SECTION 3.1.5 - OTHER ORGANIC COMPOUNDS

Codified File

Acetaldehyde (ethanal) (Type-IV)

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.

  1. Apparatus

 

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

  1. 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.

  1. 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.

Ethyl Acetate (GC) (Type-IV)

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.

  1. 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:

Ethyl Acetate (titrimetry) (Type-IV)

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.

  1. 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.

Malvidin Diglucoside (Type-IV)

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.

  1. 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.

  1. Quantitative Determination
    1.   Apparatus
      1.       Equipment for measuring fluorescence:
  • excitation wavelength 365 nm;
  • wavelength of fluorescent radiation 490 nm.
    1.       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.

Ethyl Carbamate (Type-II)

OIV-MA-AS315-04 Ethyl carbamate

Type II method

 

Ethyl carbamate analysis in alcoholic beverages: selective detection method by gas chromatography/mass spectrometry

(Applicable to the determination of ethyl carbamate concentrations between 10 and 200 μg/l).

(Caution: respect safety measures when handling chemical products, ethanol, acetone and carcinogenic products: ethyl carbamate and dichloromethane. Get rid of used solvants in a suitable way, compatible with applicable environmental rules and regulations).

  1. Principle

Propyl carbamate is added to a sample as an internal standard, the solution is diluted with water and placed in a 50 mL solid phase extraction column. Ethyl carbamate and propyl carbamate are eluted with dichloromethane. The eluate is concentrated in a rotary evaporator under vacuum. The concentrate is analyzed by gas chromatography/mass spectrometry using selected ion monitoring mode.

  1. Apparatus

 

2.1.  Gas chromatograph/mass spectrometer (GC/MS). With selected ion monitoring (SIM), and data handling system.  An autosampler is desirable.

2.2.  Capillary fused silica column: 30m[1]  0.25 mm  int., 0.25 μm of Carbowax 20M type.

2.3.  Operating conditions: injector 180°C, helium carrier gas at 1 mL/min at 25°C, splitless injection. Temperature program: 40°C for 0.75 min, then program 10°C/min to 60°C, then 3°C[2]/min to 150°C, post run: go up to 220°C and maintain for 4.25 min at 220°C. The retention time for ethyl carbamate is 23-27 min., that of propyl carbamate is 27-31 min.

GC/MS interface: transfer line 220°C. Mass spectrometer parameters set up manually with perfluorotributylamine and optimized for a lower mass sensitivity, SIM acquisition mode, solvent delay and time for the start of acquisition 22 min., dwell time/ion 100 ms.

2.4.  Rotary evaporator under vacuum or concentration system similar to Kuderna Danish. (Note: the recovery of the ethyl carbamate test sample, (3.7) must be between 90-110% during the process).

2.5.  Flask - pear-shaped, 300 mL, single neck, 24/40 standard taper joint.

2.6.  Concentrator tube - 4 mL, graduated, with a standard taper 19/22 Teflon coated joint and stopper.

 

  1. Reagents

3.1.  Acetone - HPLC quality. Note: Check each batch by GC/MS before use with regard to the absence of response for m/z 62, 74 and 89 ions.

3.2.  Dichloromethane - Note: Analyze each batch before use by GC/MS after 200 fold concentration to check the absence of response for m/z 62, 74 and 89 ions.

3.3.  Ethanol – anhydrous

3.4.  Ethyl carbamate  (EC) standard solutions

  • Stock solution - 1.00 mg/mL. Weigh 100 mg EC ( 99% purity) in a volumetric flask of 100 mL and dilute to mark with acetone.
  • (2) Standard working solution- 10.0 g/mL. Transfer 1 mL of the EC stock solution to a 100 mL volumetric flask and dilute with acetone to the mark.
    1.   n-Propyl carbamate (PC), standard solutions.
  • Stock solution - 1.00 mg/mL. Weigh 100 mg PC (reagent quality) in a 100 mL volumetric flask and dilute with acetone to the mark.
  • (2) Standard working solution- 10.0 μg/mL. Transfer 1 mL of the PC stock solution to a volumetric flask of 100 mL and dilute with acetone to the mark.
  • (3) Internal standard solution PC - 400 ng/mL. Transfer 4 mL of the standard PC working solution to a volumetric flask of 100 mL and dilute with water to the mark.
    1.   EC - nPC standard calibration solutions - Dilute the standard working solutions of EC, 3.4 (2), and PC 3.5 (2), with dichloromethane in order to obtain:
  • (1) 100 ng EC and 400 ng nPC/mL,
  • (2) 200 ng EC and 400 ng nPC/mL,
  • (3) 400 ng EC and 400 ng nPC/mL,
  • (4) 800 ng EC and 400 ng nPC/mL,
  • (5) 1600 ng EC and 400 ng nPC/mL.
    1.   Practice sample - 100 ng EC/mL in 40 % ethanol. Transfer 1 mL of the standard EC working solution, 3.4 (2) in a 100 mL volumetric flask and dilute with 40 % of ethanol to the mark.
    2.   Solid phase extraction column - Disposable material, pre-packed with diatomaceous earth, capacity 50 mL.

