lactic acid

6.1.7.     Sodium dihydrogenophosphate

6.1.8.     Sodium gluconate

6.1.9.     Sodium chlorate

6.1.10. Dipicolinic acid

6.1.11. Cethyltrimethyl ammonium bromure

6.1.12. Acetonitrile for HPLC

6.1.13. Deionized ultra filtered pure water

6.1.14. Sodium hydroxide

6.2.  Solutions

6.2.1.     Calibration stock solution

Solution in pure water (6.1.13) of different acids and sulphates to be measured (6.1.1 to 6.1.6) at exact known concentrations ranging between 800 and 1200 mg l^-1

Solution to be kept at +5° C for a maximum of 1 month

6.2.2.     Internal standard solution

Solution of sodium chlorate (6.1.9) at approximately 2 g l^-1  in pure water (6.1.13)

Solution to be kept at +5° C for a maximum of 1 month

6.2.3.     Calibration solution to be injected

In a graduated 50-ml class "A" flask using class "A" pipettes, deposit:

• 2 ml of calibration solution (6.2.1)
• 1 ml of internal standard solution (6.2.2)
• Adjust solution to 50 ml with pure water (6.1.13)

Homogenize by agitation

Solution to be prepared each day

6.2.4.     Sodium hydroxide solutions

6.2.4.1.           Sodium hydroxide solution M

In a 100-ml flask place 4g of sodium hydroxide (6.1.14)

Adjust with pure water (6.1.13)

Shake until completely dissolved.

6.2.4.2.           sodium hydroxide solution 0.1M

In a 100 ml flask place 10 ml of sodium hydroxide M (6.2.4.1)

Adjust with pure water (6.1.13)

Homogenise.

6.2.5.     Electrophoretic buffer solution

In a graduated 200-ml class "A" flask, place:

• 0.668 g of dipicolinic acid (6.1.10)
• 0.364 g of cethyltrimethyl-ammonium bromide. (6.1.11)
• 20 ml of acetonitrile (6.1.12)
• Approximately 160 ml of pure water (6.1.13)
• Shake until complete dissolution (if need be, place in ultrasound bath to eliminate any aggregated material)
• Bring M sodium hydroxide solution M (6.2.4.1) to pH 5.64 and then 0.1M sodium hydroxide (6.2.4.2)
• Make up to 200 ml with pure water (6.1.13)
• Homogenize by agitation
• Solution to be prepared each month.
• Store at laboratory temperature.

This buffer can be replaced by equivalent commercial product.

7. Apparatus

The capillary electrophoresis apparatus required for these determinations basically comprises:

• A sample changer
• Two bottles (phials) containing the buffer
• A non-coated silica capillary tube, internal diameter 50 µm, length 60 cm, between the inlet of the capillary tube and the detection cell. Depending on the apparatus, an additional 7 to 15 cm are required so that the outlet of the capillary tube is immersed in the centre of another bottle
• A high voltage DC power supply capable of outputting voltages of -30 to + 30 kV. The electrodes immersed in the two bottles where the outlets of the capillary tube emerge are connected to the terminals of the generator
• A pressurization system capable of circulating the buffer in the capillary tube and enabling the injection of the test specimen
• A UV detector
• A data acquisition system

8. Preparation of samples for tests

8.1.  Degassing and filtration

The samples rich in carbon dioxide are degassed for 2 min with ultra-sound. Turbid samples are filtered on a membrane with an average pore diameter of 0.45 µm.

8.2.  Dilution and addition of internal standard

Place 2 ml of sample in a graduated flask of 50 ml. Add 1 ml of internal standard solution (6.2.2). Adjust to 50 ml with pure water (6.1.13)

 Homogenize.

9. Procedure

9.1.  Conditioning of a new capillary tube (for example)

• Circulate pure water (6.1.13) in the opposite direction (from the outlet of the capillary tube towards the inlet flask) for 5 min at a pressure of approximately 40 psi (2.76 bar or 276 kPa)
• Circulate 0.1M sodium hydroxide (6.2.4.2) in the opposite direction for 5 min at the same pressure
• Circulate pure water (6.1.13) in the opposite direction (from the outlet of the capillary tube towards the inlet flask) for 5 min at the same pressure
• Repeat the cycle of circulating pure water, 0.1M sodium hydroxide , pure water
• Circulate electrophoretic buffer (6.2.5) in the opposite direction for 10 min

9.2.  Reconditioning a capillary tube in the course of use (optional)

• When the quality of the separations becomes insufficient, new conditioning of the capillary tube is essential. If the results obtained are still not satisfactory, change capillary tube and condition it.

9.3.  Checking the quality of the capillary tube (optional)

• Analyse 5 times the calibration solution under the recommended analysis conditions.

9.4.  Separation and detection conditions (for example)

• Light the detector lamp 1 hour before the start of the analyses
• Rinse the capillary tube by circulating the buffer for 3 min in the opposite direction at a pressure of 40 psi
• Pressure inject the samples (prepared at 8.1) at 0.5 psi for 6 to 15 seconds
• The polarity is regulated such that the anode is on the detector side
• Apply a voltage from 0 to 16 kV in 1 min then 16 kV for approximately 18 min (the duration of separation can slightly vary depending on the quality of the capillary tube)
• Maintain the temperature at + 25 C°
• Detection in the ultraviolet is at 254 Nm
• Rinse the capillary tube by circulating the electrophoretic buffer (6.2.5) for 2 min in the opposite direction at a pressure of 40 psi
• Change the electrophoretic buffer (6.2.5) contained in the inlet and outlet flasks at least every 6 injections
  5.   Order that the analyses are to be carried out (for example)

Change the electrophoretic buffer (6.2.5) for every new series of analyses

The sequence of analysis in order contains:Analysis of reference material (external concentration sample known for different acids to be measured)

Analysis of samples prepared in 8.2,chromatograms should look like those presented in appendix A

At the end of analysis, rinse with pure water (6.1.13) 10 mm in opposite direction (outlet of capillary tube toward the inlet)

Switch off detector lamp

10. Calculation of results

The calculations are based on the surface areas of the peaks obtained after integration.

The surface areas of the peaks of the aqueous solutions of the calibration solution (6.2.3) are corrected by taking into account the variations in the surface areas of the peaks of the internal standard. The response factor for each acid is calculated.

The surface areas of the peaks of the internal standard and the peaks of the aqueous solutions are read off for each sample. The surface areas of the aqueous solutions to be assayed are recalculated by taking into account variations in the surface areas of the peaks of the internal standard a second time in order to obtain "corrected" surface areas.

The corrected surface areas are then multiplied by the value of the corresponding response factor.

It is possible to use an automatic data management system, so that they can be controlled in accordance with the principles described above as well as with the best practices (calculation of response factor and / or establishment of a calibration curve).

Calculation formula

The abbreviations used to calculate the concentration in an acid are given in the following table:

Surfaces are expressed by the whole numbers of integration units.

The concentrations are given in g/L (only indicate to two decimal places).

ABBREVIATIONS
	REFERENCE SOLUTION	SAMPLE
SURFACE AREAS OF TITRATED PEAKS
INTERNAL STANDARD PEAKS
CONCENTRATION

The calculation formula is:

Whenever possible, a duplicate analysis is used to highlight a possible error in the recognition of the peaks or inaccuracy of integration. The sample changer makes it possible to carry out the analyses in automatic mode day and night.

11. Precision

11.1.        Organization of the tests

Interlaboratory trials and correspondent results are described in appendix B1 and B2

11.2.        Measurement of precision

Assessement of precision by interlaboratory trials

Number of laboratories involved: 5

12. Appencides

Appendix A: Electrophoregram of a standard solution of ACI

Electropherogram of a wine

Appendix B1

Statistic data obtained from the results of the interlaboratory trials (2006)

According to ISO 5725-2:1994, the following parameters have been defined during an interlaboratory trial. This trial has been conducted by the laboratory « Direction Générale de la Consommation et de la Répression des Fraudes de Bordeaux (France). »

Year of interlaboratory trial: 2006

Number of laboratories: 5

Number of samples: 8 double-blind (2 dry white wines, 2 sweet white wines, 2 rosé wines and 2 red wines)

Appendix B2

Statistic data obtained from the results of the interlaboratory trials (sulphates 2010)

According to ISO 5725-2:1994, the following parameters have been defined during an interlaboratory trial. This trial has been conducted by the laboratory “Instituto dos Vinhos do Douro e do Porto (Portugal)”

Year of interlaboratory trial: 2010-2011

Number of laboratories: 7 (one laboratorysent two sets of results obtained by means of  two different instruments)

Number of samples: 6 double-blind

13. Bibliography

• ARELLANO  M., COUDERC  F. and PUIG .L  (1997): Simultaneous separation of organic and inorganic acids by capillary zone electrophoresis. Application to wines and fruit juices. Am. J. Enol. Vitic., 48, 408-412.
• KANDL T. and KUPINA  S. (1999): An improved capillary electrophoresis procedure for the determination of organics acids in grape juices and wine. Am. J. Enol. Vitic., 50, 155-161.
• KLAMPF C.F. (1999): Analysis of organic acids and inorganic anions in different types of beer using capillary zone electrophoresis. J. Agric. Food Chem., 47, 987-990.

