OIV-MA-AS315-01 Acetaldehyde

Type IV method

1. Principle

Acetaldehyde (ethanal) in carbon decolorized wine, reacts with sodium nitroferricyanide and piperidine and causes a green to violet color change whose intensity is measured at 570 nm.

2. Apparatus

 

Spectrophotometer permitting measurement of absorbance at a wavelength of 570 nm with a 1 cm optical cell path.

3. Reagents

3.1.   Piperidine solution, ( 10% (v/v).

Prepare just before use by mixing 2 mL of piperidine with 18 mL of distilled water.

3.2.   Sodium nitroferricyanide solution, 0.4% (m/v).

In a 250 mL glass volumetric flask, dissolve 1 g of pulverized sodium nitroferricyanide, in distilled water and make up to volume.

3.3.   Activated carbon

3.4.   Dilute hydrochloric acid, 25% (v/v)

3.5.   Alkaline solution

Dissolve 8.75 g of boric acid in 400 mL sodium hydroxide solution, 1 M.  Make up to 1 L with distilled water.

4. Procedure
   1.    Sample

Place approx. 25 mL of wine in a 100 mL Erlenmeyer flask, add 2 g of activated charcoal. Shake vigorously for a few seconds, allow to stand for 2 minutes and filter through a fluted slow filter to obtain a clear filtrate.

Place 2 mL of the clear filtrate into a 100 mL Erlenmeyer flask, add, while shaking, 5 mL of the sodium nitroferricyanide solution (3.2) and 5 mL of the piperidine solution (3.1). Mix and place the mixture immediately into a 1 cm optical cell. The coloration produced, which varies from green to violet, is measured with reference to air at a wavelength of 570 nm.  This color change increases then decreases rapidly; measure immediately and record the maximum value of the absorbance that is obtained after about 50 seconds. The concentration of acetaldehyde in the liquid analyzed is obtained using a calibration curve.

Note: If the liquid analyzed contains excess free acetaldehyde, it will be necessary, before beginning the total acetaldehyde determination, to first combine it with sulfur dioxide.  To achieve this, add a small amount of excess free SO₂ to a portion of the liquid to be analyzed and wait for an hour before proceeding.

4.2.   Preparation of the calibration curve

4.2.1. Solution of acetaldehyde combined with sulfur dioxide

Prepare a solution of between 5 to 6% (m/v) sulfur dioxide and determine the exact strength by titrating with 0.05 M iodine solution.

In a 1 L glass volumetric flask, add a volume of this solution which corresponds to 1500 mg of sulfur dioxide.  Introduce into the flask, using a funnel, about 1 mL of acetaldehyde distillate recently distilled and collected in a cooling mixture. Make up to 1 liter with distilled water.  Mix and allow to stand overnight.

The exact concentration of this solution is determined as follows:

Place in a 500 mL Erlenmeyer flask, 50 mL of the solution; add 20 mL of dilute hydrochloric acid (3.4) and 100 mL water.  Titrate the free sulfur dioxide using a solution of 0.05 M iodine with starch as indicator, stopping at a faint blue end point. Add 100 mL of the alkaline solution, and the blue coloration will disappear.  Titrate the combined sulfur dioxide and acetaldehyde with 0.05 M iodine until a faint blue end point is reached: let n be the volume used.

The acetaldehyde solution combined with SO₂ contains 44.05 n mg of acetaldehyde per liter.

4.2.2. Preparation of the calibration standards

In five 100 mL glass volumetric flasks, place respectively 5, 10, 15, 20 and 25 mL of the stock solution.  Make up to volume with distilled water. These solutions correspond to acetaldehyde concentrations of 40, 60, 120, 160 and 200 mg/L.  The exact concentration of the dilutions must be calculated from the acetaldehyde concentration of the stock solution (4.2.1) previously determined.

Proceed with the determination of acetaldehyde on 2 mL of each of these dilutions as indicated in 4.1. The graph of the absorbance of these solutions as a function of acetaldehyde content is a straight line that does not pass through the origin.

Bibliography

• REBELEIN H., Dtsch. Lebensmit. Rdsch., 1970, 66, 5-6.

OIV-MA-AS315-02A Ethyl acetate

Type IV method

 

1. Principle of the method

Ethyl acetate is determined by gas chromatography on wine distillate using an internal standard.

2. Method
   1.   Apparatus (see chapter Volatile Acidity).
   2.   Procedure

Prepare an internal standard solution of 4-methyl-2-pentanol, 1 g/L, in ethanol solution, 10% (v/v).

Prepare the sample solution to be determined by adding 5 mL of this internal standard solution to 50 mL of wine distillate obtained as indicated in the chapter on Alcoholic Strength.

Prepare a reference solution of ethyl acetate, 50 mg/L, in ethanol, 10% (v/v). Add 5 mL of the internal standard to 50 mL of this solution.

Analyze 2 μL of the sample solution and the reference solution using gas chromatography.

Oven temperature is 90°C and the carrier gas flow rate is 25 mL per minute.

2.3.  Calculation

S = the peak area of ethyl acetate in the reference solution.

= the peak area of the ethyl acetate in the sample solution.

I = the peak area of the internal standard in the sample solution.

I = the peak area of the internal standard in the reference solution.

The concentration of ethyl acetate, expressed in milligrams per liter, is given by:

OIV-MA-AS315-02B Ethyl acetate

Type IV method

 

1. Principle of the method

 

Ethyl acetate is separated by distillation of wine brought to pH 6.5. After saponification and suitable concentration in an alkaline environment, the distillate is acidified and the vapor condensed to separate the acetic acid liberated by saponification; the acid portion is titrated with the alkaline solution.

2. Method

2.1 Reagents

2.1.1.      Sodium hydroxide solution, 1 M

2.1.2.      pH 6.5 Buffer solution

Potassium di-hydrogen phosphate,	5 g
Sodium hydroxide solution 1 M	50 mL
Water to	1 L

2.1.3.      Crystalline tartaric acid

2.1.4.      Sodium hydroxide solution, 0.02 M

2.1.5.      Neutral phenolphthalein solution, 1%, in alcohol, 96% (v/v).

2.2.  Usual method

Into a 500 mL volumetric flask, place 100 mL of non-decarbonated wine neutralized with n mL of 1 M sodium hydroxide solution, n being the volume of sodium hydroxide solution, 0.1 M, used for measuring the total acidity of 10 mL of wine. Add 50 mL of pH 6.5 buffer solution and distill. The distillation must be conducted using a tapered tube into a 500 mL round-bottom flask containing 5 mL of 1 M sodium hydroxide solution, on which a mark has been made indicating a volume of approximately 35 mL. Collect 30 mL of distillate.

Stopper the flask and allow to stand for one hour.  Concentrate the contents of the flask to approximately 10 mL by placing it in a boiling water bath and blowing a rapid stream of air into the bowl of the flask.  Allow to cool. Add 3 g tartaric acid (2.1.3).  Eliminate carbon dioxide by shaking under a vacuum. Transfer the liquid from the concentrating flask to the bubbling chamber of a steam distillation apparatus and rinse the flask twice with 5 mL of water.  Steam distill and recover at least 250 mL of distillate.

Titrate with a 0.02 M sodium hydroxide solution, in the presence of phenolphthalein.

2.3.  Calculation

Let n be the number of milliliters of sodium hydroxide solution, 0.02 M (2.1.4) used. 1 mL corresponds to 1.76 mg ethyl acetate.

The concentration of ethyl acetate in milligrams per liter is given by:

Bibliography

 

Usual method:

• PEYNAUD E., Analyse et contrôle des vins, Librairie Polytechnique Ch.- Béranger, 1958.

OIV-MA-AS315-03 Malvidin diglucoside

Type IV method

1. Principle

Malvidin diglucoside, oxidized by nitric acid, is converted to a substance that, in an ammonium medium, emits a vivid green fluorescence in ultraviolet light.

The intensity of the fluorescence of the compound formed is measured by comparison with the fluorescence of a solution titrated with quinine sulfate whose intensity of fluorescence is standardized with the malvidin diglucoside reference.

Free sulfur dioxide, which attenuates the fluorescence, must previously be combined with excess acetaldehyde.

2. Qualitative Examination
   1.   Apparatus
      1.       Ultraviolet lamp permitting measurement at 365 nm.

2.2.  Reagents

2.2.1.      Acetaldehyde solution

Crystallizable paraldehyde	10 g
Ethanol 96% (v/v)	100 mL

2.2.2.      Hydrochloric acid, 1.0 M.

2.2.3.      Sodium nitrate solution, 10 g/L.

2.2.4.      Ethanol, 96% (v/v), containing 5% concentrated ammonia solution (ρ₂₀ = 0.92 g/mL).

2.2.5.      Control wine containing 15 mg of malvidin diglucoside per liter.

2.2.6.      Wine containing no malvidin diglucoside.

2.3.  Method

Into a test tube add:

• 10 mL of wine
• 1.5 mL of acetaldehyde solution

wait 20 minutes.

Into a 20 mL centrifuge tube place:

• 1 mL of wine reacted with acetaldehyde
• 1 drop of hydrochloric acid
• 1 mL sodium nitrate solution

Stir; wait 2 minutes (5 minutes maximum); add:

• 10 mL ammoniacal ethanol

Treat similarly 10 mL of wine containing 15 mg/L malvidin diglucoside (The control wine). Stir. Wait 10 minutes and centrifuge.

Decant the clear liquids from the top into calibrated test tubes. Observe the difference in green fluorescence between the test wine and the control wine under ultraviolet light at 365 nm.

For rose wines, it is possible to increase the sensitivity using:

• 5 mL of wine treated with acetaldehyde (2.3)
• 0.2 mL hydrochloric acid, 1 M (2.2.2)
• 1 mL sodium nitrate solution, 10 g/L (2.2.3)
• 5.8 mL ammoniacal ethanol (2.2.4)

Treat the control wine in a similar manner.

2.4.  Interpretation

Wines that do not fluoresce, or have a distinctly lower fluorescence, than the control, may be considered to have no malvidin diglucoside.   Those whose fluorescence is slightly less than, equal to, or greater than the control should have a quantitative determination.

3. Quantitative Determination
   1.   Apparatus
      1.       Equipment for measuring fluorescence:

• excitation wavelength 365 nm;
• wavelength of fluorescent radiation 490 nm.
  2.       Optical quartz cell (1 cm path length)

3.2.  Reagents

3.2.1.      See qualitative examination

3.2.2.      2 mg/L quinine sulfate solution

Prepare a solution containing 10 mg very pure quinine sulfate in 100 mL sulfuric acid, 0.1 M.  Dilute 20 mL of this solution to 1 liter with sulfuric acid solution, 0.1 M.

3.3.  Procedure

Treat the wine by the method described in Qualitative examination (2), except that the aliquot of acetaldehyde treated wine is each case (red wines and roses) 1 mL.

Place the 2 mg/L solution of quinine sulfate in the cell, adjust the fluorometer to the full range (transmission T, equal to 100%) by adjusting the slit width or the sensitivity.

Replace this tube with the one containing the test wine: this is the T₁ value.

If the percentage of transmission, T₁ is greater than 35, dilute the wine with wine without malvidin diglucoside whose fluorescence must be less than 6% (this should be ascertained by previous testing.)

Remarks:

• Salicylic acid (sodium salicylate) added to the wine for stabilization before analysis, causes a spurious fluorescence which can be eliminated by an extraction with ether.
• Spurious fluorescence is caused by the addition of caramel.

3.4.  Calculation

A fluorescence intensity of 1, for wine without SO₂, for the operating conditions above with the exception of the acetaldehyde treatment, corresponds to 0.426 mg malvidin diglucoside per liter of wine.

On the other hand, red and rose wines, containing no malvidin diglucoside, give fluorescence corresponding to a T value of the order of 6%.

The amount of malvidin diglucoside in wine in milligrams per liter is therefore:

If the wine is diluted, multiply the result by the dilution factor.

3.5.  Expression of the Results

The amount of malvidin diglucoside is expressed in milligrams per liter of wine to the nearest whole number.

Bibliography

• Dorier P., Verelle L., Ann. Fals. Exp. Chim., 1966, 59, 1.
• Garoglio P.G., Rivista Vitic. Enol., 1968, 21, 11.
• Bieber H., Deutsche Lebensm. Rdsch., 1967, 44-46.
• Clermont Mlle S., Sudraud P., F.V., O.I.V., 1976 n° 586.

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OIV-MA-AS315-05A Hydroxymethylfurfural (HMF)

Type IV method

 

1. Principle of the method

 

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

Free sulfurous acid interferes with the determination. When its amount exceeds 10 mg/L, it must be previously eliminated by combining it with acetaldehyde whose excess does not interfere with the determination.

2. Colorimetric method

 

2.1.   Apparatus

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

2.1.2. Glass cells with optical paths of 1 cm.

2.2.   Reagents

2.2.1. Barbituric acid solution, 0.5% (m/v)

Dissolve 500 mg of barbituric acid in distilled water by heating slightly over a water bath at 100°C. Make up to 100 mL with distilled water. This solution keeps for about a week.

2.2.2.      Para-toluidine solution, 10% (m/v).

Place 10 g of para-toluidine in a 100 mL volumetric flask; add 50 mL of iso-propanol,, and 10 mL of glacial acetic acid,  (ρ₂₀ = 1.05 g/mL). Make up to 100 mL with iso-propanol. This solution should be renewed daily.

2.2.3. Acetaldehyde (ethanal) solution, 1% (m/v).

Prepare just before use.

2.2.4.      Hydroxymethylfurfural solution, 1 g/L.

Prepare dilutions of the above solution to containing 5, 10, 20, 30 and 40 mg hydroxymethylfurfural/L. The 1 g/L solution and its dilutions must be freshly prepared.

2.3.   Procedure

2.3.1. Preparation of sample

• Free sulfur dioxide less than 10 mg/L:

Perform the analysis on 2 mL of wine or must. If necessary filter the wine or must before analysis.

• Free sulfur dioxide greater than 10 mg/L:

15 mL of the test samples are placed in a 25 mL spherical flask with 2 mL acetaldehyde solution (2.2.3). Stir. Wait 15 minutes. Bring to volume with distilled water. Filter if necessary. Perform the analysis on 2 mL of this solution.

2.3.2. Colorimetric determination

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

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

2.3.3. Preparation of the calibration curve

Place 2 mL of each of the hydroxymethylfurfural solutions of 5, 10, 20, 30 and 40 mg/L into two sets of 25 mL flasks, a and b, and treat them as described in 2.3.2.

The graph representing the variation of absorbance with the hydroxymethylfurfural concentration in mg/L should be a straight line passing through the origin.

2.4.   Expression of results

The hydroxymethylfurfural concentration is obtained by plotting on the calibration curve the absorbance determined on the sample analyzed, taking into account any dilution carried out.