(Note: Before analysis, check each batch of extraction columns for the recovery of EC and nPC and the absence of response for ions of m/z 62,74 and 89.) Prepare 100 ng EC/mL of test sample 3.7.

Analyze 5.00 mL of the test sample as described in 4.1, 4.2, 5, and 6. The recovery of 90-110 ng of EC/mL is satisfactory. Adsorbents whose particle diameter is irregular can lead to a slow flow that affects the recovery of EC and nPC.

If, after several trials, 90-110 % of the test sample value is not obtained, change the column or use a corrected calibration recovery curve to quantify EC.

To obtain the corrected calibration curve, prepare standard solutions as described in 3.6 by using 40 % ethanol instead of dichloromethane.

Analyze 1 mL of the standard calibration solution as described in 4, 5, and 6.

Establish a new standardization curve by using the EC/nPC ratio of the extracted standards.

 

  1. Preparation of the test sample

 

Place the test material in 2 separate 100 mL beakers using the following quantities:

4.1.  Wines containing over 14 % vol.  alcohol: 5.00 mL 0.01 mL.

4.2.  Wines containing maximum 14% vol. of alcohol: 20.00 mL 0.01 mL.

In each beaker, add 1 mL of internal standard PC solution, 3.5 (3) and water, in order to obtain a total volume of 40 mL (or 40 g).

 

  1. Extraction

 

(Note: Carry out the extraction under a fume hood with adequate ventilation.)

Transfer diluted test portion from 4 to the extraction column.

Rinse the beaker with 10 mL of water and transfer the rinsing water to the column. Let the liquid be absorbed in the column for 4 minutes.  Elute with 2 x 80 mL of dichloromethane.

Collect the eluate in a 300 mL pear-shaped flask.

Evaporate the eluate to 2 to 3 mL in a rotary evaporator in a water bath at 30°C (Note: do not let extract evaporate to dryness). 

Transfer the concentrated residue to a 4 mL graduated concentrator tube, with a 9 in Pasteur pipette.

Rinse the flask with 1 mL of dichloromethane and transfer the rinsing liquid to the tube.

Concentrate the sample to 1 mL under a slight nitrogen stream.

If an autosampler is used, transfer the concentrate to a vial for GC/MS analysis.

 

  1. GC/MS Analysis

6.1.  Calibration curve - Inject 1 μl of each calibration standard solution 3.6, into GC/MS. Plot the graph of the EC-nPC area ratio for the response to m/z 62 ion on the y-axis and the quantity of EC in ng/mL on the x-axis (i.e., 100, 200, 400, 800, 1600 ng/mL).

6.2.  EC quantification - Inject 1 μl of concentrated extract from 5 in the GC/MS system and calculate the EC-nPC area ratio for m/z 62 ion. Determine the concentration of EC (ng/mL) in the extract by using the internal standard standardization curve. Calculate the EC concentration in the test sample (ng/mL) by dividing the quantity of EC (ng/mL) in the extract by the test sample volume 3.7.

6.3.  Confirmation of EC identity.  Determine if the response for m/z 62, 74 and 89 ions appear at the EC retention time. These responses characteristic respectively of the main fragments (M - and (M - and molecular ion (M). The presence of EC is confirmed if the relative ratio of these ions does not exceed 20% of the ratios of the EC standard. The extract may need to be further concentrated in order to obtain a sufficient response for the m/z 89 ion.

 

  1. Method performance.

Sample

Mean EC

found, ng/g

Recovery of added EC,  %

sr

SR

RSDr %

RSDR%

Wine over 14 % alcohol (v/v)

40

1.59

4.77

4.01

12.02

80

89

3.32

7.00

4.14

8.74

162

90

8.20

11.11

5.05

6.84

Wine under 14% alcohol (v/v)

11

0.43

2.03

3.94

18.47

25

93

1.67

2.67

6.73

10.73

48

93

1.97

4.25

4.10

8.86

 


[1] For certain wines which are particularly rich, it may be desirable to use a 50m long capillary column

[2] For certain wines which are particularly rich, it may be desirable to carry out a temperature program of 2°C per minute.