OIV-MA-AS313-20 Determination of sorbic, benzoic and salicylic acid content in wine by the use of high-performance liquid chromatography

Type IV method

 

1. Introduction

Sorbic acid and its potassium salt constitute an antiseptic that can be used in wine-making, although some countries will not tolerate even traces of it, the main reason being the smell of geraniums that develops when sorbic acid is broken down by lactic acid bacteria. Benzoic acid and salicylic acid are still prohibited in wine, but are used in other beverages.

2. Scope

All wines and grape musts, especially those likely to contain only traces of sorbic, benzoic or salicylic acid (demonstration from 1 mg/l).

3. Principle

The antiseptics are determined using HPLC by direct injection of the sample into a column functioning by isocratic reversed-phase partition chromatography with ultraviolet detection at a wavelength of 235 nm.

4. Products

4.1.  Micro-filtered fresh water (e.g. resistivity greater than 18.2 M)

4.2.  Pure tetrahydrofuran

4.3.  Pure methanol

4.4.  0.1 M hydrochloric acid (prepared by means of dilution funnels)

4.5.  Water with a pH of 2: adjust the pH of 650 ml of water (4.1) to pH2 using a pH meter (5.5) and by adding 0.1 M hydrochloric acid drop by drop without stirring (4.4)

4.6.  Elution solution: mix 650 ml of water at pH2 (4.5) with 280 ml of methanol (4.3) and 7 ml of tetrahydrofuran (4.2)

Note: it is likewise possible to use other elution solvents, for example: 80% ammonium acetate 0.005M (0.38 g/l) adjusted to pH 4 with pure acetic acid + 20% acetonitrile.

4.7.  Pure sorbic acid

4.8.  Pure benzoic acid

4.9.  Pure salicylic acid

4.10.        Absolute alcohol

4.11.        50% vol. hydro-alcohol solution: put 500 ml of absolute alcohol (4.10) into a 1-litre flask and dilute to volume with distilled water (4.1)

4.12.        Stock solution of sorbic acids at 500 mg/l: dissolve 50 mg of sorbic acids (4.7), benzoic (4.8) and salicylic (4.9) acids in 100 ml of the 50% vol. hydro-alcohol solution (4.11)

4.13.        Sorbic, benzoic and salicylic acid surrogate solutions: dilute the stock solution (4.12) in the hydro-alcohol solution (4.11) in such a way as to obtain the final concentrations required. For example, for a solution of

• 200 mg/l: put 20 ml of stock solution (4.12) into a 50-ml flask and top up to the filling mark with 4.11.
• 1 mg/l: put 2 ml of stock solution (4.12) into a 50-ml flask and top up to the filling mark with 4.11.

Intermediate solutions may be produced in the same way to satisfy calibration requirements.

5. Apparatus

5.1.  Laboratory glassware, especially pipette and volumetric flasks

5.2.  Ultrasonic bath

5.3.  Vacuum filtration device for large volumes (1 litre) using membrane filters with a pore diameter of under 1 μm (generally 0.45 μm)

5.4.  Mini-filter for samples (1 to 2 ml) using membrane filters with a pore diameter of under 1 μm (generally 0.45 μm)

5.5.  pH meter

5.6.  Isocratic-mode liquid phase chromatograph equipped with an injection system for small volumes (for example), 10 or 20- μl loop valve.

5.7.  Detector capable of functioning at an ultraviolet rating of 235 nm and fitted with a circulating tank for HPLC (for example, 8 μl for 1 cm of optical thickness)

5.8.  A 5- μm stationary phase HPLC column of the silica-type with immobilisation by octadecyl groups (C18), length 20 cm, inside diameter 4 mm

5.9.  Data acquisition system

6. Preparation of samples and the elution solvent

6.1.  Filter the samples to be analysed using the mini-filter (5.4)

6.2.  Degas the elution solvent (4.6) for 5 minutes using the ultrasonic bath (5.2)

6.3.  Filter the solvent using the device in (5.4)

7. Procedure

7.1.  Column conditioning. Prior to injection, start the pump and rinse the column with the solvent for at least 30 minutes.

7.2.  Inject one of the surrogate solutions (4.13) to check system sensitivity and ensure the resolution of the peaks of the substances to be analysed is satisfactory.

7.3.  Inject the sample to be analysed. It is possible to analyse an identical sample, to which the acids sought have been added (adapt the amount added to the quantity observed during the previous analysis - for 1 mg present, add 1 mg, and so on).

Check the resolution of the peaks of the acids sought with the peaks of the wines (normally, there are none in this zone)

8. Calculation

Having located the peaks of the acids to be determined in the sample, compare the peak area with those of the acids of a surrogate solution (4.13) with a known concentration C.

For example, let s be the peak area of the acid to be determined, and S is the peak area of the solution (4.13) with concentration C

9. Characteristics of the method

	Sorbic acid	Benzoic acid	Salicylic acid
Linearity range	0 to 200 mg/l	0 to 200 mg/l	0 to 200 mg/l
Accuracy (rate of recuperation)	> 90 %	> 90 %	> 90 %
Répétabilité : r*	2%	3%	8%
Reproducibility: R*	8%	9%	12%
Detection limit	3 mg/l	3 mg/l	3 mg/l
Quantification limit	5 mg/l	6 mg/l	7 mg/l
Uncertainty	11%	12%	13%

Bibliography

• Dosage de l'acide sorbic dans les vins par chromatographie en phase gazeuse. 1978. BERTRAND A. et SARRE Ch., Feuillets Verts O.I.V., 654-681.
• Dosage de l'acide salicylic dans les vins par chromatographie en phase gazeuse. 1978. BERTRAND A. et SARRE Ch., Feuillets Verts O.l.V., 655-682.
• Dosage de l'acide benzoic, dans les sodas et autres produits alimentaires liquides, par chromatographie en phase gazeuse. 1978. BERTRAND A. et SARRE Ch. Ann. Fals. Exp. Chim. 71, 761, 35-39.

OIV-MA-AS313-21 Determination of the presence of metatartaric acid

 

Type IV method

1. Introduction

Metatartaric acid added to the wine to avoid tartaric precipitation is traditionally proportioned by the difference between the total tartaric acid following hot hydrolysis of metatartaric acid and natural tartaric acid preceding hydrolysis. However, taking into account the precision of the determination of tartaric acid, traces of metatartaric acid are not detectable by this method, and the additive, which is not accepted in certain countries, must therefore be characterised using a more specific method.

2. Scope

Wines likely to contain traces of metatartaric acid.

3. Principle

In relatively acid mediums, metatartaric acid forms an insoluble precipitate with cadmium acetate; it is the only one of all the elements present in must and wine to give such a precipitate .

Note: Tartaric acid is also precipitated with cadmium acetate, but only in the presence of an alcohol content greater than 25% vol. The precipitate redissolves in water, unlike the precipitate obtained with metatartaric acid.

The cadmium precipitate of metatartaric acid breaks down by heating with sodium hydroxide and releases tartaric acid. The latter produces a specific orange colour with ammonium metavanadate.

4. Reagents

4.1.  Cadmium acetate solution at 5 p.100

4.1.1.      Dihydrated cadmium acetate at 98%

4.1.2.      Pure acetic acid

4.1.3.      Distilled or demineralized water

4.1.4.      Cadmium acetate solution: dissolve 5 g of cadmium acetate (4.1.1) in 99 mL of water (4.1.3) add 1 mL of pure acetic acid (4.1.2)

4.2.  Sodium hydroxide 1M

4.3.  Sulfuric acid 1M

4.4.  Solution of ammonium metavanadate 2% w/v

4.4.1.      Ammonium metavanadate

4.4.2.      Trihydrated sodium acetate at 99%

4.4.3.      Sodium acetate solution at 27 p. 100: dissolve 478 g of sodium acetate (4.4.2) in 1 liter of water (4.1.3)

4.4.4.      Solution of ammonium metavanadate: dissolve 10 g of ammonium metavanadate (4.4.1) in 150 mL of sodium hydroxide 1 M (4.2) add 200 of the sodium acetate solution at 27 p. 100 (4.4.3) and fill to 500 mL with water (4.1.3)

4.4.5.      Ethanol at 96% vol.

5. Apparatus

5.1.  Centrifuge with a rotor capable of housing 50-mL bottles

5.2.  Spectrometer capable of operating in the visible spectrum and of housing cuvets with an optical thickness of 1 cm.

6. Operating method

6.1.  Centrifuge 50 mL of wine for 10 minutes at 11000 rpm

6.2.  Take 40 mL of limpid wine using a test-tube and place the sample in a centrifuge flask

6.3.  Add 5 mL of ethanol at 96% vol (4.5)

6.4.  Add 5 mL of the cadmium acetate solution (4.1.4)

6.5.  Mix and leave to rest for 10 minutes

6.6.  Centrifuge for 10 minutes at 11000 rpm

6.7.  Decant by completely reversing the flask (once) and throw away the supernatant.

In the presence of metatartaric acid, a lamellate precipitate is formed at the bottom of the tube.