The result is expressed in milligrams per liter (mg/L) to one decimal point.

OIV-MA-AS315-05B Hydroxymethylfurfural (HMF)

 

Type IV method

 

1. Principle of the method

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

Procedures described below are given as examples.

2. High-performance liquid chromatography
   1.   Apparatus
      1. High-performance liquid chromatograph equipped with:

• a loop injector, 5 or 10 μL
• spectrophotometric detector allowing measurement at 280 nm
• column of octadecyl-bonded silica (e.g.Bondapak C₁₈-Corasil, Waters Ass)
• a recorder, preferably an integrator
• Flow rate of mobile phase: 1.5 mL/minute
• Membrane filtration system with a pore diameter of 0.45 μm.

2.2.  Reagents

2.2.1. Double distilled water

2.2.2. Methanol, distilled or HPLC quality

2.2.3. Acetic acid (ρ₂₀= 1.05 g/mL)

2.2.4. Mobile phase: water + methanol + acetic acid previously filtered through a 0.45 µm membrane filter, (40 mL + 9 mL + 1 mL)

The mobile phase must be prepared daily and degassed before using.

2.2.5. Hydroxymethylfurfural reference solution, 25 mg/L (m/v)

Into a 100 mL volumetric flask, place 25 mg of hydroxymethylfurfural accurately weighed, and bring to volume with methanol. Dilute this solution 1/10 with methanol and filter through a 0.45 μm membrane filter.

If the solution is kept refrigerated in a hermetically sealed brown glass bottle it should keep for two to three months.

2.3.  Procedure

Inject 5 (or 10) μL of the sample prepared as described above and 5 (or 10) μL of hydroxymethylfurfural reference solution into the chromatograph. Record the chromatogram.

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

2.4.  Expression of the Results

The hydroxymethylfurfural concentration is expressed in milligrams per liter (mg/L) to one decimal point.

OIV-MA-AS315-06 Cyanide derivatives

 

Type II method

1. Principle

Free and total hydrocyanic acid is liberated by acid hydrolysis and separated by distillation. After reaction with chloramine T and pyridine, the glutaconic dialdehyde formed is determined by colorimetry, due to the blue coloration it gives with 1.3-dimethyl barbituric acid.

2. Equipment

 

2.1.   Distillation apparatus: Use the distillation apparatus described for the determination of alcohol in wine.

2.2.   Round-bottomed 500 mL flask with standard taper joint.

2.3.   Water bath, thermostated at 20° C.

2.4.   Spectrophotometer permitting the measurement of absorbance at a wavelength of 590 nm.

2.5.   Glass cuvette or disposable cuvettes for one use only, with 20 mm optical path.

3. Reagents

3.1.   Phosphoric acid (H₃PO₄) at 25 p. 100 (w/v)

3.2.   Solution of chloramine T ( 3% (w/v)

3.3.   Solution of 1,3-dimethylbarbituric acid: dissolve 3.658 g of 1,3-dimethylbarbituric acid () in 15 mL of pyridine and 3 mL of hydrochloric acid (ρ₂₀ = 1.19 g/mL) and bring to 50 mL with distilled water.

3.4.   Potassium cyanide (KCN)

3.5.   Solution of potassium iodide (KI) 10% (w/v)

3.6.   Solution of silver nitrate (AgNO₃), 0.1 M

4. Procedure
   1.   Distillation:

In the 500 mL round-bottomed flask (2.2), place 25 mL of wine, 50 mL of distilled water, 1 mL of phosphoric acid (3.1) and some glass beads.

Immediately place the round-bottomed flask on the distillation apparatus. Collect the distillate through a delivery tube connected to a 50 mL volumetric flask containing 10 ml of water. The volumetric flask is immersed in an iced water bath. Collect 30-35 mL of distillate (a total of about 45 mL of liquid in the volumetric flask). Wash the delivery tube with a few milliliters of distilled water, bring the distillate to 20°C and dilute with distilled water to the mark.

4.2.  Measurement:

Place 25 mL of distillate in a 50 mL glass-stoppered Erlenmeyer flask, add 1 mL of chloramine T solution (3.2) and stopper tightly. After exactly 60 seconds, add 3mL of 1,3-dimethylbarbituric acid solution (3.3), stopper tightly and let stand for 10 minutes. Then measure the absorbance relative to the reference blank (25 mL of distilled water instead of 25 mL of distillate) at a wavelength of 590 nm in cuvettes of 20 mm optical path.

5. Establishing the standard curve
   1.    Argentimetric titration of potassium cyanide.

In a 300 mL volumetric flask, dissolve about 0.2 g of KCN (3.4) precisely weighed in 100 mL of distilled water. Add 0.2 mL of potassium iodide solution (3.5) and titrate with the solution of 0.1 M silver nitrate (3.6) until obtaining a stable yellowish color.

In calculating the concentration of KCN in the sample, 1 mL of 0.1 M silver nitrate solution corresponds to 13.2 mg of KCN.

5.2.   Standard Curve.

5.2.1.      Preparation of the standard solutions:

Knowing the KCN concentration determined in accordance with 5.1, prepare a standard solution containing 30 mg/L of hydrocyanic acid (30 mg HCN = 72.3 mg of KCN). Dilute this solution to 1/10.

Introduce 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the diluted standard solution in 100 mL volumetric flasks and bring to the mark with distilled water. The prepared standard solutions correspond to 30, 60, 90, and 150 μg/L of hydrocyanic acid, respectively.

5.2.2.      Determination:

Using 25 mL of the solutions, continue as indicated above in 4.1 and 4.2.

The values obtained for the absorbance with these standard solutions, reported according to the corresponding levels of hydrocyanic acid, form a line passing through the origin.

6. Expression of the results

Hydrocyanic acid is expressed in micrograms per liter (μg/L) without decimal.

6.1.  Calculation:

Determine the concentration of hydrocyanic acid from the standard curve. If a dilution was done, multiply the result by the dilution factor.

Repeatability (r) and Reproducibility (R)

White wine	r=3.1 μg/L	i.e. approximately 6% .
	R= 12 μg/L	i.e. approximately 25% .
Red wine	r=6.4 μg/L	i.e. approximately 8% .
	R=23 μg/L	i.e. approximately 29% .

Xi  = average concentration of HCN in the wine.

 

Bibliography

 

• JUNGE C., Feuillet vert N° 877 (1990)
• ASMUS E. GARSCHLAGEN H., Z Anal. Chem. 138, 413-422 (1953)
• WÜRDIG G., MÜLLER TH., Die Weinwissenschaft 43, 29-37 (1988)

OIV-MA-AS315-07A Examination of artificial sweeteners

Type IV method

 

1. Principle of the method

Examination of saccharine (benzoic sulfimide), Dulcin (p-ethoxyphenylurea), cyclamate (cyclohexylsulfamate) and P‑4000 (5-nitro-2-propoxyaniline or 1propoxy-2-amino-4-nitrobenzene).

After concentration of the wine, the saccharine, Dulcin and P‑4000 are extracted in an acid medium with benzene; the cyclamate is extracted from the wine after the benzene extraction using ethyl acetate (the order of extraction is important). The residues after solvent evaporation are submitted to thin layer chromatography.

Saccharine and cyclamate are identified by chromatography on cellulose plates (solvent: acetone-ethyl acetate-ammonium hydroxide), the first the benzene extract, the second in the extract by the ethyl acetate after purification by washing with ether.

These sweeteners are developed by spraying with a solution of benzidine; aniline; cupric acetate, and have the following Rf: 0.29 for cyclamate, 0.46 for saccharine.

The P‑4000 and Dulcin from the benzene extract are separated by chromatography on polyamide plates, (solvent: toluene; methanol; glacial acetic acid). These sweeteners are developed by spraying a solution of p-dimethylaminobenzaldehyde, and have the following Rf: 0.60 for Dulcin, 0.80 for P‑4000.

2. Method

 

Examination of saccharine, cyclamate, Dulcin and the P‑4000.

2.1.  Apparatus

2.1.1.      Chromatography tank

2.1.2.      Micrometry syringes or micropipettes

2.1.3.      Separator tube 15 mm in diameter and 180 mm long, with a stopcock

2.1.4.      Water bath at 100°C

2.1.5.      Regulatable oven, able to reach 125°C

2.2.  Reagents

2.2.1.      Extraction solvent:

• benzene
• ethyl acetate
  2.       aChromatography solvents:

Mixture No.1:

acetone	60 parts
ethyl acetate	30 parts
ammonium hydroxide (ρ20= 0.92 g/mL)	10 parts

Mixture No 2.:

toluene	90 parts
methanol	10 parts
Glacial acetic acid (ρ20= 1.05 g/mL)	10 parts

2.2.3.      Chromatography plates (20 x 20 cm):

• with layer of cellulose powder (for ex., Whatman CC 41 or Macherey-Nagel MN300)
• with layer of polyamide powder (for ex., Merck)
  4.       Indicating reagent for saccharine and cyclamate

Prepare:

• alcoholic solution of benzidine at 250 mg in 100 mL ethanol 
• saturated solution of cupric acetate, Cu(.H₂O
• freshly distilled aniline

Mix: 15 mL of benzidine solution, 1 mL of aniline and 0.75 mL saturated cupric acetate solution.

This solution must be freshly prepared. It corresponds to the volume required for development of a 20 x 20 cm plate.

2.2.5.      Hydrochloric acid 50% (v/v),

2.2.6.      Nitric acid solution, 25% (v/v),

2.2.7.      Indicator reagent for the P‑4000 and Dulcin: dissolve 1 g of 1,4-paradimethylaminobenzaldehyde in 50 mL methanol; add 10 mL 25% nitric acid; bring to 100 mL with methanol. Use 15 mL of this reagent for the development of a 20 x 20 cm plate.

2.2.8.      Cyclo-hexylsulfamic acid in water-ethanol solution, 0.10 g/100 mL

Dissolve 100 mg of the sodium or calcium salt of cyclo-hexylsulfamic acid in 100 mL of an equal part mixture of water and ethanol.

2.2.9.      Saccharine aqueous solution, 0.05 g/100 mL

2.2.10. Dulcin, 0.05 g/100 mL of methanol.

2.2.11. P‑4000, 0.05 g/100 mL of methanol.

2.3.  Procedure

2.3.1.      Extraction

100 mL of wine, placed in a beaker, are rapidly evaporated by boiling until the volume is reduced to 30 mL, while directing a current of cold air to the surface of the flask. Allow to cool. Acidify with 3 mL 50% hydrochloric acid (v/v). Transfer to a 500 mL conical flask with a ground stopper, add 40 mL of benzene and stir with a mechanical stirrer for 30 min.  Transfer to a separating funnel to separate the organic phase. If an emulsion is formed, it must be separated by centrifugation. Place the organic phase in a conical flask with a ground glass stopper.

Decant the wine previously extracted with benzene, which corresponds to the lower layer in the separating funnel, into a 500 mL conical flask with a ground stopper containing 40 mL of ethyl acetate. Agitate for 30 minutes and separate the organic phase as before taking care to recover only the organic fraction and not the wine.

On a 100°C water bath, evaporate each extraction solvent in 50‑60 mm diameter evaporation dishes, in small amounts while directing a stream of cold air on the surface of the dishes. Continue the evaporation until the residue has a syrupy consistency, stopping before the evaporation is complete.

Re-dissolve the benzene extract residue in the evaporation dish with 0.5 mL ethanol-water (1:1) solution (it is advisable to re-dissolve the residue once with 0.25 mL ethanol-water solution and then to rinse the dish with another portion of 0.25 mL of the same solution). Place the ethanol-water extract into a small tube with a ground stopper (extract B).

The residue of the dish in which the ethyl acetate (containing the cyclamate) has been evaporated, is dissolved with 0.5 mL of water and is poured into a small separator tube.  Wash the dish with 10 mL ether and add the ether to the contents of the separator tube.  Mix vigorously for 2 minutes and separate the lower layer into a small test tube that contains 0.5 mL ethanol.  This comprises a total of 1 mL of ethanol-water solution that contains the possible cyclamate (extract A).

2.3.2.      Chromatography

2.3.2.1.                        Saccharine and cyclamate

For examination of the saccharine and cyclamate, use a cellulose plate, with half of the plate for the identification of cyclamate and the other half for saccharine.

To do this, spot 5 to 10 μL of extract A and 5 μL of the standard cyclamate solution. On the second part of the plate spot 5 to 10 μL of extract B and 5 μL of the standard saccharine solution. Place the prepared plate in the chromatography bath containing solvent No.1 (acetone; ethyl acetate; ammonium hydroxide); allow to migrate until the solvent front reaches 10 to 12 cm. Remove the plate from the bath and dry with warm air. Spray the plate

evenly and gently with the benzidine reagent (17‑18 mL for each plate). Dry the plate with cold air. Place the plate in an oven maintained at 120-125°C for 3 minutes. The spots appear dark gray on a light chestnut background; they turn brownish with time.

2.3.2.2.                        P-4000 and Dulcin

Deposit 5 μL of extract B and 5 μL of the standard solutions of Dulcin and P-4000 on a polyamide plate. Place the prepared plate in the chromatography tank containing solvent No. 2 (toluene; methanol; acetic acid). Let the solvent front reach a height of 10 to 12cm.

Remove the plate from the tank; dry in cold air. Spray with 15 mL of the p­dimethylaminobenzaldehyde reagent, then dry with cold air until the orange-yellow colored spots appear which correspond to Dulcin and P-4000.

2.3.2.3.                        Sensitivity

The benzidine reagent allows detection of spots corresponding to 2 μg of saccharine and 5 μg of cyclamate. The p-dimethylaminobenzaldehyde reagent reveals 0.3 μg of Dulcin and 0.5 μg of P‑4000.

This method allows determination of (depending upon the efficiency of the extractions):

saccharine	2-3 mg/L
cyclamate	40-50 mg/L
Dulcin	1 mg/L
P-4000	1-1.5 mg/L

Bibliography

• TERCERO C., F.V., O.I.V., 1968, n° 277 and F.V., O.I.V., 1970, n° 352.
• Wine Analysis Commission of the Federal Health Department of Germany, 1969, F.V., O.I.V., n° 316.
• International Federation of Fruit Juice Manufacturers, 1972, F.V., O.I.V., n° 40.
• SALO T., ALRO E. and SALMINEN K., Z. Lebensmittel Unters. u. Forschung, 1964, 125, 20.

OIV-MA-AS315-07B Examination of artificial sweeteners

Type IV method

 

1. Principle of the method

Examination of saccharine, Dulcin and cyclamate.

These sweeteners are extracted from wine using a liquid ion exchanger, then re-extracted with dilute ammonia hydroxide, and are separated by thin layer chromatography using a mixture of cellulose powder and polyamide powder (solvent: xylene; n-propanol; glacial acetic acid; formic acid). These sweeteners have a blue fluorescence on a yellow background under ultraviolet light after spraying with a 2,7-dichlorofluorescein solution.