Hydroxymethylfurfural (colorimetry) (Type-IV)

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.

  1. 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.

Hydroxymethylfurfural (HPLC) (Type-IV)

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.

  1. 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.

Cyanide Derivatives (Type-II)

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.

  1. 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.

  1. 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

  1. 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.

  1. 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.

  1. 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)

Artificial sweeteners (TLC : saccharine, cyclamate, Dulcin and P‑4000 ) (Type-IV)

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.

  1. 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
    1.       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)
    1.       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 dimethylaminobenzaldehyde 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.

Artificial sweeteners (TLC: saccharine, cyclamate and Dulcin) (Type-IV)

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 hydroxybenzoic acid which migrate with the same Rf.

  1. 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.
    1. 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.

Artificial Colorants (Type-IV)

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.

  1. Apparatus

 

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

2.2.   Chromatography tank

  1. 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

  1. 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.

Diethylene glycol (Type-IV)

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.

 

  1. 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.

 

  1. 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.
    1.   Centrifuge

 

  1. 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.

 

  1. 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.

  1. 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.

Ochratoxin A (Type-II)

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.

  1. 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.

  1. 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.

  1. 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

  1. 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

  1. 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)

 

  1. 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)

  1. 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).

  1. 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.

  1. 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

 

  1. 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.

  1. 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.

  1. 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.

  1. 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 xi 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 yi 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).

  1. 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.

  1. 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.

HPLC-Determination of nine major Anthocyanins in red and rosé wines (Type-II)

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.

  1. 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].

 

  1. 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

  1. 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

  1. 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

  1. Expression of results

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

  1. 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:

hmax = 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
  1. 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)

Plant proteins (Type-IV)

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.

  1. 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)
    1.                         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).
    1.                    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.
    1.                    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.
    1.       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.

  1. 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%.

  1. 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.

  1. 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.

Determination of Lysozyme by HPLC (Type-IV)

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.

  1. 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.

  1. 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.

  1. 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.

 

  1. 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.

  1. 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

  1. 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.

  1. 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
    1.   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

  1. 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

  1. 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).

  1. 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.

Determination of 3-methoxypropane-1,2-diol and cycli diglycerols (by-products of technical glycerol) in wine by GC-MS- Description of the method and collaborative study

OIV-MA-AS315-15 Determination of 3-methoxypropane-1,2-diol and cyclic diglycerols (by products of technical glycerol) in wine by GC-MS- Description of the method and collaborative study

Type II method

 

  1. Introduction

This is an internationally validated method for the determination of 3-methoxypropane-1,2-diol (3-MPD) and cyclic diglycerols (CycDs) - both being recognised as impurities of technical glycerol - in different types of wine. It is known that glycerol produced by transesterification of plant and animal triglycerides using methanol contains considerable amounts of 3-MPD. The synthesis of glycerol from petrochemicals leads to impurities of CycDs. One of the published methods [1, 2, 3[i]] was adopted, modified and tested in an collaborative study. Here we present the optimized method and report the results of the collaborative study [2]. Design and assessment of the validation study followed the O.I.V. Resolution 8/2000 “Validation Protocol of Analytical Methods”.

  1. Scope

The described method is suitable for the determination of 3-MPD and 6 cyclic diglycerols (cis-, trans-2,6-bis(hydroxymethyl) 1,4-dioxane; cis-, trans-2,5-bis(hydroxymethyl) 1,4-dioxane; cis-, trans-2,-hydroxymethyl-6-hydroxy-1,4-dioxepane) in white, red, sweet and dry wines. The study described covers the concentration range of 0.1 to 0.8 mg/L for 3-MPD and 0.5 to 1.5 mg/L for the CycDs.

  1. Definitions

3-MPD

3-methoxypropane-1,2-diol

ANOVA

Analysis of Variance

C

Concentration

CycDs

Cyclic diglycerols

GC-MS

Gas chromatography – mass spectrometry

H2

Hydrogen

IS

Internal standard

m/z

mass/charge ratio

ML

Matrix calibration level

S0

Standard dilution 1000 ng/ μL

S1

Standard dilution 100 ng/ μL

S2

Standard dilution 10 ng/ μL

  1. Principle

The analytes and the internal standard are salted-out by addition of , and extracted using diethyl ether. Extracts are analyzed directly by GC-MS on a polar column. Detection is then carried out in selected ion monitoring mode.

  1. Reagents and Materials

5.1.  Chemicals

5.1.1.      p.A .