In the absence of any precipitate, the sample will be regarded as free from metatartaric acid. In the contrary case, or if the presence of a light precipitate is to be established with certainty, proceed as follows:

6.8.  Wash the precipitate once with 10 mL of water (4.1.3) in the form of an energetic jet towards the bottom of the tube in order to detach the precipitate from the bottom

6.9.  Add 2 mL of cadmium acetate solution (4.1.4)

6.10.        Centrifuge at 11000 rpm for 10 minutes then throw away the supernatant by completely reversing the tube (once)

6.11.        After adding one mL of sodium hydroxide 1M (4.2), plunge the tube to be centrifuged for 5 minutes in a water bath at 100° C

6.12.        After cooling, add 1 mL of sulfuric acid 1M (4.3) and 1 mL of ammonium metavanadate solution (4.4.4)

6.13.        Wait 15 minutes

6.14.        Centrifuge for 10 minutes at 11000 rpm

6.15.        Pour the supernatant into a spectrophotometer tank and measure the absorbance at 530 nm, after determining the zero point with water (4.1.3)

Standard. In parallel, produce a standard comprising the same wine as that analyzed but heated beforehand for 2.5 minutes using a microwave generator set to maximum power or with a water bath at 100° C for 5 minutes.

7. Calculation

The presence of metatartaric acid in the wine is established when, at 530 nm:

OIV-MA-AS313-22 Simultaneous determination of L-ascorbic acid and D-iso-ascorbic acid (erythorbic acid) in wine by HPLC and UV-detection

Type II method

 

1. Introduction

Ascorbic acid is an antioxidant that is naturally occurring in a wide range of foods. The natural amount of ascorbic acid in grapes decreases during must and wine production, but it can be added to musts and to wines within certain limits.

The method described has been validated in a collaborative study by the analyses of wine samples with spiked amounts of 30 mg/L to 150 mg/l for L-ascorbic acid and 10 mg/L to 100 mg/l for D-isoascorbic acid respectively.

2. Scope

This method is suitable for the simultaneous determination of L-ascorbic acid and D-iso-ascorbic acid (erythorbic acid) in wine by high performance liquid chromatography and UV-detection in a range of 3 mg/L to 150 mg/l.

For contents above 150 mg/l, sample dilution is necessary.

3. Principle

 

The samples are directly injected into the HPLC system after membrane filtration. The analytes are separated on a reversed phase column and UV-detection at 266 nm. The quantification of L-ascorbic acid and D-iso-ascorbic acid is done with reference to an external standard.

Note: The columns and operating conditions are given as example. Other types of columns may also give a good separation.

4. Reagents and Material

 

Reagents

• N-octylamine, puriss. 99.0 %
• Sodium acetate, 3, puriss 99.0 %
• Pure acetic acid, 100 %
• Phosphoric acid, approx. 25%
• Oxalic acid, puriss. 99.0 %

Ascorbate oxidase

• L-ascorbic acid, ultra 99.5 %
• D-iso-ascorbic acid, puriss. 99.0 %
• Bi-distilled water
• Methanol, p.A. 99.8 %

Preparation of the mobile phase

Solutions for the mobile phase

For the mobile phase prepare the following solutions:

• 12.93 g n-octylamine in 100 ml methanol
• 68.05 g sodium acetate, 3 in 500 ml bi-distilled water
• 12.01 g pure acetic acid in 200 ml bi-distilled water

Buffer solution (pH 5.4) : 430 ml sodium acetate solution (4.2.1.2) and 70 ml acetic acid solution (4.2.1.3)

Preparation of the mobile phase

Add 5 ml of n-octylamine solution (4.2.1.1) to approximately 400 ml bi-distilled water in a beaker. Adjust this solution to a pH of 5.4 to 5.6 by adding 25% phosphoric acid (4.1.4) drop by drop. Add 50 ml of the buffer solution (4.2.1.4), transfer the composite mix to a 1000 ml volumetric flask and fill up with bi-distilled water. Before use, the mobile phase has to be filtered through a membrane (regenerated cellulose, 0.2 μm) and if possible degassed with helium (approximately 10 minutes) depending on the needs of the HPLC system used.

Preparation of the standard solution

Note: All standard solutions (stock solution 4.3.1. and working solutions 4.3.2) have to be prepared daily and preferably stored cold in a refrigerator prior to injection.

Preparation of the stock solution (1 mg/ml)

Prepare a 2% aqueous oxalic acid solution and eliminate dissolved oxygen by blowing through nitrogen.

Weigh exactly 100 mg each of L-ascorbic acid and D-iso-ascorbic acid in a 100 ml volumetric flask and make to the mark with the 2% aqueous oxalic acid solution.

Preparation of the working solutions

For the working solutions dilute the stock solution (4.3.1) to the desired concentrations with the 2% oxalic acid solution. Concentrations between 10 mg/l and 120 mg/l are recommended, e.g. 100 μl, 200 μl, 400 μl, 800 μl, 1200 μl to 10 ml, corresponding to 10, 20, 40, 80 and 120 mg/l.

5. Apparatus

Usual laboratory equipment, in particular the following:

5.1.  HPLC-puml

5.2.  Loop injector, 20 μl

5.3.  UV-detector

6. Sampling

 

Wine samples are fltered through a membrane with pore size 0.2 μm before injection.

For contents above 150 mg/L, it is necessary to dilute the sample.

7. Procedure

7.1.  Operating conditions for HPLC

Inject 20 μl of the membrane-filtered sample into the chromatographic apparatus.

• Precolumn: e.g. Nucleosil 120 C18 (4cm x 4 mm x 7 μm)
• Column: e.g. Nucleosil 120 C18 (25 cm x 4 mm x 7 μm)
• Injection Volume: 20 μl
• Mobile Phase:  see 4.2.2, isocratic
• Flow rate: 1ml/min
• UV-detection: 266 nm
• Rinse cycle: at least 30ml bi-distilled water followed by 30ml methanol and 30ml acetonitrile

7.2.  Identification/Confirmation

Identification of peaks is done by the comparison of retention times between standards and samples. With the chromatographic system described as an example, the retention times are: for L-ascorbic acid 7.7 min. and for D-iso-ascorbic 8.3 min. respectively. (See figure 1, chromatogram A).

For further confirmation of positive findings these samples should be treated with a spatula of ascorbate oxidase and measured again (see figure 1, chromatogram B).

As a result of the degradation of L-ascorbic acid and D-iso-ascorbic acid caused by the ascorbate oxidase, no signal should be found at the retention time of L-ascorbic acid and D-iso-ascorbic acid. If interfering peaks are detected, their peak area should be taken into account for the calculation of the concentration of the analytes.

Figure 1: Example of a chromatogram of white wine: A: prior to treatment with ascorbate oxidase; B: after treatment

Note: It is recommended to analyse the ascorbate oxidase treated samples at the end of a sequence, followed by the rinse cycle for removing remaining ascorbate oxidase from the column. Otherwise the L-ascorbic acid and the D-iso-ascorbic acid may be converted by the remaining ascorbate oxidase during the HPLC-measurement and the result may be altered.

8. Calculation

Prepare a calibration curve from the working solutions (4.3.2). Following the method of external standard the quantification of L-ascorbic acid and D-isoascorbic acid is performed by measuring the peak areas and comparing them with the relevant concentration in the calibration curve.

Expression of results

The results are expressed in mg/l L-ascorbic acid and D-isoascorbic acid respectively with one decimal (e.g. 51,3 mg/l).

For contents above 150 mg/L, take into account the dilution.

9. Precision

 

The method was tested in a collaborative study with 27 laboratories participating, organised by the former Bundesgesundheitsamt (Germany) in 1994.  The design of the collaborative trial followed the § 35 of the German Food Law that has been accepted by the O.I.V until the new protocol (OENO 6/2000) was introduced.