Subsequent spraying with 1,4-dimethylaminobenzaldehyde solution allows differentiation of Dulcin, which gives only one orange spot, from vanillin and the esters of p­hydroxybenzoic acid which migrate with the same Rf.

2. Method

Examination of saccharine, cyclamate and Dulcin.

2.1.  Apparatus

2.1.1.      Apparatus for expression by thin layer

2.1.2.      Glass plate 20 x 20 cm

Preparation of the plates: mix thoroughly 9 g of dry cellulose powder and 6 g of polyamide powder. Add, while stirring, 60 mL methanol. Spread on the plates to a thickness of 0.25 mm.  Dry for 10 minutes at 70°C. The quantities prepared are sufficient for the preparation of 5 plates.

2.1.3.      Water bath with a temperature regulator or a rotary evaporator,

2.1.4.      UV lamp for examination of the chromatography plates.

2.2.  Reagents

2.2.1.      Petroleum ether (40‑60°)

2.2.2.      Ion exchange resin, for example: Amberlite LA‑2

2.2.3.      Acetic acid diluted to 20% (v/v)

2.2.4.      Ion exchange solution: 5 mL of ion exchanger is vigorously agitated with 95 mL   petroleum ether and 20 mL of 20% acetic acid.  Use the upper phase.

2.2.5.      Nitric acid in solution, 1 M

2.2.6.      Sulfuric acid, 10 % (v/v)

2.2.7.      Ammonium hydroxide diluted to 25% (v/v)

2.2.8.      Polyamide powder, for example: Macherey-Nagel or Merck

2.2.9.      Cellulose powder, for example: Macherey-Nagel MN 300 AC

2.2.10. Solvent for chromatography:

Xylene	45 parts
n-Propanol	6 parts
Glacial acetic acid (ρ20= 1.05 g/mL)	7 parts
Formic acid 98‑100%	2 parts

2.2.11. Developers:

• solution of 2,7-dichlorofluorescein, 0.2 % (m/v), in ethanol,
• solution of 1,4-dimethylaminobenzaldehyde: dissolve 1 g of dimethylamino-benzaldehyde placed in a 100 mL volumetric flask with about 50 mL ethanol.  Add 10 mL of nitric acid, 25% (v/v), and bring to volume with ethanol.
  12. Standard solution:

• solution of Dulcin, 0.1 % (m/v), in methanol, 
• solution of saccharine at 0.1 g per 100 mL in a mixture of equal parts methanol and water,
• cyclamate solution: solution containing 1 g of the sodium or calcium salt of cyclohexylsulfamic acid in 100 mL of a mixture of equal parts methanol and water,
• solution of vanillin at 1 g /100 mL in a mixture of equal parts methanol and water,
• solution of the ester of p-hydroxybenzoic acid at 1 g /100 mL in methanol.

2.3 Procedure:

50 mL of wine is placed in a separatory funnel, acidified with 10 mL dilute sulfuric acid (2.2.6) and extracted with two aliquots of the ion exchange solution using 25 mL each time. The 50 mL of ion exchange solution is washed three times using 50 mL of distilled water each time, which is discarded, then three times with 15 mL of dilute ammonium hydroxide (2.2.7). The ammonia solutions recovered are then carefully evaporated at 50°C until dry on a water bath or in a rotary evaporator. The residue is recovered with 5 mL of acetone and 2 drops 1 M nitric acid solution, filtered, and again evaporated dry at 70°C on a water bath. It is necessary to avoid heating for too long and above 70°C. The residue is recovered with 1 mL of methanol.

5 to 10 μL of this solution and 2 μL of the standard solutions are spotted on the plate. Let the solvent migrate (xylene: n-propanol: acetic acid: formic acid) (2.2.10) to a height of about 15 cm, which takes about 1 hour.

After air-drying, the dichlorofluorescein solution is thoroughly sprayed on the plate. The saccharine and the cyclamate appear immediately as light spots on a salmon colored background. Under examination in ultraviolet light (254 or 360 nm), the three sweeteners appear as a fluorescent blue on a yellow background.

The sweeteners separate, from the bottom to the top of the plate, in the following order: cyclamate, saccharine, Dulcin.

The vanillin and the esters of p-hydroxybenzoic acid migrate with the same Rf as the Dulcin. To identify Dulcin in the presence of these substances, the plate then must be sprayed with a solution of dimethylaminobenzaldehyde. The Dulcin appears as an orange spot, whereas the other substances do not react.

Sensitivity - The quantity limitation shown on the chromatography plate is 5 µg for the three substances.

This method permits detection of:

Saccharin	10 mg/L
Cyclamate	50 mg/L
Dulcin	10 mg/L

BIBLIOGRAPHY

• TERCERO C., F.V., O.I.V., 1968, n° 277 and F.V., O.I.V., 1970, n° 352. 
• Wine Analysis Commission of the Federal Health Department of Germany, 1969, F.V., O.I.V., n° 316.
• International Federation of Fruit Juice Manufacturers, 1972, F.V., O.I.V., n° 40.
• SALO T., ALRO E. and SALMINEN K., Z. Lebensmittel Unters. u. Forschung, 1964, 125, 20.

OIV-MA-AS315-08 Examination of artificial colorants

Type IV method

1. Principle

The wine is concentrated to 1/3 its original volume, made alkaline with a solution of dilute sodium hydroxide and extracted with ether.  The ether phase, after being washed with water, is extracted with a dilute acetic acid solution; this acetic solution, alkalinized with ammonia, is brought to boiling in the presence of a piece of wool thread treated with aluminum sulfate and potassium tartrate.  The colorant, if any, is fixed on the wool.  The wool on which it is fixed is then placed in a dilute acetic acid solution.  After evaporation of the acetic solution, the residue is recovered with a water-alcohol solution and analyzed by thin layer chromatography for characterization of the colorant.

The aqueous phase remaining after the ether extraction contains the acid colorants that may be present. They are extracted by using their affinity for animal fibers that markedly absorb the color: they are fixed on a wool plug in a mineral acid medium.

To concentrate the coloring material, carry out a double fixation and/or several successive fixations on increasingly smaller wool plugs.

Coloring of the wool plug indicates that an artificial colorant was added to the wine; the colorant is then identified by thin layer chromatography.

2. Apparatus

 

2.1.   20 x 20 glass plates covered with cellulose powder,

2.2.   Chromatography tank

3. Reagents

 

3.1.   Ethyl ether

3.2.   Sodium hydroxide solution, 5% (m/v)

3.3.   Glacial acetic acid (ρ₂₀= 1.05 g/mL)

3.4.   Dilute acetic acid, containing one part glacial acetic acid to 18 parts water

3.5.   Dilute hydrochloric acid: to one part hydrochloric acid (ρ₂₀ = 1.19 g/mL), add 10 parts distilled water

3.6.   Ammonium hydroxide (ρ₂₀ = 0.92 g/mL)

3.7.   White wool threads, previously washed, degreased with ether and dried

3.8.   White wool threads, previously washed, degreased with ether, dried and acidified

Acidulant: Dissolve 1 g crystallized aluminum sulfate and 1.2 g acid potassium tartrate in 500 mL water. Place 10 g of the white wool threads, previously washed, degreased with ether and dried in the solution and stir about 1 hour. Let stand 2 to 3 hours; drain, let dry at room temperature.

3.9.  Solvent No.1 for chromatography of colorants with basic characteristics:

n-Butanol	50 mL
Ethanol	25 mL
acetic acid (ρ20 = 1.05 g/mL)	10 mL
distilled water	25 mL

3.10.        Solvent No.2 for chromatography of colorants with acidic characteristics:

n-Butanol	50 mL
ethanol	25 mL
ammonium hydroxide (ρ20 = 0.92 g/mL)	10 mL
distilled water	25 mL

4. Procedure

4.1.   Examination of colorants with basic characteristics.

4.1.1. Extraction of the coloring materials.

Place 200 mL of wine in a 500 mL glass conical flask and boil until reduced to 1/3 its volume.

After cooling, neutralize with 5% sodium hydroxide solution until the natural color of wine shows a marked change.

Extract twice using 30 mL ether. The ether phases are recovered, containing basic colorants to be determined; the extraction residue must be saved for the analysis of acidic colorants.

Wash the extracted ether twice with 5 mL of water to eliminate the sodium hydroxide; mix with 5 mL dilute acetic acid.  The acidic aqueous phase obtained is colored in the presence of a basic colorant.

The presence of the colorant may be confirmed by fixation on acidified wool.  Make the acidic aqueous phase obtained alkaline using 5% ammonia.  Add 0.5 g acidified wool and boil for about 1 minute.  Rinse the wool under running water.  If the wool is colored, the wine contains some basic colorant.

4.1.2. Characterization by thin-layer chromatography.

The aqueous acetic phase containing the basic colorant is concentrated to 0.5 mL. If the colorant is fixed on the acidic wool, the wool plug is treated by boiling with 10 mL distilled water and a few drops of acetic acid (ρ₂₀ = 1.05 g/mL).  Remove the wool fragment after wringing out liquid.  Concentrate the solution to 0.5 mL.

Deposit 20 μL of this concentrated solution on the cellulose plate 3 cm from the lateral edge and 2 cm from the lower edge of the plate.

Place the plate in the tank containing solvent No.1 so that the lower edge is immersed in the solvent to a depth of 1 cm.

When the solvent front has migrated to a height of 15 to 20 cm, remove the plate from the tank.  Allow to air dry.

Identify the colorant by means of a solution of known artificial colorants of basic characteristics deposited simultaneously on the chromatogram.

4.2.   Examination of colorants with acidic characteristics

4.2.1. Extraction of the coloring material.

Use the residue from the wine used for examining colorants with basic characteristics, concentrated to 1/3 and neutralized after extraction with ether.

If the first part of the procedure has not been conducted, start with 200 mL wine, place in a conical flask, boil until reduced to 1/3.

In either case, add 3 mL of dilute hydrochloric acid and 0.5 g of white wool: boil for 5 minutes, decant the liquid and wash the wool under running water.

In the conical flask which contains the wool, add 100 mL water and 2 mL dilute hydrochloric acid; boil for 5 minutes, separate the acidic liquid and repeat this procedure until the liquid used to wash is colorless.

After the wool has been thoroughly washed to eliminate the acid completely, recover in a conical flask with 50 mL distilled water and a few drops of ammonium hydroxide (ρ₂₀ = 0.92 g/mL): bring to a gentle boil for 10 minutes in order to dissolve any artificial coloring matter fixed on the wool.

Remove the wool from the flask, bring the liquid volume to 100 mL and boil until the ammonia completely evaporates.  Acidify with 2 mL of dilute hydrochloric acid (check that the reaction of the liquid is definitely acidic by placing 1 drop of this liquid on indicator paper).

Add to the flask 60 mg (about 20 cm of standard thread) of white wool and boil for 5 minutes; remove the wool and rinse it under running water.

If, after this procedure, the wool is colored red, when it involves red wine, or yellow if it pertains to white wine, the presence of artificial organic coloring matter of an acidic nature is proven.

If the color is weak or uncertain, repeat the ammonia treatment and do a second fixation using a 30 mg wool thread.

If, during the course of the second fixation a weak but distinct pink color is obtained, assume the presence of an acidic colorant.

If necessary for a more definite determination, carry out new fixations-elutions (up to 4 or 5) using a procedure identical to that used for the second fixation until a faint but distinct pink color is obtained.

4.2.2. Characterization by thin layer chromatography.

The plug of colored wool is treated by boiling with 10 mL distilled water and few drops of ammonium hydroxide (ρ₂₀ = 0.92 g/mL). Recover the piece of wool after wringing. Concentrate the ammonium hydroxide solution to 0.5 mL.

Deposit 20 μL of this solution on a cellulose plate to within 3 cm of the lateral edge and 2 cm of the lower edge of the plate.

Put the plate in place in the tank so that the lower edge is immersed in the solvent to a depth of 1 cm.

When the solvent front has migrated to a height of 15 to 20 cm, remove the plate from the tank and let dry in the air.

Identify the colorant by means of known artificial coloring solutions deposited simultaneously on the chromatogram.

Bibliography

• TERCERO C., F.V., O.I.V., 1970, n° 356.
• Arata P., Saenz-Lascano-Ruiz, Mme I., F.V., O.I.V., 1967, n° 229.

OIV-MA-AS315-09 Diethylene glycol (2-hydoxy-ethxyethanol)

Type IV method

 

1. Objective

 

The detection of diethylene glycol,, in wine where its concentration is equal to or greater than 10 mg/L.

 

2. Principle

 

Separation of diethylene glycol from other constituents in wine by gas chromatography using a capillary column, after extraction with ether.

Note: The operating conditions described below are provided as an example.

 

3. Apparatus

 

3.1.  Gas chromatograph equipped with:

• split-splitless injector,
• flame ionization detector,
• capillary column coated with a film of polyethyleneglycol (Carbowax 20 M), 50 m x 0.32 mm I.D.

Operating conditions:

• Injector temperature: 280°C.
• Detector temperature: 270°C.
• Carrier gas: hydrogen.
• Flow rate of carrier gas: 2 mL/min.
• Flow rate: 30 mL/min.
• Injection: splitless.
• Injection volume: 2 μL.
• Injection 35°C - flow closed after 40 seconds.
• Temperature program: 120°C to 170°C at 3°C/min.
  2.   Centrifuge

 

4. Reagents

4.1.  1,3-propanediol, 1 g/L, in alcohol, 20% (v/v), (internal standard).

4.2.  Aqueous solution of diethyleneglycol 20 mg/L.

 

5. Procedure

Into a 50 mL flask, place:

• 10 mL of wine
• 1 mL of 1,3-propanediol solution
• 25 mL diethyl ether.

Shake and add sufficient quantity of neutral potassium carbonate to saturate the mixture.  Shake. Separate the two phases by centrifugation.

Carry out a second extraction. Eliminate the diethyl ether by evaporation and recover the residue with 5 mL ethanol.

The yield of the extraction must be at least 90%.

Carry out the chromatography according to the conditions given in 3.1.

6. Results

 

The diethylene glycol is identified by comparing its retention time to the time of the reference solution, analyzed under the same conditions as the wine.

The amount is determined by comparison to the reference solution using the internal standard method.

It is recommended, if the concentration is equal to or less than 20 mg/l, to confirm the presence by mass spectrometry.

Bibliography

• Bandion F., VALENTA M. & KOHLMANN H., Mitt. Klosterneuburg, Rebe und Wein, 1985, 35, 89.
• Bertrand A., Conn. vigne vins, 1985, 19, 191.
• Laboratoire de la répression des fraudes et du contrôle de la qualité de Montpellier, F.V., O.I.V., 1986, n° 807.