5.1.2.      Diethyl ether Uvasol  for spectroscopy

5.1.3.      Molecular sieve (2 mm diameter, pore size 0.5 nm)

5.1.4.      Ethanol (Absolute)

5.2.  Standards

5.2.1.      Cyclic diglycerol mixture (6 components) Solvay Alkali GmbH[1], 89.3 %

cis-, trans-2,6-bis(hydroxymethyl) 1,4-dioxane; cis-, trans-2,5-

bis(hydroxymethyl) 1,4-dioxane; cis-, trans-2,-hydroxymethyl-6-hydroxy-1,4-dioxepane

5.2.2.      3-Methoxypropane-1,2-diol (3-MPD)  98% (CAS 623-39-2)

5.2.3.      Butane-1,4 -diol-1,1,2,2,3,3,4,4-(2H)8  98% (CAS 74829-49-5)

5.3.  Preparation of standard solution

5.3.1.      S0 stock solutions

Accurately weigh 10.0 mg 0.05 mg of each standard substance (11.2 mg are weighed for the CycDs, corresponding to 89.3 % purity) and transfer them to a 10 mL volumetric flask (one for each). Add exactly 10 mL of ethanol and mix thoroughly. The concentration of this solution is 1000 ng/ μL.

5.3.2.      S1 working solutions

Volumetrically transfer 1000 μL of the S0 stock solution (6.3.1) to a 10 mL volumetric flask, dilute the contents to volume with ethanol, thoroughly stopper the flask and invert to mix. The concentration of this solution is 100 ng/ μL.

5.3.3.  S2 working solutions

Volumetrically transfer 100 μL of the S0 stock solution (6.3.1) to a 10 mL volumetric flask, dilute the content to volume with ethanol, thoroughly stopper the flask and invert to mix. The concentration of this solution is 10 ng/ μL.

Overview of required standard solutions:

CycDs mixture (6 components)

Solution

Concentration

S0

1000

ng/ μL

S1

100

ng/ μL

3-Methoxypropane-1,2-diol (3-MPD)

Solution

Concentration

S0

1000

ng/ μL

S1

100

ng/ μL

S2

10

ng/ μL

1,4 Butane-1,4-(2H)8 (internal standard IS)

Solution

Concentration

S0

1000

ng/ μL

S1

100

ng/ μL

5.4.  Preparation of the matrix calibration curve

Matrix-matched calibration solutions are prepared in an uncontaminated wine. It is necessary to analyze this wine first to check that it is not contaminated with 3-MPD or CycDs. If the concentrations of the analytes in the sample are outside the range of the calibration curve, additional levels must be prepared.  To ensure that the internal standard does not interfere with any wine components, a blank should be included.

Table 1. Pipetting scheme of matrix calibration

Matrix calibration level

Volume Wine

C Wine

C Wine

Spike μl

ml

μg/L

mg/L

Blank

IS

-

10

0

0

3-MPD

-

CycDs

-

ML0

IS

100

S1

10

1000

1.00

3-MPD

-

CycDs

-

ML1

IS

100

S1

10

1000

1.00

3-MPD

100

S2

100

0.10

CycDs

50

S1

500

0.50

ML2

IS

100

S1

10

1000

1.00

3-MPD

25

S1

250

0.25

CycDs

100

S1

1000

1.00

ML3

IS

100

S1

10

1000

1.00

3-MPD

50

S1

500

0.50

CycDs

20

S0

2000

2.00

ML4

IS

100

S1

10

1000

1.00

3-MPD

100

S1

1000

1.00

CycDs

30

S0

3000

3.00

ML5

IS

100

S1

10

1000

1.00

3-MPD

200

S1

2000

2.00

CycDs

40

S0

4000

4.00

  1. Apparatus

 

6.1.  Analytical balance.0.0001 g readability.

6.2.  Lab centrifuge (at least 4000 rpm/min)

6.3.  Gas chromatograph.-With mass spectrometric detector, split-splitless injector,

6.4.  Diverse precision pipettes and volumetric flasks

6.5.  Pasteur pipettes

6.6.  40 mL centrifugation vials

6.7.  GC-vials (1.5 –2.0 mL)

6.8.  Thermostat

6.9.  Shaking machine

  1. Sampling

Wine samples for the analysis should be taken in a sufficient size. Volume needed for one test sample is 10 mL. The wine used for the preparation of the matrix-calibration (5.4) shall be free of analyte.