The study included four different samples of wine - two white wines and two red wines - of which five repetitions of each were requested. Due to the fact that it was not possible to prepare samples with a sufficient stability of the analytes (different degradation rates) it was decided to send defined amounts of pure standard substances together with the wine samples to the participants. The laboratories were advised to transfer the standards quantitatively to the wine samples and to analyse them immediately. Amounts of 30 to 150 mg/l for L-ascorbic acid and 10 to 100 mg/l for D-iso-ascorbic acid were analysed. In the Annex the detailed study results are presented. Evaluation was done following the DIN/ISO 5725 (Version 1988) standard.

The standard deviations of repeatability( ) and reproducibility () were coherent with the L-ascorbic acid and D-iso-ascorbic acid concentrations. The actual precision parameters can be calculated by the following equations:

L-ascorbic acid:

• = 0.011x + 0.31
• = 0.064x + 1.39

x: L-ascorbic acid concentration (mg/l)

D-iso-ascorbic acid

•   = 0.014 x + 0.31
• = 0.079 x + 1.29

x: D-iso-ascorbic acid concentration (mg/l)

Example:

D-iso-ascorbic acid 50 mg/l

• = 1.0 mg/l
• = 5.2 mg/l

10. Other characteristics of the analysis

10.1.        Limit of detection

The limit of detection of this method was estimated at 3mg/l for L-ascorbic acid and D-iso-ascorbic acid

10.2.        Trueness

The mean recovery calculated from the collaborative trial over four samples (see Annex) was:

• 100.6 % for L-ascorbic acid
• 103.3 % for D-iso-ascorbic acid

11. Annex: Collaborative Trial

• L-Ascorbic Acid

		Red Wine I	White Wine II	Red Wine III	White Wine IV
X	mg/l	152.7	119.8	81.0	29.9
Amount spiked	mg/l	150	120	80	30
Recovery	%	101.8	99.8	101.3	99.7
n		25	23	25	23
Outliers		1	3	1	3
Repeatability sr	mg/l	1.92	1.55	1.25	0.58
RSDr	%	1.3	1.3	1.5	1.9
HorRat		0.17	0.17	0.19	0.20
r	mg/l	5.4	4.3	3.5	1.6
Reproducibility SR	mg/l	10.52	10.03	6.14	3.26
RSDR	%	6.9	8.4	7.6	10.9
Horwitz RSDR	%	7.5	7.8	8.3	9.6
HorRat		0.92	1.08	0.92	1.14
R	mg/l	29.5	28.1	17.2	9.1

D-Isoascorbic Acid

		Red Wine I	White Wine II	Red Wine III	White Wine IV
X	mg/l	102.4	79.8	11.3	29.4
Amount Spiked	mg/l	100	80	10	30
Recovery	%	102.4	99.8	113.0	98.0
n		25	23	24	22
Outliers		1	3	2	4
Repeatability sr	mg/l	1.71	1.49	0.47	0.70
RSDr	%	1.7	1.9	4.1	2.4
HorRat		0.21	0.23	0.37	0.25
r	mg/l	4.8	4.2	1.3	2.0
Reproducibility SR	mg/l	9.18	7.96	2.394	3.23
RSDR	%	9.0	10.0	21.2	11.0
Horwitz RSDR	%	8.0	8.3	11.1	9.6
HorRat		1.12	1.21	1.91	1.14
R	mg/l	25.7	22.3	6.7	9.0

12. Bibliography

• B. Seiffert, H. Swaczyna, I. Schaefer (1992): Deutsche Lebensmittelrundschau, 88 (2) p. 38-40
• C. Fauhl: Simultaneous determination of L -ascorbic acid and D –iso-ascorbic acid (erythorbic acid) in wine by HPLC and UV-detection – OIV FV 1228, 2006

OIV-MA-AS313-23 Identification of L-tartaric acid as being of plant or fossil origin by measuring its activity

 

Type IV method

 

1. Purpose and scope

The method can be used to identify tartaric acid as being of plant or fossil origin, and in cases of a mixture of the two, to determine the respective proportions of the two types. In these situations, the method enables the detection of fossil-derived L(+)-tartaric acid quantities below 10%.

2. Principle

In the majority of cases, commercially available tartaric acid of plant origin is a product of winemaking. The potassium hydrogénotartrate present in the lees is extracted and marketed in the form of L-tartaric acid. The concentration in the acid is therefore related, as with ethanol from wine, to the  concentration in the carbon dioxide in wines from the same year of production. This concentration is relatively high as a result of the human activity involved.

Synthetic tartaric acid on the other hand, derived from fossil fuel by-products, has a much lower or even negligible concentration of .

Measuring the  activity in DPM/gram of carbon (Disintegrations Per Minute) using liquid scintillation therefore allows the origin to be determined as well as any combination of the types.

3. Reagents and products

3.1.  Reagents

3.1.1.      Scintillation fluid such as Instagel Plus

3.1.2.      toluene reference with activity certified by  laboratory for callibration, for calculating the sensitivity and efficiency of the machine by the definition of a quench curve

3.1.3.      and standards and toluene for the background noise, for calibrating the scintillation counter

3.1.4.      Nitromethane 99%

3.1.5.      Ultrapure water (>18 MΩ )

3.1.6.      toluene solution with activity of approx. 430 DPM/ml obtained by diluting stock reference solution in toluene.

3.2.  Standards

3.2.1.      Defining the quench curve

Once the scintillator has been calibrated using the three certified , and toluene standards, plot a quench curve using the following procedure.

Prepare a dozen vials with 10 ml of a solution of 500 g/l of fossil-derived L-tartaric acid in water, then add the quantity of toluene standard needed for approx. 400-1000 DPM in total per vial (if necessary, make up an intermediate solution of standard solution in toluene), then add increasing quantities of nitromethane, e.g. for 12 vials: 0, 0, 0, 5, 10, 15, 20, 35, 50, 100, 200 and 400 μL followed by 10 m of scintillation fluid. There must be at least 3 samples containing no nitromethane.

Define a quench curve once a year, analysing the vials in increasing order of nitromethane content.

The quench curve can then be used to determine the sensitivity or mean efficiency.

3.2.2.      Determination of background noise (test blank)

Using fossil-derived L-tartaric acid, such as that used for calculating the efficiency, determine the background noise, or test blank value. This test should be performed immediately after defining the quench curve, then roughly every three months.

3.2.3.      Defining the calibration curve

The purity of the plant and fossil-derived L-tartaric acids must be checked using HPLC before the scintillation test is done.

Calibration using a mixture of tartaric acid (which is known with certainty to be of plant origin) containing between 0% and 100 % of this type in combination with the fossil-derived type.

Preparation of 500 g/l solutions
	Blank or background noise	Standards	Internal standard
Weighing	respectively in 50 ml volumetric flasks
	25 g fossil-derived L-tartaric acid	25 g known combinations of fossil and plant L-tartaric acid	Use the blank
Dissolution	Seal
	Homogenise the mixture well by shaking and/or tumbling
Preparation of scintillation mixtures
	In plastic vials, add respectively
Sample taken from the 500 g/l solutions	10 ml using volumetric pipettes
Added concentration	/////////////////////////	/////////////////////////	100 μL
Added scintillation fluid	10 ml using an automatic burette
	Screw the cap on
	Wait 5 min. then analyse for 500 min.

3.3.  Internal control

3.3.1.      Nature of product used for internal control

A 500 g/l solution of fossil-derived L-tartaric acid is enriched with a quantity of toluene (DPM<100)

The background noise should be determined using the same fossil-derived L-tartaric acid solution.

3.3.2.      Nature of internal control

Measurement of the added concentration provides verification that there is no spectral interference in the medium being studied.

3.3.3.      Internal control limits

The control limits depend on the equipment used: a 5% value is acceptable.

3.3.4.      Inspection frequency and procedure

Once a month during frequent use, or at each analysis sequence, an internal control is performed on the scintillator. The same check is also carried out at every change of scintillation fluid batch or after a new quench curve has been defined.

3.3.5.      Decision rules to be taken depending on the results of the internal control

If the results fall outside the internal control limits, calibrate the scintillator after checking the protocol, then repeat the internal control.

If the calibration is accurate but the new internal control measurement is not, make a new quench curve and carry out a new control.

4. Apparatus
   1.   Liquid scintillation spectrometre with computer and printer previously calibrated with quenching curve established with nitromethane
   2.   Low content potassium identical bottles (40K) with screw top stopper, and low background noise
   3.   10 ml 2 graduations pipettes
   4.   Automatic distribution burette adapted to screw top for liquid scintillating bottle
   5.   Glass laboratory

5. Samples

The purity of the samples can be checked using HPLC if required, before running the scintillation analysis.