OIV-MA-AS315-10 Measuring ochratoxine A in wine after going through an immunoaffinity column and HLPC with fluorescence detection

Type II method

1. Field of application

This document describes the method used for determining ochratoxine A (OTA) in red, rosé, and white wines, including special wines, in concentrations ranging up 10 µg/l using an immunoaffinity column and high performance liquid chromatography (HPLC) [1].

This method was validated following an international joint study in which OTAs were measured in white and red wines during the analysis of naturally contaminated wines and wines with toxins ranging from 0.01 µg/l to 3.00 μg/l.

This method can apply to semi-sparkling wines and sparkling wines as long as the samples have been degassed beforehand, through sonication, for example.

2. Principle

Wine samples are diluted with a solution containing polyethylene glycol and sodium hydrogen carbonate. This solution is filtered and purified on the immunoaffinity column.

OTA is eluted with methanol and quantified by HPLC in inverse state with fluorimetric detection.

3. Reagents

3.1.  Reagents for separation of the OTA on an immunoaffinity column

The reagents listed below are examples. Suppliers of immunoaffinity columns may offer dilution solutions and eluents suitable for their products. If so, it is preferable to use these products.

3.1.1.      Sodium hydrogen phosphate dihydrate() CAS [10028-24-7]

3.1.2.      Sodium dihydrogen phosphate monohydrate ( ) CAS [10049-21-5]

3.1.3.      Sodium chloride (NaCl) CAS [7647-14-5]

3.1.4.      Purified water for laboratories, for example EN ISO 3696 quality (water for analytical laboratory use – Specification and test method [ISO 3696:1987]).

3.1.5.      Phosphate buffer (dilution solution)

Dissolve 60g of (3.1.1) and 8.8g of (3.1.2) in 950ml of water and add more water to make up to 1 litre.

3.1.6.      Phosphate buffer saline (washing solution)

Dissolve 2.85g of (3.1.1), 0.55g of (3.1.2) and 8.7g of NaCl in 950ml of water and add more water to make up to 1 litre.

3.1.7.      Methanol () CAS [67-56-1]

3.2.  Reagents for HPLC

3.2.1.      Acetonitrile for HPLC () CAS [75-05-8]

3.2.2.      Glacial acetic acid (COOH) CAS [64-19-7]

3.2.3.      Mobile phase: water: acetonitrile: glacial acetic acid, 99:99:2, v/v/v

Mix 990 ml of water with 990 ml of acetonitrile (3.2.2) and 20 ml of glacial acetic acid (3.2.3). In the presence of undissolved components, filter through a 0.45µm filter. Degas (with helium, for example) unless the HPLC equipment used includes a degassing step.

3.3.  Reagents for the preparation of the OTA stock solution

3.3.1.      Toluene () CAS [108-88-3]

3.3.2.      Mixture of solvents (toluene: glacial acetic acid, 99:1, v/v).

Mix 99 parts in volume of toluene (3.3.1) with one part volume of glacial acetic acid (3.2.2).

3.4.   OTA stock solution

Dissolve 1 mg of OTA or the same content in a bulb, if the OTA was obtained in the form of film after evaporation, in the solvent mixture (3.12) to obtain a solution containing approximately 20 to 30 μg/ml of OTA.

To determine the exact concentration, record the absorption spectrum between 300 and 370 nm in a quartz space with 1 cm of optical path while using the solvent mixture (3.12) as a blank. Identify maximum absorption and calculate the concentration of OTA (c) in µg/ml by using the following equation:

In which:

= Absorption determined by the longest maximum wave (about 333 nm)

M = OTA molecular mass = 403,8 g/mole

ε = coefficient d'extinction molaire de l'OTA dans le mélange de solvant (3.12) ( = 544/mole)

δ = optical pathway (cm)

This solution is stable at -18°C for at least 4 years.

3.5.   Standard OTA solution (2 µg/ml in toluene: acetic acid, 99:1, v/v)

Dilute the stock solution (3.13) with the solvent mixture (3.12) to obtain a standard solution of OTA with a concentration of 2 μg/ml.

This solution can be stored at + 4 °C in a refrigerator. The stability should be tested regularly.

4. Equipment

Usual laboratory equipment and in particular, the following equipment:

4.1.   Glass tubes (4 ml)

4.2.   Vacuum pump to prepare the immunoaffinity columns.

4.3.   Reservoir and flow tube adapted to immunoaffinity columns.

4.4.   Fibre glass filters (for example Whatman GF/A).

4.5.   Immunoaffinity columns specifically for OTA.

The column should have the total link capacity of at least 100 ng OTA. This will allow for a purification yield of at least 85% when a diluted solution of wine containing 100 ng OTA is passed through.

4.6.   Rotating evaporator

4.7.   Liquid chromatography, a pump capable of attaining a constant flow of 1 ml/mn isocratic, as with the mobile phase.

4.8.   Injection system must be equipped with 100 μl loop.

4.9.   Column of analytical HPLC in steel 150  4.6 mm (i.d.) filled with a stationary phase (5 μm) preceded with a pre-column or a pre-filter (0,5 μm) containing an appropriate phase. Different size columns can be used provided that they guarantee a good base line and background noise enabling the detection of of OTA peaks, among others.

4.10.         Fluorescence detector is connected to the column and the excitation wavelength is set at 333 nm and the emitting wavelength at 460 nm.

4.11.         Information retrieval system

4.12.         U.V. spectrometer

5. Procedure

5.1.   Preparation of samples

Pour 10 ml of wine in a 100 ml conical flask. Add 10 ml of the dilution solution (3.8). Mix vigorously. Filter through fibreglass filter (4.4). Filtration is necessary for cloudy solutions or when there is precipitation after dissolving.

5.2.   Purification by immunoaffinity column

Set up the by immunoaffinity column (4.5) to the vacuum pump (4.2), and attach the reservoir (4.3).

Add 10 ml (equivalent to 5 ml of wine) of the diluted solution in the reservoir. Put this solution through the immunoaffinity column at a flow of 1 drop per second. The immunoaffinity column should not become dry. Wash the immunoaffinity column with 5 ml of cleaning solution (3.9) and then with 5 ml of water at a flow of 1 to 2 drops per second.

Blow air through to dry column. Elute OTA in a glass flask (4.1) with 2 ml of methanol (3.4) at the rate of 1 drop per second. Evaporate the eluate to dryness at 50° C with nitrogen. Dissolve again immediately in 250 μl of the HPLC mobile phase (3.10) and keep at 4° C until the HPLC analysis.

5.3.   HPLC analysis

Using the injection loop, inject 100 μl of reconstituted extract (equivalent to 2 ml of wine) in the chromatography.

Operating conditions

 

Flow	1 ml /min.
Mobile phase	acetonitrile: water: glacial acetic acid (99:99:2, v/v/v)
Fluorescence detector	Excitation wavelength = 333 nm
	Emitting wavelength = 460 nm
Volume of injection	100 μl

6. Quantification of ochratoxine A (OTA)

The quantification of OTA should be calculated by measuring the area or the height of the peaks at the OTA retention time and compared to the calibration curve

6.1.   Calibration curve

Prepare a calibration curve dayly and every time chromatographical conditions change. Measure out 0.5 ml of the standard OTA solution (3.14) at 2 μlg/ml in a glass flask and evaporate the solvent using nitrogen.

Dissolve again in 10 ml in the HPLC mobile phase (3.10) which was previously filtered using a 0.45 100 μm filter. This produces an OTA of 100 ng/ml solution.

Prepare 5 HPLC calibration solutions in five 5 ml graduated flasks following Table 1.

Complete each 5 ml standard solution with HPLC mobile phase. (3.10).

Inject 100 μl of each solution in the HPLC.

Table 1

	Std 1	Std 2	Std 3	Std 4	Std 5
µl of mobile phase filtered HPLC (3.10)	4970	4900	4700	4000	2000
µl of OTA solution at 100 ng/ml:	30	100	300	1000	3000
OTA concentration (ng/ml)	0.6	2.0	6.0	20	60
Injected OTA (ng)	0.06	0.20	0.60	2.00	6.00

 

NOTE:

If the quantity of OTA in the samples is outside the calibration range, an appropriate dilution should occur or smaller volumes should be injected. In these cases, the final (7) should be reviewed on a case by case basis.

Due to the great variations in concentrations, it is recommended to pass the linear calibration by zero in order to obtain an exact quantification for low concentrations of OTA. (less than 0.1 μg/l)

 

7. Calculations

Calculate the quantity of OTA in the aliquot of the solution testes and injected in the HPLC column.

Calculate the concentration of OTA () in ng/ml (equivalent to µg/l) by using the following formula:

Where:

is the volume of ochratoxin A (in ng) in the aliquot part of the template injected on the column and evaluated from the calibration curve.

F:is the dilution factor

is the sample volume to be analysed (10 ml)

the volume of the solution tested and injected in the column (100 μl)

is the volume of solution used to dissolve the dry eluate (250 μl)

8. Performances using this method in laboratories

Table 2 regroups performances of the method applied to white, rosé and red wines in laboratories

participating in the validation of this method.

Table 2. Recovery of ochratoxin A from wines overweighted with different concentrations of added ochratoxin A
	Red wine		Rosé wine		White wine
Addition (µg/l)	Yield ± SD* (%)	RSD# (%)	Yield ± SD* (%)	RSD# (%)	Yield ± SD* (%)	RSD# (%)
0.04	96.7 ± 2.2	2.3	94.1 ± 6.1	6.5	91.6 ± 8.9	9.7
0.1	90.8 ± 2.6	2.9	89.9 ± 1.0	1.1	88.4 ± 0.2	0.2
0.2	91.3 ± 0.6	0.7	88.9 ± 2.1	2.4	95.1 ± 2.4	2.5
0.5	92.3 ± 0.4	0.5	91.6 ± 0.4	0.4	93.0 ± 0.2	0.2
1.0	97.8 ± 2.6	2.6	100.6 ± .,5	2.5	100.7 ± 1.0	1.0
2.0	96.5 ± 1.6	1.7	98.6 ± 1.8	1.8	98.0 ± 1.5	1.5
5.0	88.1 ± 1.3	1.5	-	-	-	-
10,0	88,9 ± 0,6	0,7	-	-	-	-
Average of averages	92.8 ± 3.5	3.8	94.5 ± 5.2	5.5	94.5 ± 4.1	4.3

* SD =   Spread type (Standard deviation) (n = 3 replicates) ;

^#  RSD = Relative spread type (Variation percentage).

9. Group work

The method was validated by a group study with the participation of 16 laboratories in 8 countries, following the protocol recommendations harmonised for validating the analysis methods. [2]. Each participant analysed 10 white wines, 10 red wines, representing 5 random duplicate wines; naturally contaminated or with OTA added. The performances of the method which resulted from this work are found in appendixes I and II, outlining critical points of the method are found in appendix III.

10. Participating laboratories

Unione Italiana Vini, Verona Istituto Sperimentale per l’Enologia, Asti Istituto Tecnico Agraria, S. Michele all’Adige (TN) Università Cattolica, Piacenza Institute for Health and Consumer Protection, JRC – Ispra Neotron s.r.l., S. Maria di Mugnano (MO) Chemical Control s.r.l., Madonna dell’Olmo (CN) Laboratoire Toxicologie Hygiène Appliquée, Université V. Segalen, Bordeaux Laboratoire de la D.G.C.C.R.F. de Bordeaux, Talence National Food Administration, Uppsala Systembolagets Laboratorium, Haninge Chemisches Untersuchungsamt, Trier State General Laboratory, Nicosia Finnish Customs Laboratory, Espoo Central Science Laboratory, York E.T.S. Laboratories, St. Helena, CA

ITALY ITALY ITALY ITALY ITALY ITALY ITALY FRANCE FRANCE SWEDEN SWEDEN GERMANY CYPRUS FINLAND UNITED KINGDOM UNITED STATES

 

11. References

[1] A. Visconti, M. Pascale, G. Centonze. Determination of ochratoxin A in wine by means of immunoaffinity column clean-up and high-performance liquid chromatography. Journal of Chromatography A, 864 (1999) 89-101.

[2] AOAC International 1995, AOAC Official Methods Program, p. 23-51.

Appendix I

The following data was obtained in inter-laboratory tests, according to harmonised protocol recommendations for joint studies in view of validating an analysis method.

WHITE WINE		Added OTA (μg/l)
Sample	White	0.100	1.100	2.000	n.c.
Inter-laboratory test year	1999	1999	1999	1999	1999
Number of laboratories	16	16	16	16	16
Number of laboratories retained after eliminating  absurd findings	14*	13*	14	14	15
Number of eliminated laboratories	-	1	2	2	1
Number of accepted results	28	26	28	28	30
Average value (μg/l)	<0,01	0,102	1,000	1,768	0,283
Spread-type/Repeatabilityr (μg/l)	-	0.01	0.07	0.15	0.03
Relative spread-type (Variation percentage) /Repeatability RSDr (%)	-	10.0	6.6	8.5	10.6
Repeatability limit r (μg/l)	-	0.028	0.196	0.420	0.084
Spread-type/capacity of being reproduced sR (μg/l)	-	0.01	0.14	0.23	0.04
Relative spread-type (variation percentage) /capacity of being reproduced RSDR (%)	-	14.0	13.6	13.3	14.9
Capacity of being reproduced limit  R  (μg/l)	-	0.028	0.392	0.644	0.112
Extraction yield  %	-	101.7	90.9	88.4	-

* 2 laboratories were excluded from the statistical 'evaluation due to high detection limit (= 0,2 μg/l).

n.c. = sample naturally contaminated

 

Appendix II

The following data was obtained in inter-laboratory tests, according to harmonised protocol recommendations for joint studies in view of validating an analysis method.

RED WINE		Added OTA (μg/l)
samples	White	0.200	0.900	3.000	n.c.
Inter-laboratory test year	1999	1999	1999	1999	1999
Number of laboratories	15	15	15	15	15
Number of laboratories retained after eliminating absurd findings	14*	12*	14	15	14
Number of eliminated laboratories	-	2	1	-	1
Number of accepted results	28	24	28	30	28
Average value (μg/l)	<0.01	0.187	0.814	2.537	1.693
Spread-type/Repeatabilityr (μg/l)	-	0.01	0.08	0.23	0.19
Relative spread-type (Variation percentage) /Repeatability RSDr (%)	-	5.5	9.9	8.9	10.9
Repeatability limit r (μg/l	-	0.028	0.224	0.644	0.532
Spread-type/capacity of being reproduced sR (μg/l )	-	0.02	0.10	0.34	0.23
Relative spread-type (variation percentage) /capacity of being reproduced RSDR (%)	-	9.9	12.5	13.4	13.4
Capacity of being reproduced limit R (μg/l)	-	0.056	0.280	0.952	0.644
Extraction yield  %	-	93.4	90.4	84.6	-

* 1 laboratory was excluded from the statistical evaluation because of high detection limits (= 0,2 µg/l).

n.c. = naturally contaminated sample

 

Appendix III

Guide to the critical points of the method of measuring ochratoxin A by immunoaffinity column, type II.