  1. Procedure

8.1.  Extraction

Add 100 μL internal standard solution S1 (6.3.2) to 10 mL wine to a suitable centrifugation vial e.g. 40 mL.  (This corresponds to a concentration of 1 mg/L butane-1,4-(2H)8).  Carefully add 10 g of K2CO3 and mix.  Take care during this addition as heat is produced due to the release of CO2. After cooling the solution to approximately 20 °C in a water bath, add 1 mL diethyl ether.  Homogenise the mixture for 5 minutes using a vertical-shaking machine.  Centrifuge the vials at 4000 rpm for 5 min.  For better removal of the organic phase, the extract can be partially transferred into a vial with a smaller diameter.  Using a Pasteur pipette, transfer the upper organic phase, composed of diethyl ether and ethanol, into a GC vial.  Add approximately 120 mg of molecular sieve into the vial.  Close the vial, leave for at least 2 h and shake well from time to time.  The clear supernatant is transferred to a second GC vial for the GC-MS analysis.

8.2.  GC-MS Analysis

Specific parameters for the GC-MS analysis are provided below.  Alternative systems may be used, if they provide a similar chromatographic performance and adequate sensitivity. The chromatographic system must be able to separate the internal standard from phenylethanol, a potential interference.

Typical GC conditions

Gas chromatograph: HP 5890 or equivalent

DB-Wax (J&W) column 60 m, 0.32 mm internal diameter, 0.25 μm film thickness, 2 m capillary containment same dimensions or equivalent

Carrier gas: H2

Flow: Pressure 60 k Pa column head

Temperature program:

90° C, 2 min., ramp at 10°C/min. up until 165° C, held for 6 min., ramp at 4° C/min to 250°C, held for 5 min.

Injection temperature: 250° C; Injected volume; 2 μL, 90 sec splitless for 90 s.

Specificl MS conditions

Mass spectrometer: Finnigan SSQ 710 or equivalent

Transfer line: 280° C

Source: 150° C

MS detection:

window1.: 0-25 min.:

14.3 min. 3-MPD: m/z 75, m/z 61

16.7 min IS: m/z 78, m/z 61

Acquisition time for each mass is 250 µs (dwell time).

Monitor for m/z 91 the separation of the internal standard (IS) peak from phenylethanol, which also produces a fragment m/z 78.

window 2. 25-40 min.:

32-34.5 min. CycDs:  m/z 57, m/z 117

Acquisition time for each mass is 250 µs (dwell time).

It has been observed that the analysis may degrade chromatographiccolumn. In particular, the injection of the high boiling CycDs mixture is suspected to cause irreversible damage. Injections of reference standard solutions should be avoided; analysis should be restricted to salted-out solutions with low analyte concentrations. In addition it is recommended to use a 1-2 m pre column in order to protect the analytical column. Nevertheless, the analytical column has to be considered as a consumable and must be replaced quite regularly.

  1. Evaluation

9.1.  Identification

Record the relative retention time of each analyte to the IS. Calculate the mean relative retention time of the analytes in the calibration standards. The relative retention time of the analyte should be the same as that of the standard within a margin of 0.5 %. As a confirmation criterion, an ion ratio can be calculated for each analyte from the selected ion monitoring.  This ratio is 117/57 for CycDs, 75/61 for 3-MPD and 78/61 for the IS.  The ratio should be within 20 % of that which is found in the spiked sample. Confirmation of the identity of substances by full scan using ionsn can also be used.

9.2.  Quantification

The quantification is done by a matrix calibration curve prepared according to appropriate section. The analyte/IS area ratios of the indicated mass ratios are correlated by linear regression against the concentration of the analyte.  Quantification of the CycDs is achieved by summing the peak area of all six peaks and calculating the total content, to allow for other distributions of the six characteristic CycDs than in the standard.  The following m/z values are used for quantification:

3-MPD: m/z 75

IS:m/z 78

CycDs:  m/z 117

9.3.  Expression of results

Results should be expressed in mg/L for 3-MPD and CycDs with two decimals (e.g. 0.85 mg/L).

9.4.  Limit of Detection and limit of quantification

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 limit of detection (LD) and the limit of quantification (LQ) were estimated using the instrumentation and conditions mentioned exemplarily above (s. 8) following the instructions in the resolution OENO 7-2000 (E-AS1-10-LIMDET) “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 ion trace (m/z) chromatogram is drawn extendedly enclosing a range of a tenfold peak width at mid-height (w½) of an analyte peak in a relevant part of the chromatogram. Furthermore two parallel lines are drawn which just enclose the maximum amplitude of the signal window.

The distance of these two lines gives hmax, expressed in abundance units is multiplied by 3 for LD, by 10 for LQ and finally converted into concentration units by implementing the individual response factor.