Make up a 500 g/solution of the sample to be analysed in ultra-pure water.

Preparation of 500 g/l solutions
	Test blank or background noise	Standards	Internal standard	Sample
Weighing	respectively in 50 ml volumetric flasks
	25 g fossil-derived L-tartaric acid	25 g known combinations of fossil-derived and plant L- tartaric acid	Use the blank	25 g
Dissolution	Seal
	Homogenise the mixture well by shaking and/or tumbling
Preparation of scintillation mixtures
	In plastic vials, add respectively
Sample taken from the 500 g/l solutions	10 ml using volumetric pipettes
Added concentration	//////////////////////	////////////////	100 μL	//////////////
Added scintillation fluid	10 ml using an automatic burette
	Screw the cap on and shake hard
	Wait 5 min. then analyse for 500 min.
Notes	Every 5 to 10 test samples, run a sample with 0 % plant tartaric acid, i.e. 10 ml fossil tartaric acid and 10 ml scintillation fluid.
	Measure the background noise at the end of each analysis sequence

6. Calculation

Measurements are given directly in Counts Per Minute CPM, but these must be converted to DPM/gram of carbon.

6.1.  Results:

Calculation of the specific ¹⁴C radioactivity of the sample in DPM/gram of carbon:

A: radioactivity in disintegrations/minute and per gram of carbon

X: CPM of the sample

X’: CPM for the fossil L-tartaric acid used for the background noise

m: mass of the tartaric acid in the 10 ml sample from the 500 g/l solution, i.e. in 5 g  of acid

Rm: the mean efficiency expressed as a percentage

 

₍₁₎ There are 3.125 grams of tartaric acid to each gram of carbon (ratio of the molar mass of the acid (150 g/mol) to the total mass of carbon (or 4 *x 12 = 48 g/mol)

The result is expressed to one decimal place.

6.2.  Verification of the results using internal controls:

The check should be carried out by comparing the value obtained at § 3.5.1 with the result given by the added concentration method. If the difference is significant (> 5 %), recalculate the DPM value from the CPM value as below:

with the mean efficiency being obtained from the quench curve.

The two results must not differ by more than 5% from their mean value. If they do, repeat the analysis on the sample, doubling the quantity of the internal standard. Compare the 2 results obtained with the standards: if they do not differ by more than 5 % from the mean of the 2, give the mean result.

Note: in this case, that would mean that the quenching of the sample is so great that direct analysis cannot be used.

6.3.  Uncertainty

The uncertainty value obtained under standard test conditions is +/- 0.7 DPM/gram of carbon.

7. Validation by comparison with a reference method

 

7.1.  Principle

Tartaric acid is converted to by combustion then converted to benzene;

Measurement is then carried out using liquid scintillation.

After undergoing a pre-treatment designed to eliminate any contamination, the from the sample is converted to benzene following the reaction chain below:

800°C

(1) Organic sample: the carbon flushed with oxygen plus a heat source (or by combustion in the presence of pressurised oxygen) produces carbon dioxide from the sample ().

(2) Mineral sample (marine or continental carbonates, water, etc.): The carbonate is attacked by pure hydrochloric acid (HC) to produce the carbon dioxide () from the sample plus water and ionised calcium.

(3) The action of the on lithium metal heated to between +600°C and +800°C produces lithium carbide and lithium oxide ( O).

(4) The action of water (hydrolysis) on the lithium carbide produces acetylene (), lithium hydroxide,. Non-tritiated, radon-free water must be used.

(5) Trimerisation of the acetylene over a chrome-plated aluminium-based catalyst support at approx. 185 °C produces benzene ().

7.2.  Procedure:

The carbon dioxide () from a sample, obtained either by burning, combustion or acid attack, is preserved in a storage cylinder. The necessary quantity of lithium (lithium = catalyst for a chemical transformation) is placed in a nickel capsule, which is then placed at the bottom of a heat reaction chamber. A vacuum is created inside the chamber and its lower part is heated while its upper part is cooled at the sides with the help of a water circulation partition.

7.2.1.      Carburisation.

After approximately one hour of heating, the temperature reaches 650°C. The CO₂ can then be brought into contact with melted lithium. The quantity of lithium is always higher in relation to the quantity of carbon in the sample. The excess amount of lithium to use in relation to the stoechiometric conditions varies from 20% to 100% according to different sources.

The chemical reaction (carburisation or "pickup") is almost instantaneous and the first few minutes of pickup are the most crucial in the carburisation process.

The reaction is exothermic (an increase of 200°C). Carburisation is quite rapid and is considered to be at the carburised stage after the first 20 minutes, but heating continues for 45 to 50 minutes in order to any eliminate traces of radon (a by-product of uranium), which could be mixed in with the carbon dioxide.

7.2.2.      Cooling

Once the treatment period (heating) is complete, the reaction chambers are allowed to cool until they reach room temperature (25-30°C).

7.2.3.      Hydrolysis of Lithium Carbide

Water is introduced into the reaction chambers, in a much higher quantity than that required by the reaction (1.5 L). The chemical reaction is instantaneous and the acetylene is released at the same time. This reaction is also exothermic (temperature increase between +80°C and +100°C).

The acetylene produced is then brought to a vapour state (sublimation) and trapped over the chrome-plated (Cr3+) aluminium catalyst support. This is previously air dried for a minimum of three hours, then vacuum dried for two hours under heat at +380°C. Drying is vital in order to eliminate any water remaining in the catalyst support balls.

7.2.4.      Trimerisation - Polymerisation of acetylene to benzene by catalysis

Before trimerisation, the temperature of the catalyst support must have dropped to between +60°C and +70°C, and since this reaction is also exothermic, automatic temperature maintenance is needed. The catalyst support is then reheated to +180°C for 1½ hours and the vaporised benzene is desorbed then trapped in a trap tube surrounded by liquid nitrogen. Desorption takes place under dynamic vacuum. At the end of the experiment, the crystallised benzene is left to reheat to room temperature so that it regains its liquid state before being used for the counting.

7.3.  Benchtop arrangement for the synthesis of Benzene

7.4.  Reference Chemical solution for the Counting

A solution volume set at 4 ml is used as the reference for the liquid scintillation counting.

The solution comprises a target base of 3.52g benzene from the sample (solvent) + the scintillation fluid (solute) made up of 2 scintillation fluids, one main and one secondary.

Since the mass per volume of benzene is 0.88 g/litre, 0.88 x 4ml = 3.52 g.

Main scintillation fluid	Buthyl-PBD
Chemical composition	(2-(4-Biphenylyl)-5-(4-tert-buthyl-phenyl)-1,3,4-oxadiazole)
Maximum wavelength fluorescence	367 nanometers
Secondary scintillation fluid	bis-MSB
Chemical composition	1,4-Di-(2-Methylstyryl)-Benzene
Maximum wavelength fluorescence	415 nanometers
Optical absorption and coupling emission of the two fluids:
Maximum absorption wavelength	409 nanometers
Maximum absorption wavelength	412 nanometers

7.5.  Delta correction for Isotope Fractionation

The measurement involves a correction for isotope fractionation using the standardisation procedure with a stand PDB with a value of - 25 o/oo.

8. Characteristics of the method

 

8.1.  Procedure

One sample of wine-derived tartaric acid and one sample of synthetic acetic acid were used to prepare test tartaric acid solutions at 500 g/l.

The concentrations of the wine-derived tartaric acid in the solutions varied between 0°C and 100%.

The origin and purity of the two starter samples had been previously checked using the reference method.

8.2.  Results:

The results are given in the table and diagram below:

% of wine-derived tartaric acid
Actual concentrations	Results from the alternative method	Results from the reference method
0	0 and 0	0
10	3.5 and 6.0	12
20	11.4 and 12	22
30	24.6 and 25.4	31
40	34.7 and 38	40
50	41.4 and 50.6	50
60	57.8 and 58.8	63
70	60 and 63.3	70
80	81	81
85	84	86
90	88	91
95	94	96
100	100	100

 

 

8.3.  Accuracy,trueness:

Accuracy is 6.9%.

The standard deviation of repeatability for the alternative method is: 2.86 % of plant tartaric acid.