The critical points to observe are listed below for information purposes only and are a guide to applying the method. Numbering refers to paragraphs of the resolution.

1. Field of application

For information purposes only the method can be applied to grape musts, partially fermented grape musts, and new wines still under fermentation. The validation parameters concern wines only.

2. Principle

The method is broken down into two steps. The first step involves purification and concentration of the OTA in the wine or the must by capture on an immunoaffinity column followed by elution. The second step involves quantification of the eluate by HPLC using fluorescence detection.

3. Reagents

3.1.  OTA stock solution

The use of OTA in solid form in not recommended; it is recommended to use a standard solution of OTA (point 3.5)

3.2.  Standard OTA solution

Use of a commercial solution of standard concentration (around 50 µg/ml) with an analysis certificate stating the reference value and uncertainty of the concentration.

In theory the volume of these solutions is not certified, and they must be sampled with certified pipettes to constitute stock solutions from 0.25 to 1 mg/l in pure ethanol or in the mobile phase of the HPLC method (see 3.2.3).

This solution is stable at -18°C for at least 4 years.

4. Equipment

4.1.  Recommendations for assessment of the performance of immunoaffinity columns (optional)

The step of concentration on an immunoaffinity column is a major source of inaccuracy in the analysis method. Experience shows that the various columns offered on the market could have recovery rates of between 70 and 100%.

It is therefore recommended to check the performance of a batch of columns before use. This step is recommended where there has been a change in supplier or column references.

4.2.  Characterisation of the batch of columns (measure of recovery rate):

Select around 10 columns representative of the types of column routinely used in the laboratory, and all from different batch numbers. Prepare the same number of wines representing different matrices, with zero OTA concentrations, with known additions x_i of between 0.5 and

2 μg∙kg^-1. After the known additions quickly analyse these n samples with the batch of selected columns. Let y_i be the values found.

The recovery rate data are calculateed, the rate being the measured quantity in relation to the known added quantity.

	Recovery rate with column
	Average recovery rate
	Standard deviation of the recovery rate

The standard deviation of the recovery rate calculated in this way represents not only the variability of the recovery rate of the columns, but also the standard uncertainty of the measurement system used after use of the columns (HPLC). It is nevertheless possible to establish a reasonable estimate of the standard deviation of the recovery rate of the columns by deducting the standard uncertainty of the HPLC system from the calculated recovery error:

• Estimate the standard uncertainty (expressed as the standard deviation) of the measurement system in the strict sense of the word (without considering the the immunoaffinity column step).

For this it is possible to use a fidelity study on the OTA solutions.

The standard deviation of the recovery rate Sp is estimated as follows:

For a fairly wide concentration range, it is preferable to express this value as the coefficient of variation of the standard deviation (RSDR).

5. Procedure

The procedure outlined in point 5 is an example. The composition of dilution and washing solutions may differ from one column manufacturer to another. Likewise, the concentration of the diluted wine sample may be adjusted as needed.

6. Quantification of ochratoxine A (OTA)

6.1.  Calibration curve

Prepare a calibration curve daily or each time that the chromatographic conditions change. Prepare the curve using solutions produced by diluting the stock solution in the mobile phase (see 3.2.3). The values chosen must provide the working range taking into account the concentration factor of the wine.

OIV-MA-AS315-11 HPLC-Determination of nine major anthocyanins in red and rosé wine

Type II method

1. Field of application

The analytical method concerns the determination of the relative composition of anthocyanins in red and rosé wine. The separation is performed by HPLC with reverse phase column and UV-VIS detection.

Many authors [3, 6-17] have published data on the anthocyanin composition of red wines using similar analytical methods. For instance Wulf et al. [18] have detected and identified 21 anthocyanins and Heier et al. [13] nearly 40 by liquid chromatography combined with mass spectrometry. The anthocyanin composition may be very complex, so it is necessary to have a simple procedure. Consequently this method only determines the major compounds of the whole anthocyanin fraction.

Member states are encouraged to continue research in this area to avoid any non scientific evaluation of the results.

2. Principle

Separation of the five most important non acylated anthocyanins (see Figure 1, peaks 1-5) and four major acylated anthocyanins (see Figure 1, peaks 6-9).

Analysis of red and rosé wine by direct separation by HPLC by using reverse phase column with gradient elution by water/formic acid/acetonitrile with detection at 518 nm [1.2].

 

3. Reagents and material

Formic acid (p.a. 98 %) (CAS 64-18-6);

Water, HPLC grade;

Acetonitrile, HPLC grade (CAS 75-08-8);

HPLC solvents:

Solvent A:  Water/Formic acid/Acetonitrile 87 : 10 : 3 (v/v/v)

Solvent B:  Water/Formic acid/Acetonitrile 40 : 10 : 50 (v/v/v)

Membrane filter for HPLC solvent degassing and for sample preparation to be analysed.

Reference products for peak identification.

The HPLC analysis of anthocyanins in wine is difficult to perform due to the absence of commercially available pure products. Furthermore, anthocyanins are extremely unstable in solution.

The following anthocyanin pigments are commercially available:

Cyanidol-3-glucoside (also couromanin chloride); M = 484.84 g/mol

Peonidol-3-glucoside; M = 498.84 g/mol

Malvidol-3-glucoside (also Oeninchloride); M = 528.84 g/mol

Malvidol-3,5-diglucoside (also Malvinchloride); M = 691.04 g/mol

4. Apparatus

 

HPLC system with:

binary gradient pump, injection system for sample volumes ranging from 10 to 200 μl,

diode array detector or a UV detector with a visible range,

integrator or a computer with data acquisition software,

furnace for column heating at 40°C,

solvent degassing system,

analytical column, for example:

LiChrospher 100 RP 18 (5 μm) in LiChroCart 250-4 guard column: for example RP 18 (30-40 mm) in a cartridge 2 mm in diameter x 20 mm long

5. Procedure

5.1.  Preparation of samples

Clear wines are poured directly without any preparation into the sample vials of the automatic sample changer. Cloudy samples are filtered using a 0.45 μm membrane filter for HPLC sample preparation. The first part of the filtrate should be rejected.

Since the range of the linearity of absorption depending on the concentration of anthocyanins is large, it is possible to modulate the injection volumes between 10 and 200 μl depending on the intensity of the wine colour. No significant difference between the results obtained for different injection volumes was observed.

5.2.  Analysis

HPLC conditions

The HPLC analysis is carried out in the following conditions:

Injection Volume:	50 μl (red wine) up to 200 μl (rosé wine)
Flow:	0.8 ml/minute
Temperature:	40°C
Run time:	45 minutes
Post time:	5 minutes
Detection:	518 nm

Gradient elution:	Time (min)	Solvent A % (v/v)	Solvent B % (v/v)
	0	94	6
	15	70	30
	30	50	50
	35	40	60
	41	94	6

To check the column efficiency, the number of theoretical plates (N) calculated according to malvidol-3-glucoside should not be below 20,000, and the resolution (R) between peonidol-3-coumaryl glucoside and malvidolin-3-coumaryl glucoside should not be lower than 1.5. Below these values, the use of a new column is recommended.

A typical chromatogram is given in Figure 1, where the following anthocyanins are separated:

		Peak-N°
Group 1: “Nonacylated anthocyanidin-3-glucosides”:	delphinidol-3-glucoside cyanidol-3-glucoside petunidol-3-glucoside peonidol-3-glucoside malvidol-3-glucoside	1 2 3 4 5
Group 2: “Acetylated anthocyanidin-3-glucosides”:	peonidol-3-acetylglucoside malvidol-3-acetylglucoside	6 7
Group 3: “Coumarylated anthocyanidin-3-glucosides”:	peonidol-3-coumarylglucoside malvidol-3-coumarylglucoside	8 9

6. Expression of results

Note that the values are expressed as relative amounts of the sum of the nine anthocyanins defined in this method.

7. Limit of detection and limit of quantification

The limit of detection (LD) and the limit of quantification (LQ) are estimated following the instructions in the resolution OENO 7-2000 “Estimation of the Detection and Quantification Limits of a Method of Analysis“. Along the line of the ”Logic Diagram for Decision-Making” in N° 3 the graph approach has to be applied following paragraph 4.2.2.

For this purpose a part of the chromatogram is drawn out extendedly enclosing a range of a tenfold mid-height width (w½) from an anthocyan relevant peak.

Furthermore two parallel lines are drawn which just enclose the maximum amplitude of the signal window. The distance of these two lines gives , expressed in milli Absorption Units (mAU).

The limit of detection (LD) and the limit of quantification (LQ) depend on the individual measurement conditions of the chemical analysis and are to be determined by the user of the method. The Annex gives an example of its determination with the following results:

h_max = 0,208 [mAU]; LD = 3 x 0,208 [mAU] = 0,62 [mAU].

LQ = 10 x 0, 208 [mAu] = 2,08 [mAU].

Recommendation:

With combined data out of the whole Anthocyanin composition such as the sum of Acylated Anthocyanins or the ratio of Acetylated to Coumarylated Anthocyanins the calculation should not be carried out in cases where one of the components is below the limit of quantification (LQ).

On the other hand measurements below the limit of quantification (LQ) are not devoid of information content and may well be fit for purpose [1].

Bibliography:

• Thompson, M.; Ellison, S.L.R. ; Wood, R., Harmonized Guidelines for Single-Laboratory Validation of Methods of Analysis, Pure Appl. Chem. (2002) 74: 835- 855

8. Fidelity parameters

The repeatability (r) and the reproducibility (R) values for the nine anthocyanins are given in Table 2 and depend on the amount of the peak area. The uncertainty measurement of a particular peak area is determined by the value of r and R which corresponds to the nearest value given in Table 2.

The values made up of validation data can be calculated by following the appropriate statistical rules. To calculate the total error (sr) for example of the sum of acetylated anthocyanins, the variances (sr2) of specific the total error of ratios, for example, that of acetylated to coumarylated anthocyanins the square of relative errors (=sr/ai) are to be added. By using these rules, all the fidelity values can be calculated by using the data in Table 2.

Annex A : Bibliography

• Marx, R., B. Holbach, H. Otteneder; Determination of nine characteristic Anthocyanins in Wine by HPLC; OIV, F.V.N° 1104 2713/100200
• Holbach, B., R. Marx, M. Ackermann; Bestimmung der Anthocyanzusammensetzung von Rotwein mittels Hochdruckflüssigkeitschromatographie (HPLC). Lebensmittelchemie (1997) 51: 78 – 80
• Eder, R., S. Wendelin, J. Barna; Auftrennung der monomeren Rotweinanthocyane mittels. Hochdruckflüssigkeitschromatographie (HPLC).Methodenvergleich und Vorstellung einer neuen Methode. Mitt. Klosterneuburg (1990) 40: 68-75
• ISO-5725-2: 1994 “Accuracy (trueness and precision) of measurement methods and results - Part 2: Basic method for the determination of repeatability and reproducibility”
• Otteneder, H., Marx, R., Olschimke, D.; Method-performance study on the determination of nine characteristic anthocyanins in wine by HPLC. O.I.V. F.V.N° 1130 (2001)
• Mattivi F.; Scienza, A.; Failla, O.; Vika, P.; Anzani, R.; Redesco, G.; Gianazza, E.; Righetti; P. Vitis vinifera - a chemotaxonomic approach: Anthocyanins in the skin. Vitis (special issue) 1990, 119-133
• Roggero, I.P.; Larice, I.L.; Rocheville-Divorne, C.; Archier, P.; Coen, V. Composition Antocyanique des cepages. Revue Francaise d’Oenologie 1998, 112, 41-48
• Eder, R.; Wendelin, S; Barna, J. Classification of red wine cultivars by means of anthocyanin analysis. Mitt. Klosterneuburg 1994, 44, 201-212
• Arozarena, I.; Casp, A.; Marin, R.; Navarro, M. Differentiation of some Spanish wines according to variety and region based on their anthocyanin composition. Eur. Food Res. Technol. 2000, 212, 108-112
• Garcia-Beneytez, E.; Revilla, E.; Cabello, F. Anthocyanin pattern of several red grape cultivars and wines made from them. Eur. Food Res. Technol. 2002, 215, 32-37
• Arozarena, I.; Ayestarán, B.; Cantalejo, M.J.; Navarro, M.; Vera, M.; Abril, K.; Casp, A. Eur. Food Res. Technol. 2002, 214, 313-309
• Revilla, E.; Garcia-Beneytez, E.; Cabello, F.; Martin-Ortega, G.; Ryan, J-M. Value of high-performance liquid chromatographic analysis of anthocyanins in the differentiation of red grape cultivars and red wines made from them. J. Chromatogr A 2001, 915, 53-60
• Heier, A.; Blaas, W.; Droß, A.; Wittkowski, R.; Anthocyanin Analysis by HPLC/ESI-MS, Am.J.Enol.Vitic, 2002, 53, 78-86
• Arozarena, I.; Casp, A.; Marin, R.; Navarro, M. Multivariate differentiation of Spanish red wines according to region and variety. J. Sci. Food Agric, 2000, 80, 1909-1917
• Anonymous. Bekanntmachung des Bundesinstituts für gesundheitlichen Verbraucherschutz und Veterinärmedizin. Bundesgesundheitsbl. Gesundheitsforsch. Gesundheitsschutz, 2001, 44, 748
• Burns, I.; Mullen, W.; Landrault, N.; Teissedre, P.-L.; Lean, M.E.I.; Crozier, A. Variations in the Profile and Content of Anthocyanins in Wines made from Cabernet Sauvignon and hybrid grapes. J. Agric. Food Chem. 2002, 50, 4096-4102
• Otteneder, H.; Holbach, B.; Marx, R.; Zimmer, M. Rebsortenbestimmung in Rotwein mittels Anthocyanspektrum. Mitt. Klosterneuburg, 2002, 52, 187-194
• L.W. Wulf and C.W. Nagel; High-Pressure liquid chromatographic separation of Anthocyanins of Vitis vinifera.
  Am.J.Enol.Vitic 1978, 29, 42-49

 

Annex B Statistical results

 

Method performance study and evaluation

17 laboratories from 5 European Nations participated in the validation study of the method under the coordination of the German Official State Laboratory for Food Chemistry in Trier. The participants are listed in Table 3. An example of a chromatogram is presented in Figure 1 and the detailed results are given in Table 2.

The statistical evaluation followed the Resolution 6/99 and the Standard ISO 5725-1944 [4.5].

The chromatograms sent back with the results sheets fulfilled all requirements concerning the performance of the analytical column. No laboratory had to be completely eliminated, for example, because of a wrong peak identification.