3-MPD:

LD: 0,02 mg/l

LQ: 0,06 mg/l

CycDs (sum):

LD: 0,08 mg/l

LQ: 0,25 mg/l

(Note: Since the CD are a mixture of six single compounds with the same response factor - due to their chemical equality - and with hmax constant in the relevant part of the chromatogram the LD and LQ for each single compound are one sixth of the figures above)

  1. Precision (interlaboratory validation)

Eleven laboratories participated in the collaborative study. The participating laboratories have proven experience in the analysis of the by-products. All of them participated in the pre-trial.

Repeatability (r) and reproducibility (R) and the respective standard deviations (Sr and SR) were found to be correlated statistically significantly with the concentration of the analytes (ANNEX: Figures 1 and 2), r with more than 95% probability and R with more than 99% probability for each of the analytes using the linear regression model.

The actual performance parameters can be calculated by:

3-MPD

Sr=0,060x

x=concentration of 3-MPD [mg/L]

SR=0,257x

r = 0,169 x

x=concentration of 3-MPD [mg/L]

R = 0,720 x

CycDs

Sr  = 0,082 x

x=concentration of CycDs [mg/L]

SR = 0,092 x + 0,070

r  = 0,230 x

x=concentration of CycDs [mg/L]

R = 0,257 x + 0,197

Annex (Interlaboratory Study)

 

Participants

11 international laboratories participated in the collaborative study (5). The participating laboratories have proven experience in the analysis of the by-products. All of them participated in the pre-trial:

  • CSL, York, UK
  • Unione Italiana Vini, Verona, Italy
  • BfR, Berlin, Germany
  • BLGL, Würzburg, Germany
  • Istituto Sperimentale per l'enologia, Asti, Italy
  • LUA, Speyer, Germany
  • Labor Dr. Haase-Aschoff, Bad Kreuznach, Germany 
  • CLUA, Münster, Germany
  • Kantonales Laboratorium, Füllinsdorf, Switzerland
  • LUA, Koblenz, Germany
  • ISMAA, S. Michele all Adige, Italy

Samples

In November 2002, participating laboratories were sent 11 wine samples consisting of five sets of blind duplicates and one further single test material. Dry white wines, dry red wines and a sweet red wine were used for test materials. The samples were subjected to homogeneity testing previously ([ii]).

Data analysis

Statistical analysis was carried out according to the “Protocol for the Design, Conduct and Interpretation of Method Performance Studies” ([iii]) using a blind duplicate model.

Determination of outliers was assessed by Cochran, Grubbs and paired Grubbs tests.

Statistical analysis was performed to obtain repeatability and reproducibility data.

Horrat values were calculated.

Table 2. Results for 3-MPD

Sample A

White

wine

Sample B

Red

wine a

Sample C

White

wine

Sample F

Sweet red

wine

Sample G

White

wine

Mean mg/L

0.30

0.145

0.25

0.48

0.73

Spiked mg/L

0.30

0.12

-

-

0.80

Recovery %

100

121

-

-

91

n

10

10 a

10

10

10

nc

1

1 a

1

1

1

outliers

2

0

0

1

1

n1

7

9 a

9

8

8

r

0.03

-

0.05

0.08

0.13

sr

0.01

-

0.02

0.03

0.05

RSDr %

3.20

-

7.20

5.80

6.57

Hor

0.30

-

0.60

0.50

0.59

R

0.13

0.13

0.15

0.31

0.59

sR

0.05

0.05

0.05

0.11

0.21

RSDr %

15.50

32.67

21.20

22.70

28.91

HoR

0.80

1.53

1.10

1.30

1.72

 

a Single test sample; n, nc and n1 are single results

mean: arithmetic mean of the data used in the statistical analysis

n: total number of sets of data submitted

nc: number of results (laboratories) excluded due to non-compliance

outliers: number of results (laboratories) excluded due to determination as outliers by either Cochran’s or Grubbs’ tests

n1: number of results (laboratories) retained in statistical analysis

Sr: the standard deviation of the repeatability

RSDr: the relative standard deviation of the repeatability (Srx100/mean)

r: repeatability  (2.8 x Sr)

Hor: the Horrat value for repeatability is the observed RSDr divided by the RSDr value estimated from the Horwitz equation using the assumption r = 0.66R

R: reproducibility (between laboratory variation) (2.8 x SR)

SR: the standard deviation of the reproducibility

RSDR: the relative standard deviation of the reproducibility (SRx100/mean)

HoR: the Horrat value for reproducibility is the observed RSDR value divided by the RSDR value calculated from the Horwitz equation

Figure 1. Correlation between 3-MPD concentration and r and R.