 

9. Bibliography

• Compendium of international methods of analysis of spirits and alcohols and of the aromatic fraction of beverages, Office Internationale de la Vigne et du Vin, Edition officielle, juin 1994, page 201, 204, 210 et 307.
• Methods of analysis for neutral alcohol applicable to the wine sector, EEC Regulation no. 625/2003, 2 April 2003, Journal Officiel des communautés européennes 15 May 1992, n°L130, p18. (Journal Officiel, 8 April 2003, N° L90, p4).
• J. GUERAIN and S. TOURLIERE, Radioactivité carbone et tritium dans les alcools, Industries Alimentaires et Agricoles – 92^nd year, July – August 1975, N° 7-8
• S. COHEN, B. CRESTO, S. NACHAMPASSAK, T. PAYOT, B. MEDINA, S. CHAUVET, Détermination de l’origine de l’acide tartrique L(+): naturelle ou fossile par la détermination de son activité C¹⁴ - Document OIV FV 1238, 2006

OIV-MA-AS313-24 Determination of totla ethanol in wine by high-performance liquid chromatography

Type IV method

Scope of application

The method described is suitable for the determination of total (free and sulphur-dioxide-bound) ethanal in wine for concentrations between 0.2 and 80 mg/L.

1. Principle

The analyte is quantified after derivatisation of the molecule with 2,4-Dinitrophenylhydrazine (DNPH) followed by elution using HPLC. Detection is based on the retention time at the wavelength of 365 nm.

2. Reagents and products2,4-Dinitrophenylhydrazine (DNPH), CAS no. 119-26-6, purity 99.0% (HPLC)
   2.   Sulphur dioxide (), as ,CAS no. 16731-55-8, purity 98%
   3.   Sulphuric acid (), CAS no. 7664-93-9, purity 95.0-98.0%
   4.   Formic acid (), CAS no. 64-18-6, purity ≈ 98%
   5.   Acetonitrile (N), CAS no. 75-05-8, purity ≥ 99.9%
   6.   Ethanal (CHO), CAS no. 75-07-0, purity ≥ 99.5%
   7.   Ultra-pure, HPLC-grade type I water compliant with standards ASTM D1193 and ISO 3696, CAS no. 7732-18-5
   8.   Perchloric acid (HCl), CAS no. 7601-90-3, purity 70 %

Preparation of reagent solutions

2.9.  Freshly-prepared sulphur dioxide solution at a concentration of 1120 mg/L SO₂, obtained by preparing a 2 g/L solution of   (2.2) with ultra-pure, HPLC-grade water

2.10.                    25% v/v sulphuric acid solution prepared by dilution of concentrated sulphuric acid (2.3) with ultra-pure, HPLC-grade water

2.11.                    2.8% acetonitrile solution acidified with perchloric acid, obtained through the dilution of perchloric acid (2.8) in acetonitrile (2.5)

2.12.                    Freshly-prepared solution of 2,4-dinitrophenylhydrazine (2.1) in acidified acetonitrile (2.11) at a concentration of 2 g/L DNPH

2.13.                    Preparation of calibration solutions 

The stock solution is prepared by dilution of an appropriate quantity of ethanal (density = 0.785 g/mL) in ultra-pure, HPLC-grade water in order to obtain a concentration of between 300 and 400 mg/L. Given that pure ethanal is highly volatile, the stock solution should be prepared by sampling constant volumes of ethanal using calibrated flasks (3.1). To prepare the stock solution, measure 10 mL pure ethanal in a calibrated flask, transfer the pure ethanal to another 20-mL calibrated flask and make up to the mark with ultra-pure, HPLC-grade water. The solutions diluted are prepared by making up to volume with ultra-pure, HPLC-grade water in calibrated flasks of greater capacity. Calibration solutions, with concentrations of 10 mg/L, 30 mg/L, 50 mg/L, 70 mg/L and 100 mg/L, are obtained through dilution of the stock solution in 50-mL calibrated flasks. The volumes required are sampled from the stock solution using a precision micropipette (3.2), for example, and made up to volume with ultra-pure, HPLC-grade water in calibrated flasks (3.1).

Preparation of solvent A for HPLC analysis

2.14.                    0.5% (v/v) formic acid solution obtained by diluting concentrated formic acid (2.4) in ultra-pure, HPLC-grade water (2.7).

3. Apparatus
   1.   Everyday laboratory glassware, including class-A calibrated flasks of 10, 20 and 50 mL, 2-mL vials and 1-L containers for the solvents, and Pasteur pipettes
   2.   Precision micropipettes
   3.   Vortex-type stirrer
   4.   0.45- μm membrane filters for sample preparation, certified for use in HPLC
   5.   1-L vacuum flask (where the automatic degasser of the solvents is not provided with the HPLC apparatus)
   6.   Vacuum pump (where the automatic degasser of the solvents is not provided with the HPLC apparatus)
   7.   Analytical balance with precision of 0.0001 g
   8.   Natural convection oven with precision of 1 °C at 65 °C
   9.   HPLC apparatus with UV detector, equipped with two gradient pumps and an oven for the heating of the column
   10.                     C18 column (250 x 4.6 mm, particle diameter: 4 μm)

Note: Any other system may be used on the condition that the ethanal is well separated from the other derivatised carbonyl compounds. The chromatographic resolution between the ethanal peak and the greatest neighbouring peak on the chromatogram should be higher than 1.

 

4. Sampling

The wine sample should be taken and stored in a glass container sealed with a Teflon stopper in an inert atmosphere (nitrogen or argon).

5. Procedure

Derivatisation

The derivatisation takes place in 2-mL glass vials sealed with Teflon stoppers, inside which the following is successively added: 100 μL wine or standard solution filtered at 0.45 μm, 20 μL freshly-prepared sulphur dioxide solution (2.2) at a concentration of 1120 mg/L SO₂, 20 μL 25% sulphuric acid (2.3) and 140 μL freshly-prepared solution of 2,4-dinitrophenylhydrazine in acetonitrile at a concentration of 2 g/L DNPH (2.12).

After these additions, the solution is immediately vortex stirred and placed in the oven at 65 °C for 15 minutes before being cooled at room temperature.

Once the reaction is completed, the solution is cooled at room temperature for 15 minutes before being injected into the HPLC apparatus. The samples should be injected in less than 10 hours from the end of the derivatisation reaction.

HPLC analysis

The specific HPLC-analysis parameters are provided below by way of example.

Normal operating conditions:

• Injection volume: 15 μL
• Flow rate: 0.75 mL/min
• C18 column (5.10)
• Solvent for cleaning the injector: acetonitrile
• Column temperature: 35 °C
• Mobile-phase-A solvent: 0.5% formic acid in ultra-pure, HPLC-grade water
• Mobile-phase-B solvent: acetonitrile
• Detection at 365 nm

Elution gradient

The elution programme provides for:

35% eluent B (0.1 s)	65% eluent A (0,1 s)
60% eluent B (8 min)	40% eluent A (8 min)
90% eluent B (13 min)	10% eluent A (13 min)
95% eluent B (15 min)	5% eluent A (15 min)
95% eluent B (17 min)	5% eluent A (17 min)
35% eluent B (21 min)	65% eluent A (21 min)
35% eluent B (25 min)	65% eluent A (25 min)

Example chromatogram

6. Calculation

The ethanal concentration is calculated based on the equation of the calibration curve obtained after injection of the calibration solutions (2.13).

7. Precision and validation parameters

The coefficient of variation for the analyses repeated within the same laboratory should be less than 6% (for a concentration interval of between 10 mg/L and 100 mg/L). The repeatability standard deviation is 2.7% for a concentration of 14 mg/L, 2.98% for a concentration of 18 mg/L, 4.8% for a concentration of 22 mg/L and 1.3% for a concentration of 60 mg/L. The linearity range is between 0.2 and 80.0 mg/L. The limit of detection is 0.1 mg/L. The recovery rate in wine is between 92% and 102% (m/m).

8. Results

The results are expressed in mg total ethanal / L to 1 decimal point.

9. Bibliography

• Behforouz, M., Bolan, J. L., and Flynt, M. S., '2, 4-Dinitrophenylhydrazones: a modified method for the preparation of these derivatives and an explanation of previous conflicting results', The Journal of Organic Chemistry, 50 (8), 1985, pp. 1186-1189.
• Elias, R. J., Laurie, V. F., Ebeler, S. E., Wong, J. W., and Waterhouse, A. L., 'Analysis of selected carbonyl oxidation products in wine by liquid chromatography with diode array detection', Analytica chimica acta, 626 (1), 2008, pp. 104-110.
• Han, G., Wang, H., Webb, M. R., and Waterhouse, A. L., 'A rapid, one step preparation for measuring selected free plus SO 2-bound wine carbonyls by HPLC-DAD/MS', Talanta, 134, 2015, pp. 596-602.

OIV-MA-AS313-25 Determination of L-lactic acid in wines by automated enzymatic method

Type III method

 

1. Scope of application

This method makes it possible to determine L-lactic acid 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 L-lactic acid in the range from 0.06 to 1.43 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: Lactic acid – enzymatic method, OIV-MA-AS313-07,
• ISO 78-2: Chemistry – Layouts for standards.