The outlier values were searched using Dixon and Grubbs outlier testing according to the procedure for “Harmonised Protocol – IUPAC 1994” and the OIV Resolution OENO 19/2002. The values of sr, sR, r and R were calculated for 9 major anthocyanins at 5 content levels. For analytical results, the values of the closest levels should be used.

In order to have a global vision of the method performance, all the values RSDr- et RSDR- gathered are grouped by range of areas in the following table:

Table 1: Summary of the results of the method performance study

Range of relative peak areas*[%]	Range of RSDr [%]	Range of RSDR [%]
>0.4 – 1.0	6.8 - 22.4	20.6 - 50.9
>1.1 – 1.5	4.2 - 18.1	11.8 - 28.1
>1.5 – 3.5	2.1 – 7.7	10.6 - 15.6
>3.5 – 5.5	2.7 – 5.7	18.7 – 7.5
>5.5 – 7.5	2.4 – 3.9	6.5 - 10.0
>10 – 14	1.1 – 2.9	3.7 - 9.2
>14 – 17	1.0 - 3.9	3.2 - 5.4
>50 – 76	0.3 - 1.0	2.1 - 3.1
* independent of anthocyanin

This leads to the conclusion that repeatabilities and reproducibilities depend on the total sum of the relative peak areas. The higher they are, the better are RSDr and RSDR. For anthocyanin contents close to the detection limit (e.g. Cyanidin-3-glucoside) with small relatives areas (less than 1%) the RSDr et RSDR values can rise significantly. For anthocyanin whose relative areas are more than 1%, the RSDr and RSDR values are reasonable.

 

Figure 1:  Separation of 9 anthocyanins in red wine

 

Table 2: Results of the method performance study

Anthocyanin		sample 1	sample 2	sample 3	sample 4	sample 5
Delphinidol-3-glucoside
n		14	14	16	15	16
mean		6.75	14.14	3.45	16.68	3.54
sr		0.163	0.145	0.142	0.142	0.108
RSDr(%)		2.4	1.0	4.1	0.8	3.1
r		0.46	0.41	0.40	0.40	0.30
sR		0.544	0.462	0.526	0.704	0.490
RSDR(%)		8.1	3.3	15.2	4.2	13.8
R		1.52	1.29	1.47	1.97	1.37
Cyanidol-3-glucoside
n		16	17	16	15	14
mean		2.18	1.23	0.61	1.46	0.34
sr		0.086	0.053	0.043	0.110	0.031
RSDr(%)		4.0	4.3	7.1	7.5	9.2
r		0.24	0.15	0.12	0.31	0.09
sR		0.460	0.211	0.213	0.180	0.158
RSDR(%)		21.2	17.2	34.9	12.3	46.7
R		1.29	0.59	0.60	0.50	0.44
Petunidol-3-glucoside
n		15	17	16	14	15
mean		10.24	14.29	5.75	12.21	6.19
sr		0.233	0.596	0.157	0.097	0.196
RSDr(%)		2.3	4.2	2.7	0.8	3.2
r		0.65	1.67	0.44	0.27	0.55
sR		0.431	0.996	0.495	0.469	0.404
RSDR(%)		4.2	7.0	8.6	3.8	6.5
R		1.21	2.79	1.39	1.31	1.13
Peonidol-3-glucoside
n		16	15	17	17	16
mean		11.88	6.23	13.75	7.44	4.12
sr		0.241	0.166	0.144	0.232	0.174
RSDr(%)		2.0	2.7	1.0	3.1	4.2
r		0.68	0.47	0.40	0.65	0.49
sR		0.981	0.560	1.227	0.602	0.532
RSDR(%)		8.3	9.0	8.9	8.1	12.9
R		2.75	1.57	3.44	1.69	1.49
Malvidol-3-glucoside
n		16	15	17	16	16
mean		55.90	55.04	76.11	52.60	61.04
sr		0.545	0.272	0.251	0.298	0.377
RSDr(%)		1.0	0.5	0.3	0.6	0.6
r		1.53	0.76	0.70	0.83	1.06
sR		2.026	2.649	2.291	1.606	1.986
RSDR(%)		3.6	4.8	3.0	3.1	3.3
R		5.67	7.42	6.41	4.50	5.56
n	= N° of laboratories retained after eliminating outliers
sr	= standard deviation of repeatability
RSDr(%)	= relative standard deviation of repeatability
r	= repeatability
sR	= standard deviation of reproducibility
RSDR(%)	= relative standard deviation of reproducibility
R	= reproducibility

Table 2:  Results of the method performance study

Anthocyanin		sample 1	sample 2	sample 3	sample 4	sample 5
Peonidol-3-acetylglucoside
n		14	16		14	16
mean		1.16	1.44		0.59	3.74
sr		0.064	0.062		0.059	0.215
RSDr(%)		5.5	4.3		10.1	5.8
		0.18	0.17		0.17	0.60
sR		0.511	0.392		0.272	0.374
RSDR(%)		43.9	27.2		46.4	10.0
R		1.43	1.10		0.76	1.05
Malvidol-3-acetylglucoside
n		16	17		17	16
mean		5.51	4.84		3.11	15.07
sr		0.176	0.167		0.088	0.213
RSDr(%)		3.2	3.4		2.8	1.4
r		0.49	0.47		0.25	0.60
sR		0.395	0.366		0.496	0.617
RSDR(%)		7.2	7.6		16.0	4.1
R		1.11	1.02		1.39	1.73
Peonidol-3-coumarylglucoside
n		16	14		17	16
mean		1.26	0.90		0.89	1.32
sr		0.130	0.046		0.060	0.058
RSDr(%)		10.3	5.1		6.8	4.4
r		0.36	0.13		0.17	0.16
sR		0.309	0.109		0.204	0.156
RSDR(%)		24.5	12.2		23.0	11.8
R		0.86	0.31		0.57	0.44
Malvidol-3-coumarylglucoside
n		17	17		17	16
mean		4.62	2.66		4.54	4.45
sr		0.159	0.055		0.124	0.048
RSDr(%)		3.4	2.1		2.7	1.1
r		0.45	0.15		0.35	0.13
sR		0.865	0.392		0.574	0.364
RSDR(%)		18.7	14.7		12.6	8.2
R		2.42	1.10		1.61	1.02
n	= N° of laboratories retained after eliminating outliers
sr	= standard deviation of repeatability
RSDr(%)	= relative standard deviation of repeatability
r	= repeatability
sR	= standard deviation of reproducibility
RSDR(%)	= relative standard deviation of reproducibility
R	= reproducibility

 

Table 3: List of participants

ABC Labor Dahmen, Mülheim/Mosel	D
Chemisches Landes- und Staatliches Veterinäruntersuchungsamt Münster	D
Institut für Lebensmittelchemie Koblenz	D
Institut für Lebensmittelchemie Speyer	D
Institut für Lebensmittelchemie Trier	D
Institut für Lebensmittelchemie und Arzneimittel Mainz	D
Labor Dr. Haase-Aschoff, Bad Kreuznach	D
Labor Dr. Klaus Millies, Hofheim-Wildsachsen	D
Labor Heidger, Kesten	D
Landesveterinär- und Lebensmitteluntersuchungsamt Halle	D
Staatliche Lehr- und Forschungsanstalt für Landwirtschaft, Weinbau und Gartenbau, Neustadt/Weinstraße	D
Staatliches Institut für Gesundheit und Umwelt, Saarbrücken	D
Staatliches Medizinal-, Lebensmittel- und Veterinäruntersuchungsamt, Wiesbaden	D
Laboratoire Interrégional de la D.G.C.C.R.F de Bordeaux, Talence/France	F
Unidad de Nutricion y Bromotologia, Facultad de Farmacia, Universidad de Salamanca, Salamanca/Espana	E
University of Glasgow, Div. of Biochem. and Molek. Biology	UK
Höhere Bundeslehranstalt und Bundesamt für Wein- und Obstbau, Klosterneuburg	A

Laboratories

D (13); A (1); F (1); E (1); UK (1)

OIV-MA-AS315-12 Determination of plant proteins in wines and musts

Type IV method

 

The technique developed below enables to determine the quantity of proteins possibly remaining in beverages treated with proteins of plant origin after racking.

1. Principle

Wine and must proteins are precipitated with trichloroacetic acid, then they are separated by electrophoresis in polyacrylamide gel in the presence of dodecyl sodium sulphate (DSS). The addition of Coomassie blue colours the proteins. The intensity of the colouration enables to determine the protein content using a calibration curve made beforehand with the known protein concentration solutions. The antigenic capacity of musts and treated wines is determined by immunoblotting testing.

2. Protocol

2.1.  Concentration of proteins by precipitation with trichloroacetic acid (TCA)

2.1.1.      Reagents

2.1.1.1.                        Pure trichloroacetic acid (TCA)

2.1.1.2.                        TCA at 0.1% prepared using 2.1.1.1: 0.1 g in

100 ml of water.

2.1.1.3.                        TCA at 100% prepared using 2.1.1.1: 100 g in

100 ml of water.

2.1.1.4.                        Sodium hydroxide 0.5 M

2.1.1.5.                        Buffer Tris/HCl 0.25 M pH=6.8

30.27 g of Tris-(hydroxymethyl)aminomethane (Tris) are dissolved in 300 ml of distilled water. The pH is adjusted to 6.8 with concentrated hydrochloric acid for analysis. The volume is completed to 1 l with distilled water. The buffer is stored at 4°C.

2.1.1.6.                        Pure glycerol

2.1.1.7.                        Pure dodecyl sodium sulphate (DSS)

2.1.1.8.                        Pure 2-mercaptoethanol

2.1.1.9.                        Buffer solution for samples: it is made up of a buffer Tris/HCl 0.25 M, pH=6.8 (2.1.1.5); 7.5% of pure glycerol (2.1.1.6); 2% of dodecyl sodium sulphate (DSS) (2.1.1.7) and 5% of pure 2-mercaptoethanol (2.1.1.8). The percentages of different reagents correspond to the final concentration in the buffer solution.

2.1.2.      Procedure

3 ml of trichloroacetic acid at 100% (2.1.1.3) and 24 ml of wine or must (treated or untreated) are successively put in 50 ml centrifuge tubes. The final concentration in TCA thus obtained is 11%.

After 30 minutes at 4°C, the samples are centrifuged at 10,000 rpm for 30 minutes at 4°C. The pellets are washed in an aqueous solution of TCA at 0.1% (2.1.1.2), re-centrifuged and put again in suspension in 0.24 ml mixture (1:1, v/v) of sodium hydroxide 0.5 M (2.1.1.4) and buffer solution (2.1.1.9). The samples are heated at 100°C in a water bath for 10 minutes.

2.2.  Electrophoresis in Polyacrylamide Gel in the presence of DSS

2.2.1.      Reagents

2.2.1.1.                        Buffer Tris/HCl 1.5 M pH=8.8

181.6 g of Tris-(hydroxymethyl)aminomethane are dissolved in 300 ml of distilled water. The pH is adjusted at 8.8 with concentrated hydrochloric acid for analysis. The volume is completed to 1 l with distilled water. The buffer is stored at 4°C.

2.2.1.2.                        Mixture of acrylamide (30%)–bis-acrylamide (0.8%)–glycerol (75%)

Slowly add 300 g of acrylamide and 8 g of bis-acrylamide to 600 ml of a glycerol solution at 75%. After dissolution, adjust the volume to 1 l with glycerol at 75%. The mixture is stored in the dark at room temperature.

2.2.1.3.                        DSS at 10%

10 g of DSS are dissolved in 100 ml of distilled water. Store at room temperature.

2.2.1.4.                        N,N,N’,N’-tetramethylenediamine (TEMED) for electrophoresis

2.2.1.5.                        Ammonium persulfate at 10%

1 g of ammonium persulfate is dissolved in 10 ml of distilled water. Store at 4°C.

2.2.1.6.                        Bromophenol blue solution

10 mg of bromophenol blue for electrophoresis are dissolved in 10 ml of distilled water.

2.2.1.7.                        Solution for the separation gel (15% of acrylamide)

It is prepared just before use:

• 1.5 ml of Tris/HCl 1.5 M, pH=8.8 (2.2.1.1),
• 1.5 ml of distilled water,
• 3 ml of glycerol acrylamide mixture (2.2.1.2),
• 50 μl of DSS 10% (2.2.1.3),
• 10 μl of N,N,N’,N’-tetramethylenediamine (TEMED) for electrophoresis (2.2.1.4),
• 20 μl of ammonium persulfate (2.2.1.5).
• 1 drop of bromophenol blue (2.2.1.6)
  8.                         Buffer Tris/HCl 0.5 M pH=6.8

60.4 g of Tris-(hydroxymethyl)aminomethane are dissolved in 400 ml of distilled water. The pH is adjusted to 6.8 with concentrated hydrochloric acid for analysis. The volume is completed to 1 l with distilled water. The buffer is stored at 4°C.

2.2.1.9.                        Mixture of acrylamide (30%)–bis-acrylamide (0.8%)–water

Slowly add 300 g of acrylamide and 8 g of bis-acrylamide to 300 ml of water. After dissolution, adjust the volume to 1 l with distilled water. The mixture is stored in the dark at room temperature.

2.2.1.10.                   Concentration gel at 3.5% of acrylamide

It is prepared just before use:

• 0.5 ml of Tris/HCl 0.5 M pH=6.8 (2.2.1.8),
• 1.27 ml of distilled water,
• 0.23 ml of water acrylamide mixture (2.2.1.9),
• 20 μl of DSS 10% (2.2.1.3),
• 5 μl of N,N,N’,N’-tetramethylenediamine (TEMED) for electrophoresis (2.2.1.4),
• 25 μl of ammonium persulfate (2.2.1.5),
• 1 drop of bromophenol blue (2.2.1.6).
  11.                    Migration buffer

30.27 g of Tris-(hydroxymethyl)aminomethane, 144 g of glycine and 10 g of DSS are dissolved in 600 ml of distilled water. The pH should be 8.8. If necessary, it is adjusted with concentrated hydrochloric acid for analysis. The volume is completed to 1 l with distilled water. The buffer is stored at 4°C. At the time of use, the solution is diluted to 1/10 in distilled water.

2.2.1.12.                   Colouring solution

Are successively mixed:

• 16 ml of ultra-pure Coomassie brilliant blue G-250 at 5% (5 g in 100 ml of distilled water),
• 784 ml from a 1 l solution where 100 g of ammonium sulphate and 13.8 ml of orthophosphoric acid at 85% were dissolved for analysis,
• 200 ml of absolute ethanol.
  13.                    Discolouring solution

Are successively mixed:

• 100 ml of glacial acetic acid 100% for analysis,
• 200 ml of absolute ethanol for analysis.
• 700 ml of distilled water.
  2.       Procedure

The separation gel solution (2.2.1.7) is poured between two glass plates of 7x10cm. The upper surface of the gel is levelled by the addition of 2 drops of distilled water.