Table 3. Results for cyclic dyglycerols

Sample A White

wine

Sample B

Red

winea

Sample D

Red

wine

Sample F

Sweet red

wine

Sample G White

wine

Mean  mg/L

1.55

0.593

0.80

0.96

0.56

Spiked mg/L

1.50

0.53

0.50

Recovery %

103

113

112

n

11

11a

11

11

11

nc

0

0

0

0

0

outliers

2

0

1

2

1

n1

9

11a

10

9

10

r

0.37

-

0.19

0.18

0.15

sr

0.13

-

0.07

0.07

0.05

RSDr %

8.50

-

8.60

6.70

9.30

Hor

0.90

-

0.80

0.60

0.80

R

0.61

0.379

0.39

0.41

0.34

sR

0.22

0.135

0.13

0.15

0.12

RSDR %

14.00

22.827

17.30

15.20

21.50

HoR

0.90

1.319

1.00

0.90

1.20

 a Single test sample; n and nc are single results

Figure 2. Correlation between CycDs concentration and r and R.



[1] Solvay Alkali GmbH no longer provides the standard mixture; solutions of the mixture may be obtained from the BfR. Federal Institute for Risk Assessment, Thielallee 88-92, D-14195 Berlin. www.bfr.bund.de; poststelle@bfr.bund.de


(1) Bononi, M., Favale, C., Lubian, E., Tateo F. (2001)

A new method for the identification of cyclic diglycerols in wine

J. Int. Sci. Vigne Vin. 35, 225-229

(2) Thompson, M. and Wood, R. (1993)

International Harmonised Protocol for the Proficiency Testing of (Chemical) Analytical Laboratories -  J AOAC Int 76, 926-940

(3) Horwitz ,W. (1995)

Protocol for the design, conduct and interpretation of method-performance studies

Pure and Applied Chemistry 67, 331-343

Determination of releasable 2,4,6-trichloroanisole in wine (Type-IV)

OIV-MA-AS315-16 Determination of releasable 2,4,6-trichloroanisole in wine by cork stoppers

Type IV method

  1. Scope

 

The method of determination of releasable 2,4,6-trichloroanisole (TCA) by cork stoppers measures the quantity of TCA released by a sample of cork stoppers macerated in a aqueous-alcoholic solution. The aim of this method is to evaluate the risk of releasing by the lot of analyzed cork stoppers and to provide a method for controlling the quality of cork stoppers.

  1. Principle

 

The method aims to simulate 2,4,6-trichloroanisole migration phenomena susceptible of being produced between the cork stopper and wine in bottles. Cork stoppers are macerated in a wine or a aqueous-alcoholic solution, until a balance is obtained. The TCA of the head space is sampled from an appropriate part of the macerate by the solid-phase micro-extraction technique (SPME), then analyzed by gas chromatography, with detection by mass spectrometer (or by electron-capture detector).

  1. Reagents and products

 

3.1.   White wine with an alcoholic strength ranging between 10 and 12 % vol. (It can be replaced by an aqueous-alcoholic solution with an alcoholic strength of 12 % vol). The wine and/or the aqueous-alcoholic solution must be free of TCA.

3.2.   Sodium chloride 99.5 %

3.3.   Internal standard for GC/MS analysis: 2,4,6-trichloroanisole (TCA)-d5 purity 98% or 2,3,6-trichloroanisole purity 99%.

Internal standard for GC/ECD analysis; 2,6-dibromoanisole purity  99% or 2,3,6-trichloroanisole purity 99%.

3.4.   2,4,6-trichloroanisole (TCA) purity ≥ 99.0%

3.5.   Absolute ethanol

3.6.   Pure de-ionised water void of TCA (Standard EN ISO 3696)

3.7.   Aqueous-alcoholic solution at 12 % vol.

Prepared using absolute ethanol (3.5) and de-ionised water void of TCA (3.6).

3.8.   Internal standard stock solution (500 mg/L)

Add either 0.050 g of 2,4,6-trichloroanisole- (or 2,6-dibromoanisole or 2,3,6-trichloroanisole (3.3) to approximately 60 ml of absolute ethanol (3.5). After dissolution, adjust the volume to 100 mL with absolute ethanol (3.5). It can be kept in a glass bottle with a metallic or glasscover.

3.9.   Intermediate solution of internal standard (5.0 mg/L)

Add 1 mL of a solution of either 2,4,6-trichloroanisole- (or 2,6-dibromoanisole or 2,3,6-trichloroanisole) at 500 mg/L (3.8) to approximately 60 mL of absolute ethanol (3.5). Adjust the volume to 100 mL with absolute ethanol (3.5). It can be kept in a glass bottle with a metallic or glass cover.