3. Reaction principles

In the presence of nicotinamide adenine dinucleotide (NAD), L-lactic acid is oxidised to pyruvate in a reaction catalysed by L-lactate dehydrogenase (L-LDH). Since the equilibrium reaction is in favour of the lactate, it is necessary to remove the pyruvate formed which is converted into L-alanine in the presence of L-glutamate. This reaction is catalysed by glutamate pyruvate transaminase (GPT).

The reduced nicotinamide adenine dinucleotide (NADH) produced is measured based on its absorption at 340 nm. It is proportional to the quantity of L-lactic acid.

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.     Glycylglycine (CAS no. 556-50-3)

4.1.3.     Glutamic acid (CAS no. 56-86-0)

4.1.4.     NAD (nicotinamide adenine dinucleotide) (CAS no. 53-84-9)

4.1.5.     L-LDH (L-lactate dehydrogenase) (CAS no. 9001-60-9)

4.1.6.     GPT (glutamate pyruvate transaminase) (CAS no. 9000-86-6)

4.1.7.     L-lactic acid of purity ≥ 98% (CAS no. 79-33-4)

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

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

Note 1: There are commercial kits for the determination of L-lactic acid. 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’s activitiy. 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.     A pH 10 buffer (0.60 M glycylglycine, 0.1 M L-glutamic acid).

The preparation may be as follows:

• Glycylglycine (4.1.2): 4.75 g,
• glutamic acid (4.1.3): 0.88 g,
• PVP (4.1.8): 1 g,
• water for analytical usage (4.1.1): 50 mL.

The mixture is adjusted to pH 10 using a 10 M sodium hydroxide solution, then made up to 60 mL with water for analytical usage. This solution is stable for at least 4 weeks at 2-8 °C (approx.).

4.2.2.     R1 working solution (example):

• water for analytical usage (4.1.1): 12 mL,
• NAD (4.1.4): 420 mg.

This solution is stable for at least 4 weeks at 2-8 °C (approx.).

4.2.3.     R2 working solution (example):

• water for analytical usage (4.1.1): 1.2 mL,
• L-LDH (4.1.5):  7600 U,
• GPT(4.1.6): 2200 U.

This solution is stable for at least 4 weeks at 2-8 °C (approx.).

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 L-lactic acid 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 a UV detector. The reaction temperature should be tightly controlled (generally 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 NADH formed by the reaction is 340 nm.

5.2.  Balance

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

pH meter

5.3.  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 still wines

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

• Filtration or centrifugation should be used for highly turbid samples.
• Sample dilution (manual or automatic) with water for analytical usage should be used for values exceeding the measurement range.
  2.   Preparation of samples of sparkling wine containing CO₂

Sparkling 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 be strictly observed. This also applies to the various enzymatic kits available on the market

The procedure takes place as follows:

• The sample (S) is placed in a reaction cuvette.
• Working solution R1 (4.2.2) is then added to the cuvette.
• 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: 2.5 μL,
• buffer (80%) and R1 (20%): 120 μL,
• R2: 15 μL.

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

Figure 1: Reaction curve

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 ∆DO value with the desired concentration of L-lactic acid, regular calibration of the apparatus is carried out using the calibration solutions at a minimum of 3 points covering the measurement range used. In the example given in Figure 2, the calibration curve is a straight line for values between 0 to 1.22 g/L L-lactic acid. In this case, for higher values, it is preferably to carry out a dilution. Inaddition, 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 = ), yet in this method it is more generally order 2 (Concentration =). 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 with high concentrations).

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

9. Expression of results

The results are expressed in g/L of L-lactic acid to 2 d.p.

10. Precision

 

Interlaboratory reproducibility

• = 7% (from 0.5 g/L)
• % (k=2) = 2·RSD_R = 14%, (from 0.5 g/L)

 

Repeatability

• = 2% (from 0.5 g/L)
• % (k=2) = 2·RSD_r = 4% (from 0.5 g/L)

Limit of quantification

Validated LOQ = 0.06 g/L

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

Annex: Results of the interlaboratory tests

Collaborative study

A total of 16 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
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 wine

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).

Table of the results obtained Note: The results from samples B and H should be taken with caution due to the very low concentration levels, which are below the laboratories’ limit of quantification

Figure 3: R limit according to concentration

Figure 4: Interlaboratory RSDR% according to concentration. Modelling: % = 0.758·+ 7

 

OIV-MA-AS313-26 Determination of L-malic acid in wine by automated enzymatic method

Type III method

 

1. Scope of application

This method makes it possible to determine L-malic acid 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 L-malic acid in the range from 0.12 to 2.3 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: L-malic acid – enzymatic method, OIV-MA-AS313-11,
• ISO 78-2: Chemistry – Layouts for standards.

3. Reaction principles

In the presence of nicotinamide adenine dinucleotide (NAD), L-malic acid is oxidised to oxaloacetate in a reaction catalysed by L-malate dehydrogenase (L-MDH). Since the equilibrium reaction is in favour of the malate, it is necessary to remove the oxaloacetate formed which is converted into L-aspartate in the presence of L-glutamate. This reaction is catalysed by glutamate oxaloacetate transaminase (GOT).

The reduced nicotinamide adenine dinucleotide (NADH) produced is measured based on its absorption at 340 nm. It is proportional to the quantity of L-malic acid.

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.      Glycylglycine (CAS no. 556-50-3)

4.1.3.      Glutamic acid (CAS no. 56-86-0);

4.1.4.      NAD (nicotinamide adenine dinucleotide) (CAS no. 53-84-9);

4.1.5.      L-MDH (L-malate dehydrogenase) (CAS no. 9001-64-3);

4.1.6.      GOT (glutamate oxaloacetate transaminase) (CAS no. 9000-97-9);

4.1.7.      L-malic acid, purity 95% (CAS no. 97-67-6);

4.1.8.      Optional: polyvinylpyrrolidone (PVP) (CAS no. 9003-39-8) or potentially PVPP (CAS no. 25249-54-1);

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

Note 1: There are commercial kits for the determination of L-malic acid. 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. 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.      A pH 10 buffer (0.60 M glycylglycine, 0.1 M L-glutamic acid).

The preparation may be as follows:

• glycylglycine (4.1.2): 4.75 g,
• glutamic acid (4.1.3): 0.88 g,
• PVP (4.1.8): 1 g,
• water for analytical usage (4.1.1): 50 mL.

The mixture is adjusted to pH 10 using a 10 M sodium hydroxide solution, then made up to 60 mL with water for analytical usage. This solution is stable for at least 4 weeks at 2-8 °C (approx.).

4.2.2.      R1 working solution (example):

• water for analytical usage (4.1.1): 12 mL,
• NAD (4.1.4): 420 mg.

This solution is stable for at least 4 weeks at 2-8 °C (approx.).

4.2.3.      R2 working solution (example):

• water for analytical usage (4.1.1): 1.2 mL,
• L-MDH (4.1.5): 4800 U,
• GOT (4.1.6): 320 U.

This solution is stable for at least 4 weeks at 2-8 °C (approx.).

4.3.  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 L-malic acid 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 a UV detector. The reaction temperature should be tightly controlled (generally 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.      Use of a reagent blank

The results are read by comparing the light intensity absorbed at the chosen wavelength between a cuvette in which the reaction is carried out and a cuvette in which the reaction does not take place (blank reagent).

5.1.6.      Wavelength

The wavelength of maximum absorption of the NADH formed by the reaction is 340 nm.

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 still wines

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

filtration or centrifugation should be used for highly turbid samples,

sample dilution (manual or automatic) with water for analytical usage should be used for values exceeding the measurement range.

6.2.  Preparation of samples of sparkling wine containing CO₂

Sparkling 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 be strictly observed. This also applies to the various 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

4)      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: 2.5 μL,
• mixture of 80% buffer (3.2.1) and 20% R1 (3.2.2): 120 μL,
• R2 (3.2.3): 15 μL.

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

 

Figure 1: Reaction curve

 

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

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 L-malic acid, regular calibration of the apparatus is carried out using the calibration solutions (§4.3) at a minimum of 3 points covering the measurement range. In the example given in Figure 2, the calibration curve obtained is a straight line for values between 0 and 2 g/L L-malic acid. In this case, for higher values, it is preferable to dilute the sample. 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 with high concentrations).

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

Expression of results

The results are expressed in g/L L-malic acid to 2 d.p.