After polymerisation of the separation gel and the elimination of water, 1 ml of concentration gel (2.2.1.10) is deposited on the separation gel using a 1 ml pipette. Then the comb is set up whose imprints will create deposit wells.

The samples necessary for the calibration range are prepared in a mixture (1:1), v/v, 0.5% M sodium hydroxide (2.1.1.4) and the buffer solution (2.1.1.9) in order for the calibration range be between 5 μg/ml and 50 μg/ml.

20 to 30 μl of wine and calibration solution are deposited in the wells.

After migration (at a constant voltage of 90 V) at room temperature for about 3-4 hours, the gels are removed from the mould. They are immediately plunged into 50 ml of an aqueous solution of TCA 20% for 30 minutes then in 50 ml of the colouring solution (2.2.1.12).

The proteins appear in the form of blue coloured bands. The gel is then discoloured with 50 ml of discolouring solution (2.2.1.13). When the bottom of the gel is transparent, it is placed in distilled water for storage.

3. Quantitative analysis

The intensity of each spot is evaluated by using a scanner for gel with an image analyser software. The quantity of protein on the gel is determined by the calculation of the average density of the pixels of the band and by integration of the band width. The protein content of each sample is obtained using a calibration curve. The points of this curve are obtained by tracing the known concentration values of plant proteins deposited on the gel depending on the corresponding integration area.

The detection and quantification limit is about 0.030 ppm for peas and at 0.36 ppm for gluten, in an environment concentrated 100 times. The coefficient of variation is always below 5%.

4. Search by immunoblotting of the antigenic potential of wines and musts treated

The antigenic capacity of proteins that could remain in the beverages treated after racking is then evaluated.

4.1.  Principle

After electrophoresis, the gels are submitted to the immunoblotting technique. The proteins are transferred to a membrane where they are adsorbed. An antigen–antibody complex is formed by the addition of a plant anti-protein antibody (for example anti–gliadin antibodies if the plant protein is gluten). The method is revealed by the addition of an antibody directed against the plant anti-protein antibodies coupled with phosphatase. In the presence of the chromogenic substrate of the enzyme, a colouration whose intensity will be proportional to the quantity of immunocomplexes will develop. This immunoreactivity will be quantified using a calibration curve made with known concentration plant proteins solutions.

4.2.  Protocol

4.2.1.      Reagents

4.2.1.1.                        Transfer buffer

3.03 g of Tris, 14.4 g of glycine (R), 200 ml of methanol (R) are mixed and completed to 1 l with distilled water.

4.2.1.2.                        Gelatine 1%

8.77 g of sodium chloride (R), 18.6 g of ethylenediaminetetraacetic acid (EDTA) for analysis, 6.06 g of Tris and 0.5 ml of Triton X are dissolved in 800 ml of distilled water. The pH is adjusted to 7.5 with concentrated hydrochloric acid for analysis. 10 g of gelatine are added and the volume is completed to 1 l.

4.2.1.3.                        Gelatine 0.25%

8.77 g of sodium chloride (R), 18.6 g of ethylenediaminetetraacetic acid (EDTA) for analysis, 6.06 g of Tris and 0.5 ml of Triton X are dissolved in 800 ml

of distilled water. The pH is adjusted to 7.5 with concentrated hydrochloric acid for analysis. 2.5 g of gelatine are added and the volume is completed to 1 l.

4.2.1.4.                        Polyclonal antibody solution (marketed or described in the annex)

10 μl of polyclonal plant anti-protein antibodies

q.s.f. 10 ml with gelatine at 0.25% (4.2.1.3).

4.2.1.5.                        TBS buffer

29.22 g of sodium chloride for analysis and 2.42 g of tris are dissolved in 1 l of distilled water.

4.2.1.6.                        Alkaline phosphatase buffer

5.84 g of sodium chloride (R), 1.02 g of magnesium chloride (R) and 12.11 g of Tris are dissolved in 800 ml of distilled water. The pH is adjusted to 9.5 with concentrated hydrochloric acid and the volume is completed to 1 l.

4.2.1.7.                        Developer

15 g of bromochloroindol phosphate (BICP) and 30 g of nitro blue tetrazolium (NBT) are dissolved in 100 ml of alkaline phosphatase buffer (4.2.1.6).

4.2.2.      Procedure

After electrophoresis, the proteins are transferred from the gel to a membrane of polyvinylidene difluoride by electrophoretic elution: 16 hours at 4°C at 30 V in the transfer buffer (4.2.1.1). The membranes are saturated with gelatine at 1% (4.2.1.2) and washed 3 times with gelatine at 0.25% (4.2.1.3). The gelatine becomes set on free sites and inhibits non specific adsorption of immunological reagents. The membrane is then plunged into 10 ml of the plant anti-protein polyclonal antibody solution (4.2.1.4). For gluten, the anti-gliadin antibodies are purchased. The other antibody types are prepared according to the method provided for in the annex. The IgG-antigen complex is detected by the addition of 10 μl of anti-IgG rabbit antibodies marked with alkaline phosphatase. The membranes are washed twice with gelatine 0.25% (4.2.1.3) and once with the TBS buffer (4.2.1.5). After incubation in the developer (4.2.1.7), a dark purple precipitate is formed in the spot where the enzyme is attached.

4.3.  Quantitative analysis

In order to calculate the quantity of residual immunoreactivity of a marketed wine, a calibration curve is traced out: known concentrations of plant proteins deposited on the gel (and transferred to a membrane) depending on the areas obtained by integration of the intensity of the spots corresponding to the formation of immune-complex. The analysis is done with the same equipment as for analysing electrophoresis gels.

Annex Production of polyclonal anti-peas

Anti-peas polyclonal antibodies necessary for the determination of antigenic capacity of pea proteins in wine and musts treated are being carried out on animals.

1. Principle

Serums containing polyclonal antibodies are obtained from New Zealand rabbits after an intradermal injection of antigen.

2. Protocol

2.1.  Reagents

2.1.1.      PBS pH=7.4 phosphate buffer: 8 g of NaCl, 200 mg of KCl, 1.73 of Na₂HPO₄ H₂O and 200 mg of KH₂PO₄ are dissolved in 300 ml of distilled water. pH is adjusted to 7.4 with sodium hydrate 1 M. The volume is brought to 1 l with distilled water.

2.1.2.      Antigens:

10 mg of pea protein is dissolved in 5 ml of PBS phosphate buffer (2.1.1). The solution is then filtered under sterile conditions through 0.2 µm and stored at -20°C until the day of immunization.

2.2.  Procedure

1 ml of 2.1.2. solution is mixed with 1 ml of Freund complete adjuvant. 1 ml of this mixture is injected intradermically to a New Zealand rabbit weighing approximately 3 kg. This injection is repeated on day 15, day 30 and day 45.

60 days after the first injection, 100µl of blood were withdrawn from the auricular vein which was then tested for its capacity to react to antigens. Immunoblotting was used for this evaluation as described in Chapter 4.2 of the analysis method using a gel with a pea protein which migrated on the gel.

After checking the formation of an antigen-antibody complex, 15 ml of blood were withdrawn from the auricular vein. The blood is placed at 37°C for 30 minutes. The serum containing the anti-pea polyclonal antibodies is withdrawn after centrifuging the blood at 3000 rpm for 5 minutes.

OIV-MA-AS315-14 Measurement of lysozyme in wine by high performance liquid chromatography

Type IV method

1. Introduction

It is preferable to have an analysis method available for lysozyme which is not based on enzyme activity.

2. Scope

The method allows the quantification of lysozyme (mg of protein per l) present in red and white wines independently of the enzyme activity (which could be inhibited by partial denaturation or by complex formation or coprecipitation phenomena) found in the test solution.

3. Definition

HPLC provides an analytical approach based on steric, polar or adsorptive interactions betwen the stationary phase and the analyte, and is therefore not linked to the actual enzyme activity exhibited by the protein.

4. Principle

The analysis is carried out using HPLC with a spectrophotometric detector combined with a spectrofluorimetric detector. The unknown quantity in the wine sample is calculated on the chromatographic peak areas, using the external calibration method.

 

5. Reagents

5.1.  Solvents and working solutions

HPLC analysis on Acetonitrile (CN)

Pure trifluoroacetic acid (TFA)

deionised water for HPLC analysis

Standard solution: Tartaric acid 1g/L, Ethyl alcohol 10% v/v, adjusted to pH 3.2 with neutral potassium tartrate.

5.2.  Eluents

A: CN 1%, TFA 0.2 %, = 98.8%

B: CN 70%, TFA 0.2 %, = 29.8%

5.3.  Reference solutions

Quantities from 1 to 250 mg/L standard lysozyme, dissolved in standard solution by stirring continuously for at least 12 hours.

6. Equipment

6.1.  HPLC apparatus equipped with a pumping system suitable for gradient elution

6.2.  Thermostated column compartment (oven)

6.3.  Spectrophotometer combined with spectrofluorimeter

6.4.  20 μL loop injection

6.5.  Column: polymer in reverse phase with phenyl functional groups (diameter of pores = 1000 Å, exclusion limit = 1000000 Da) Toso Bioscience TSK-gel Phenyl 5PW RP, 7.5 cm x 4.6 mm ID as an example

6.6.  Pre-column in the same material as the column: Toso Bioscience TSK-gel Phenyl 5PW RP Guardgel, 1.5 cm 3.2mm ID as an example

7. Preparation of the sample

The wine samples are acidified with HCl (10M) diluted 1/10 and filtered using a polyamide with 0.22 μm diameter pores filter, 5 minutes after the addition. The chromatography analysis is carried out immediately after filtering.

8. Operating conditions
   1.   Eluent flow-rate: 1mL/min
   2.   Temperature of column: 30°C
   3.   Spectrophotometric detection: 280 nm
   4.   Spectrofluorimetric detection:

• λ ex = 276 nm;
• λ em = 345 nm;
• Gain = 10
  5.   Gradient elution sequence

Time (min)	A%	B%	gradient
0	100	0
			isocratic
3	100	0
			linear
10	65	35
			isocratic
15	65	35
			linear
27	40.5	59.5
			linear
29	0	100
			isocratic
34	0	100
			linear
36	100	0
			isocratic
40	100	0

8.6.  Average retention time of lysozyme: 25.50 minutes

9. Calculation

The reference solutions containing the following concentrations of lysozyme: 1; 5; 10; 50; 100; 200; 250 mg/L are analysed in triplicate. For each chromatogram, the peak areas corresponding to the lysozyme are plotted according to the respective concentrations, in order to obtain the linear regresssion lines expressed by the formula Y= ax+b. The correlation coefficient must be > 0.999

10. Characteristics of the method

A validation study was carried out for the purpose of assessing the suitability of the method for the purpose in question, taking into account linearity, limits of detection and quantification and the accuracy of the method. The latter parameter was determined by defining the levels of precision and trueness of the method.

10.1.        Linearity of the method

Based on the results obtained from the linear regression analysis, the method proved to be linear within the ranges shown in the table below:

	Linearity range (mg/L)	Line gradient	Correlation coefficient (r2)	LD (mg/L)	LQ (mg/L)	Repeatability (n=5) RSD%			Reproducibility (n=5) RSD%
						Std1	V.R.2	V.B.3	Std1
UV	5-250	3 786	0,9993	1,86	6,20	4,67	5,54	0,62	1,93
FLD	1-250	52 037	0,9990	0,18	0,59	2,61	2,37	0,68	2,30

Table 1: Data related to characteristics of the method: standard solution (Std 1); red wine (V.R 2); white wine (V.B 3)

10.2.        Limit of detection and limit of quantification

The detection limit (LD) and limit of quantification (LQ) were calculated as the signal equivalent to respectively 3 times and 10 times the background chromatography noise under working conditions on an actual test solution (table 1),

10.3.        Precision of the method

The parameters taken into account were repeatability and reproducibility. Table 1 shows the values of these parameters (expressed as %age St.dv. of measurements repeated in different concentrations) found for standard solution, red wine and white wine

10.4.        Trueness of the method

The percentage recovery was calculated on the standard solutions containing 5 and 50 mg/L of lysozyme, with known quantities of lysozyme added, as shown in the table below.

	Nominal initial [C]  (mg/L)	Quantity added (mg/L)	Theoretical [C] (mg/L)	[C] found	Std.Dev.	%age recovery
UV 280 nm	50	13.1	63.1	62.3	3.86	99
FD	50	13.1	63.1	64.5	5.36	102
UV 280 nm	5	14.4	19.4	17.9	1.49	92.1
FD	5	14.4	19.4	19.0	1.61	97.7

Fig.1 Chromatogram of red wine containing pure lysozyme (standard solution containing 1 000 mg/L of lysozyme was added to wine to obtain a final concentration of 125 mg/L of lysozyme). A: UV detector at 280 nm; B: UV detector at 225 nm; C: FLD detector (λ ex 276 nm; λ em 345 nm).

11. Bibliography

• Claudio Riponi; Nadia Natali; Fabio Chinnici. Quantitation of hen’s egg white lysozyme in wines by an improved HPLC-FLD analytical method. Am. J. Enol. Vit., in press.

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OIV-MA-AS315-18 Analysis of biogenic amines in musts and wines using HPLC

Type II method

 

1. Scope

This method can be applied for analysing biogenic amines in musts and wines:

• Ethanolamine: up to 20 mg/l
• Histamine: up to 15 mg/l
• Methylamine: up to 10 mg/l
• Serotonin: up to 20 mg/l
• Ethylamine: up to 20 mg/l
• Tyramine: up to 20 mg/l
• Isopropylamine: up to 20 mg/l
• Propylamine: normally absent
• Isobutylamine: up to 15 mg/l
• Butylamine: up to 10 mg/l
• Tryptamine: up to 20 mg/l
• Phenylethylamine: up to 20 mg/l
• Putrescine or 1,4-diaminobutane: up to 40 mg/l
• 2-Methylbutylamine: up to 20 mg/l
• 3-Methylbutylamine: up to 20 mg/l
• Cadaverine or 1,5-diaminopentane: up to 20 mg/l
• Hexylamine: up to 10 mg/l

2. Definition

The biogenic amines measured are:

• Ethanolamine: – CAS [141 – 43 – 5]
• Histamine:- CAS [51 – 45 – 6]
• Methylamine: – CAS [74 – 89 – 5]
• Serotonin: C₁₀H₁₂N₂O – CAS [153 – 98 – 0]
• Ethylamine: C₂H₇N – CAS [557 – 66 – 4]
• Tyramine: C₈H₁₁NO - CAS [60 – 19 – 5]
• Isopropylamine: C₃H₉N - CAS [75 – 31 – 0]
• Propylamine: C₃H₉N – CAS [107 – 10 – 8]
• Isobutylamine: C₄H₁₁N – CAS [78 – 81 – 9]
• Butylamine: C₄H₁₁N – CAS [109 – 73 – 9]
• Tryptamine: C₁₀H₁₂N₂ – CAS [61 – 54 – 1]
• Phenylethylamine: C₈H₁₁N – CAS [64 – 04 – 0]
• Putrescine  or 1,4-diaminobutane: C₄H₁₂N₂ – CAS [333 – 93 – 7]
• 2-Methylbutylamine: C₅H₁₃N - CAS [96 – 15 – 1]
• 3-Methylbutylamine: C₅H₁₃N - CAS [107 – 85 – 7]
• Cadaverine or 1,5-diaminopentane: C₅H₁₄N₂ – CAS [1476 – 39 – 7]
• 1,6-Diaminohexane: C₆H₁₆N₂ – CAS [124 – 09 – 4]
• Hexylamine: C₆H₁₅N – CAS [111 – 26 – 2]

3. Principle

The biogenic amines are directly determined by HPLC using a C₁₈ column after O-phthalaldehyde (OPA) derivatization and fluorimetric detection.