Precision

 

Interlaboratory reproducibility

= 5% (from 1 g/L)

% (k=2) = 2·= 10%, (from 1 g/L)

Repeatability

= 2% (from 1 g/L)

% (k=2) = 2·RSD_r = 4% (from 1 g/L)

Limit of quantification

Validated LOQ = 0,12 g/L

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

Annex Results of the interlaboratory tests

Collaborative study

A total of 16 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
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 wines.

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	1.12	1.09	2.16	2.21	0.08	0.08	1.38	1.39		0.16	0.16	0.05	0.04	0.07	0.06	1.31		1.35	1.24	1.27	0.07	0.07
	rep#2	1.15	1.11	2.18	2.11	0.08	0.09	1.35	1.41		0.16	0.16	0.05	0.04	0.06	0.06	1.32		1.30	1.23	1.24	0.07	0.07
Labo6	rep#1	0.99	0.95	2.28	2.31	0.04	0.02	1.44	1.47		0.05	0.08	0.04	0.04	0.06	0.04	1.38		1.46	1.34	1.19	0.04	0.03
	rep#2	0.93	0.93	2.15	2.16	0.05	0.03	1.23	1.37		0.08	0.09	0.03	0.05	0.03	0.04	1.28		1.30	1.20	1.28	0.03	0.03
Labo7	rep#1	1.10	1.10	2.28	2.33	0.01		1.44	1.52		0.12	0.10		0.01			1.38		1.41	1.33	1.30	0.01	0.02
	rep#2	1.13	1.13	2.35	2.33	0.03	0.04	1.47	1.49		0.14	0.13				0.02	1.40		1.43	1.32	1.33	0.02	0.03
Labo9	rep#1	1.14	1.10	2.27	2.21	0.06	0.10	1.43	1.48		0.19	0.19	0.02	0.02	0.05	0.06	1.35		1.38	1.27	1.28	0.05	0.08
	rep#2	1.15	1.20	2.20	2.23	0.10	0.06	1.50	1.51		0.15	0.14	0.03	0.01	0.09	0.11	1.31		1.37	1.22	1.28	0.04	0.06
Labo12	rep#1	1.12	1.12	2.34	2.32	0.06	0.02	1.47	1.54		0.14	0.14	0.04	0.03	0.03	0.03	1.37		1.37	1.29	1.31	0.06	0.04
	rep#2	1.12	1.12	2.38	2.34	0.07	0.01	1.46	1.46		0.16	0.19	0.04	0.05	0.03	0.04	1.32		1.33	1.28	1.30	0.06	0.04
Labo13	rep#1	1.11	1.09	2.12	2.26	0.04	0.02	1.44	1.45		0.12	0.14	0.01	0.01	0.03	0.03	1.45		1.33	1.29	1.29	0.02	0.02
	rep#2	1.18	1.17	2.20	2.29	0.03	0.01	1.47	1.50		0.11	0.12			0.02	0.03	1.39		1.38	1.32	1.33		0.01
Labo14	rep#1	1.32	1.30	2.66	2.68			1.69	1.67		0.08	0.08						1.65	1.60	1.51	1.52
	rep#2	1.30	1.30	2.63	2.68			1.67	1.67		0.08	0.08						1.61	1.60	1.51	1.53
Labo15	rep#1	1.18	1.19	2.29	2.42	0.06	0.05	1.41	1.50		0.16	0.16	0.04	0.04	0.08	0.08		1.33	1.39	1.26	1.30	0.07	0.07
	rep#2	1.13	1.20	2.21	2.42	0.05	0.05	1.49	1.52		0.16	0.16	0.04	0.04	0.08	0.08		1.38	1.39	1.27	1.28	0.07	0.07
Labo16	rep#1	1.22	1.22	2.52	2.48			1.63	1.62		0.09	0.09						1.50	1.53	1.47	1.45
	rep#2	1.21	1.20	2.45	2.55			1.62	1.61		0.09	0.09		0.03				1.52	1.51	1.44	1.45
Labo17	rep#1	1.14	1.14	2.22	2.22	0.07	0.08	1.51	1.50		0.17	0.17	0.07	0.07	0.11	0.11		1.34	1.35	1.28	1.27	0.09	0.09
	rep#2	1.13	1.15	2.17	2.20	0.08	0.08	1.46	1.47		0.18	0.17	0.08	0.07	0.12	0.12		1.31	1.34	1.28	1.29	0.09	0.09
Labo18	rep#1	1.10	1.11	2.14	2.13	0.18	0.18	1.42	1.52		0.28	0.28	0.16	0.16	0.19	0.19		1.33	1.38	1.27	1.31	0.17	0.18
	rep#2	1.10	1.13	2.19	2.16	0.18	0.18	1.44	1.51		0.27	0.27	0.16	0.16	0.19	0.19		1.33	1.38	1.29	1.33	0.17	0.18
Labo19	rep#1	1.13	1.19	2.20	2.23	0.08	0.08	1.47	1.44		0.17	0.19	0.07	0.08	0.11	0.11		1.36	1.30	1.27	1.26	0.10	0.10
	rep#2	1.15	1.22	2.21	2.23	0.08	0.09	1.48	1.45		0.18	0.19	0.07	0.08	0.12	0.11		1.33	1.30	1.28	1.24	0.09	0.10
Labo20	rep#1	1.17	1.20	2.27	2.27	0.04	0.05	1.50	1.48		0.17	0.17	0.04	0.03	0.07	0.07		1.33	1.34	1.28	1.29	0.07	0.06
	rep#2	1.17	1.20	2.27	2.27	0.04	0.05	1.50	1.48		0.17	0.17	0.04	0.03	0.07	0.07		1.34	1.34	1.28	1.29	0.07	0.06
Labo21	rep#1	1.10	1.12	2.30	2.47	0.05	0.05	1.45	1.49		0.11	0.10	0.05	0.05	0.05	0.05		1.46	1.44	1.33	1.41	0.05	0.05
	rep#2	1.09	1.16	2.28	2.50	0.05	0.05	1.49	1.51		0.09	0.11	0.05	0.05	0.05	0.05		1.43	1.46	1.34	1.37	0.05	0.05
Labo22	rep#1	1.08	1.07	2.35	2.31	0.04	0.03	1.46	1.45		0.11	0.12	0.01	0.02	0.03	0.03		1.35	1.35	1.26	1.31	0.03	0.02
	rep#2	1.06	1.08	2.30	2.31	0.04	0.04	1.51	1.46		0.12	0.12	0.01	0.03	0.04	0.03		1.35	1.34	1.28	1.25	0.01	0.01

 

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	15	12	14	13	13	12	12	12	12
No of repetitions	4	4	4	4	4	4	4	4	4	4
Min.	0.95	2.15	0.03	1.38	0.08	0.01	0.02	1.32	1.25	0.02
Max.	1.31	2.66	0.08	1.68	0.28	0.08	0.19	1.45	1.36	0.10
Overall average	1.14	2.30	0.05	1.49	0.14	0.04	0.07	1.36	1.29	0.05
Repeatability variance	0.001	0.003	0.000	0.001	0.000	0.000	0.000	0.001	0.000	0.000
Inter-laboratory stand. dev.	0.08	0.14	0.02	0.07	0.05	0.02	0.05	0.04	0.03	0.03
Reproducibility variance	0.006	0.021	0.001	0.006	0.003	0.000	0.002	0.002	0.001	0.001
Repeatability stand. dev.	0.03	0.05	0.01	0.03	0.01	0.01	0.01	0.03	0.02	0.01
r limit	0.07	0.15	0.04	0.08	0.02	0.02	0.01	0.07	0.06	0.02
Repeatability RSDr	2.2%	2.4%	25.0%	2.0%	6.2%	15.4%	7.4%	1.9%	1.6%	14.5%

 

Reproducibility stand. dev.	0.08	0.14	0.02	0.08	0.05	0.02	0.05	0.04	0.04	0.03
R limit	0.22	0.41	0.07	0.21	0.16	0.06	0.14	0.12	0.10	0.08
Reproducibility RSDR	6.9%	6.2%	46.9%	5.0%	39.1%	57.5%	71.2%	3.2%	2.8%	53.1%
Horwitz RSDr	3.66	3.29	5.81	3.51	5.02	6.13	5.57	3.56	3.59	5.81
Horratr	0.61	0.72	4.30	0.57	1.24	2.52	1.33	0.53	0.45	2.49
Horwitz RSDR	5.55	4.99	8.80	5.32	7.60	9.29	8.44	5.40	5.44	8.81
HorratR	1.25	1.25	5.33	0.95	5.14	6.19	8.43	0.59	0.51	6.02

 

Table of the results obtained

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

 

Figure 3: R limit according to concentration

 

Figure 4: RSDR% according to concentration Modelling: CV% = 3.763·C(-0.895)+ 5