4. Reagents and products

4.1.  High purity resistivity water (18MΩ·cm)

4.2.  Dihydrate disodium hydrogenophosphate – purity 99 %

4.3.  Acetonitrile - Transmission minimum at 200 nm - purity 99 %

4.4.  O-phthalaldehyde (OPA) - Application for fluorescence - purity 99 %

4.5.  Disodium tetraborate decahydrate - purity 99 %

4.6.  Methanol - purity 99 %

4.7.  Hydrochloric acid 32 %

4.8.  Sodium hydroxide pellets - purity 99 %

4.9.  Ethanolamine - Purity 99 %

4.10.        Histamine dichlorhydrate - Purity 99 %

4.11.        Ethylamine chlorhydrate - Purity 99 %

4.12.        Serotonin - Purity 99 %

4.13.        Methylamine chlorhydrate – Purity 98 %

4.14.        Tyramine chlorhydrate - Purity 99 %

4.15.        Isopropylamine purity 99 %

4.16.        Butylamine - Purity 99 %

4.17.        Tryptamine chlorhydrate - purity 98 %

4.18.        Phenylethylamine - Purity 99 %

4.19.        Putrescine dichlorhydrate - Purity 99 %

4.20.        2-Methylbutylamine - Purity 98 %

4.21.        3-Methylbutylamine - Purity 98 %

4.22.        Cadaverine dichlorhydrate - Purity 99 %

4.23.        1-6-Diaminohexane - Purity 97 %

4.24.        Hexylamine - Purity 99 %

4.25.        Nitrogen (maximum impurities: H₂O 3 mg/l; O₂ 2 mg/L; C_nH_ms  0.5 mg/l)

4.26.        Helium (maximum impurities: H₂O 3 mg/l; O₂ 2 mg/L; C_nH_m  0.5 mg/l)

 

Preparation of reagent solutions:

4.27.        Preparation of eluents

Phosphate solution A: Weigh 11.12 g 0.01 g of di-basic sodium phosphate (4.2) in a 50-ml beaker (5.5) on a balance (5.27). Transfer to a 2-litre volumetric flask (5.9) and make up to 2 litres with high purity water (4.1). Homogenize using a magnetic stirrer (5.30) and filter over a 0.45 μm membrane (5.17). Put in the 2-litre bottle (5.12).

Solution B: The acetonitrile (4.3) is used directly.

4.28.        OPA solution – Daily preparation

Weigh 20 mg 0.1 mg of OPA (4.4) in a 50-ml flask (5.7) on the precision balance (5.27). Make up to 50 ml with methanol (4.6). Homogenize.

4.29.        Preparation of the borate buffer (4.29) – Weekly preparation

Weigh 3.81 g 0.01 g of Na₂B₄O₇·10H₂O (4.5) in a 25-ml beaker (5.6) on the precision balance (5.27). Transfer to a 100-ml volumetric flask (5.8) and make up to 100 ml with demineralised water (4.1). Homogenize with a magnetic stirrer (5.30), transfer to a 150-ml beaker (5.4) and adjust to pH 10.5 using a pH meter (5.28 and 5.29) with 10 N soda (4.8).

4.30.        0.1 M hydrochloric acid solution: Put a little demineralised water (4.1) into a 2-litre volumetric flask (5.9). Add 20 ml of hydrochloric acid (4.7) using a 10-ml automatic pipette (5.24 and 5.25)

4.31.        Calibration solution in 0.1 M hydrochloric acid

Guideline concentration of the calibration solution - weigh at 0.1 mg

	Indicative final concentration in the calibration mix in mg/l
Ethanolamine	5
Histamine	5
Methylamine	1
Serotonin	20
Ethylamine	2
Tyramine	7
Isopropylamine	4
Propylamine	2.5
Isobutylamine	5
Butylamine	5
Tryptamine	10
Phenylethylamine	2
Putrescine	12
2- Methylbutylamine	5
3- Methylbutylamine	6
Cadaverine	13
1.6 Diaminohexane	8
Hexylamine	5

The true concentration of the calibration solution is recorded with the batch number of the products used.

Certain biogenic amines being in salt form, the weight of the salt needs to be taken into account when determining the true weight of the biogenic amine.

The stock solution is made in a 100-ml volumetric flask (5.8).

The surrogate solution is made in a 250-ml volumetric flask (5.10).

4.32.        1,6 Diaminohexane internal standard

Weigh exactly 119 mg in a 25-ml Erlenmeyer flask (5.1) on a balance (5.26). Transfer to a 100-ml volumetric flask (5.8) and top up to the filling mark with 0.1 N hydrochloric acid (4.30).

4.33.        2-Mercaptoethanol - Purity  99 %.

5. Apparatus

5.1.  25-ml Erlenmeyer flasks

5.2.  250-ml Erlenmeyer flasks

5.3.  100-ml beakers

5.4.  150-ml beakers

5.5.  50-ml beaker

5.6.  25-ml beaker

5.7.  50-ml volumetric flasks

5.8.  100-ml volumetric flasks

5.9.  2,000-ml volumetric flasks

5.10.        250-ml volumetric flask

5.11.        1-litre bottles

5.12.        2-litre bottle

5.13.        2-ml screw cap containers suitable for the sample changer

5.14.        50-ml syringe

5.15.        Needle

5.16.        Filter holder

5.17.        0.45 μm cellulose membrane

5.18.        0.8 μm cellulose membrane

5.19.        1.2 μm cellulose membrane

5.20.        5 μm cellulose membrane

5.21.        Cellulose pre-filter

5.22.        1-ml automatic pipette

5.23.        5-ml automatic pipette

5.24.        10-ml automatic pipette

5.25.        Cones for 10-ml, 5-ml and 1-ml automatic pipettes

5.26.        Filtering system

5.27.        Balances for weighing 0 to 205 g at 0.01 mg

5.28.        pH meter

5.29.        Electrode

5.30.        Magnetic stirrer 

5.31.        HPLC pump

5.32.        Changer-preparer equipped with an oven

Note: An oven is indispensable, if a changer-preparer is used for injecting several samples one after another. This operation may likewise be done manually) the results may be less precise;

5.33.        Injection loop

5.34.        5 μm column, 250 mm  4 (which must lead to a similar chromatogram as presented in annex B);

5.35.        Fluorimetric detector

5.36.        Integrator

5.37.        Borosilicic glass tube with a stopper and closure cap covered with PTFE
(ex Sovirel 15).

6. Preparation of samples

Samples are previously purged of gas with nitrogen (4.25).

6.1.  Filtering

Filter approximately 120 ml of the sample over membrane:

• for a wine: 0.45 μm (5.17),
• for a must or non-clarified wine: 0.45 (5.17) – 0.8 (5.18) – 1.2 (5.19) - 5 μm (5.20) + pre-filter (5.21), pile filters in the following order, the sample pushed by the top: 0.45 μm (5.17) + 0.8 μm (5.18) + 1.2 μm (5.19) + 5 μm (5.20) + prefiltered (5.21)
  2.   Preparation of the sample

Put 100 ml of the sample (6.1) into a 100-ml volumetric flask (5.8);

Add 0.5 ml of 1-6-diaminohexane (4.32) at 119 mg/100 ml using a 1-ml automatic pipette (5.21 and 25);

Draw off 5 ml of the sample using the pipette (5.23 and 5.25); pour this into a 25-ml Erlenmeyer flask (5.1);

Add 5 ml of methanol to this (4.6) using the pipette (5.23 and 5.25);

Stir to homogenize;

Transfer to containers (5.13);

Start the HPLC pump (5.31), then inject 1 µl (5.32 and 5.33)

6.3.  Derivatisation

In a borosilicic glass tune (5.37), pour 2 ml of OPA solution (4.28), 2 ml of borate buffer (4.29), 0,6 ml of 2-mercaptoethanol (4.33). Close, mix (5.30). Open and pour 0,4 ml of sample. Close, mix (5.30). Inject immediately, as the derivitive is not stable. Rinse recipient immediately after injection, due to odour.

Note: Derivatisation can be carried out by an automatic changer-preparer. In this case, the process will be programmed to come close to the proportion of manual derivisation

6.4.  Routine cleaning

Syringe (5.13) and needle (5.14) rinsed with demineralised water (4.1) after each sample;

filter holder (5.16) rinsed with hot water, then MeOH (4.6). Leave to drain and dry.

7. Procedure

Mobile phase (5.31)

• A: phosphate buffer (4.2)
• B: acetonitrile (4.3)

Elution gradient:

Time ( in mins)	% A	% B
0	80	20
15	70	30
23	60	40
42	50	50
55	35	65
60	35	65
70	80	20
95	80	20

 

Note: The gradient can be adjusted to obtain a chromatogram close to the one presented in annex B

Flow rate: 1 ml/min;

Column temperature: 35 °C (5.32);

Detector (5.35): Exc = 356 nm, Em = 445 nm (5.30);

Internal calibration

The calibration solution is injected for each series;

Calibration by internal standard;

Calculation of response factors:

Cci = concentration of the component in the calibration solution and

Ccis = concentration of the internal standard in the calibration solution (1-6-diaminohexane).

Area i = area of the product peak present in the sample

Area is = area of the internal standard peak in the sample

Calculation of concentrations:

Area i = area of the product peak present in the sample

Area is = area of the internal standard peak present in the sample

XF = quantity of internal calibration added to samples for analysis

XF = 119  0.5/100 = 5.95.

8. Expression of results

Results are expressed in mg/l with one significant digit after the decimal point.

9. Reliability

	r (mg/l)	R (mg/l)
Histamine	0.07x + 0.23	0.50x + 0.36
Methylamine	0.11x + 0.09	0.40x + 0.25
Ethylamine	0.34x - 0.08	0.33x + 0.18
Tyramine	0.06x + 0.15	0.54x + 0.13
Phenylethylamine	0.06x + 0.09	0.34x + 0.03
Diaminobutane	0.03x + 0.71	0.31x + 0.23
2-methylbutylamine et 3-methylbutylamine	0.38x + 0.03	0.38x + 0.03
Diaminopentane	0.14x + 0.09	0.36x + 0.12

 

The details of the interlaboratory trial with regard to reliability of the method are summarised in appendix A.

10. Other characteristics of the analysis

The influence of certain wine components: amino acids are released at the beginning of the analysis and do not impede in detection of biogenic amines.

The limit of detection (LOD) and limit of quantification (LOQ) according to an intralaboratory study

	LOD (in mg/l)	LOQ (in mg/l)
Histamine	0,01	0,03
Methylamine	0,01	0,02
Ethylamine	0,01	0,03
Tyramine	0,01	0,04
Phenylethylamine	0,02	0,06
Diaminobutane	0,02	0,06
2-methylbutylamine	0,01	0,03
3-methylbutylamine	0,03	0,10
Diaminopentane	0,01	0,03

11. Quality control

Quality controls may be carried out with certified reference materials, with wines the characteristics of which result from a consensus or spiked wines regularly inserted into analytical series and by following the corresponding control charts.

Annex A

Statistical data obtained from the results of interlaboratory trials

The following parameters were defined during an interlaboratory trial. This trial was carried out by the Oenology Institute of Bordeaux (France) under the supervision of the National Interprofessional Office of Wine (ONIVINS – France).

Year of interlaboratory trial: 1994

Number of laboratories: 7

Number of samples: 9 double blind samples

(Bulletin de l’O.I.V. November-December 1994, 765-766, p.916 to 962) numbers recalculated in compliance with ISO 5725-2:1994.

Types of samples: white wine (BT), white wine (BT) fortified = B1, white wine (BT) fortified = B2, red wine n°1 (RT), red wine fortified = R1, red wine (RT) fortified = R2, red wine n°2 (CT), red wine (CT) fortified = C1 and red wine (CT) fortified = C2. fortified in mg/l.

	HistN	MetN	EthN	TyrN	PhEtN	DiNbut	IsoamN	DiNpen
wine B1	wine BT + 0,5	vine BT + 0,12	wineBT + 0,13	wine BT + 0,36	vine BT + 0,15	wine BT + 0,5	wine BT + 0,28	wineBT + 0,25
wine B2	wine BT + 2	wine BT + 0,40	wine BT + 0,50	wine BT + 1,44	wine BT + 0,60	wine BT + 2	Wine BT + 0,1,74	wine BT + 1,04
wine C1	wine CT + 2	wine CT + 0,1	wine CT + 0,18	wine CT + 0,72	wine CT + 0,15	wine CT + 2	wine CT + 0,29	wine CT + 0,26
wineC2	wine CT + 4	wine CT + 0,41	wine CT + 0,50	wine CT + 2,90	wine CT + 0,58	wine CT + 8	wine CT + 1,14	wine CT + 1,04
wine R1	wine RT + 2	wine RT + 0,14	wine RT + 0,13	wine RT + 1,45	wine RT + 0,19	wine RT + 3	wine RT + 0,0,57	wine RT + 0,51
wine R2	wine RT + 5	wine RT + 0,41	wine RT + 0,50	wine RT + 2,88	wine RT + 0,59	wine RT + 10	wine RT + 2,28	wine RT + 2,08

HistN : histamine, MetN : methylamine, EthN : ethylamine, TyrN : tyramine,  PhEtN : phenylethylamine, DiNbut : diaminobutane, IsoamN : isoamylamine and DiNpen : diaminopentane.

 

Annex B : Chromatogram model obtained by this method

Bibliography

• TRICARD C., CAZABEIL J.-M., SALAGOÏTI M.H. (1991): Dosage des amines biogènes dans les vins par HPLC, Analusis, 19, M53-M55.
• PEREIRA MONTEIRO M.-J. et BERTRAND A. (1994): validation d'une méthode de dosage – Application à l'analyse des amines biogènes du vin. Bull. O.I.V., (765-766), 916-962.