3.10.         Internal standard solution (2.0 µg/L)

Add 40 μL of a solution of either 2,4,6-trichloroanisole-d5  (or 2,6-dibromoanisole or 2,3,6 trichloroanisole) at 5.0 mg/L (3.9) to approximately 60 mL of absolute ethanol (3.5). Adjust the volume to 100 ml with absolute ethanol (3.5). It can be kept at an ambient temperature in a glass bottle with a metallic or glass cover.

3.11.         Stock solution of TCA standard (40 mg/L)

Add 0.020g of 2,4,6-trichloroanisole to approximately 400 ml of absolute ethanol (3.5). Following dissolution, adjust volume to 500 mL with absolute ethanol (3.5).

3.12.     Intermediate solution A of TCA standard (80 μg/L)

Add 1 mL of 2,4,6-trichloroanisole solution at 40 mg/L (3.11) to approximately 400 mL of absolute ethanol (3.5). Following dissolution, adjust volume to 500 mL with absolute ethanol (3.5).

3.13.         Intermediate solution B of TCA standard (160 ng/L)

Add 1 mL of solution 2,4,6-trichloroanisole at 80 μg/L (3.12) to approximately 400 mL of pure de-ionised water (3.6). Following dissolution, adjust the volume to 500 mL with pure de-ionised water (3.6)

3.14.         Use the standard-addition technique to make up a range of standard solutions of TCA. Standard solutions in the range from 0.5 ng/L to 50 ng/L can be used, by making additions with a solution of 2,4,6-trichloroanisole at 160 ng/L (3.13) to 6 ml of absolute ethanol (3.5). Following dissolution, adjust volume to 50 mL with pure de-ionised water (3.6)

The calibration curve obtained should be evaluated regularly and in any case whenever there is a major change in the GC/MS or GC/ECD systems.

3.15.         Carrier gas: Helium, chromatographic purity ( 99.9990 %)

  1. Apparatus

4.1.   Laboratory glassware

4.1.1. Graduated 100-mL flask

4.1.2. 100- μL microsyringe

4.1.3. Wide-neck glass jar of a capacity adapted to the sample size, closed with a glass or metallic stopper or a material which does not bind TCA.

4.1.4. 20-mL glass sample bottle closed with a perforated capsule and a liner with one side Teflon-coated.

4.2.   Solid-phase microextraction system (SPME) with a fiber coated with a polydimethylsiloxane film 100 μm thick

4.3.   Heating system for sample bottle (4.1.4)

4.4.   Stirring system for sample bottle (4.1.4)

4.5.   Gas chromatograph equipped with a "split-splitless" injector and a mass spectrometer detector (MS) or an electron-capture detector (ECD)

4.6.   Data-acquisition system

4.7.   If required, an automatic sampling and injection system operating with an SPME system

4.8.   Capillary column coated with an apolar stationary phase, of the phenylmethylpolysiloxane type (e.g.: 5 % phenyl methylpolysiloxane, 30 m x 0,25 mm x 0,25 µm film thickness or equivalent.)

  1. Sample preparation

The corks are placed whole in a glass closed container. The container capacity (4.1.3), the same as the quantity of wine or aqueous-alcoholic solution (3.1 or 3.7), must be chosen in accordance to the sample size while ensuring that the corks are completely covered and immersed in the maceration container.

Example 1: 20 corks (45x24) mm, in a 1 L container;

Example 2: 50 corks (45x24) mm, in a 2 L container.

Most of the TCA released during maceration of the groups of stoppers is generally derived from a very low percentage of these stoppers. In order to obtain the best representation of a batch of stoppers, a number of appropriate analyses according to sampling rules and risk with regard to wine contamination should be carried out.

  1. Operating method

6.1.   Extraction

After macerating at ambient temperature for (24 2) hours under laboratory ambient temperature conditions, the maceration is homogenized by inversion. A part of the aliquot of the 10ml maceration solution (5) is transferred to a glass sample bottle (4.1.4)

To increase extraction efficiency and subsequent sensitivity of the method, a quantity sodium chloride (3.2) can be added.  The amount of sodium chloride can be adjusted / optimized by the users of this method, depending on the desired level of sensitivity and possible matrix effects that may occur. For example, a quantity of about 3 g of sodium chloride is suggested. 50 μL of the internal standard solution at 2.0 μg/L (3.10) are immediately added, then the bottle is closed using a perforated metal capsule fitted with a silicone / Teflon-coated liner. The capsule is crimped. The contents of the bottle are homogenized for 10 minutes by mixing using a stirring system (4.4) or by using an automatic system (4.7).

The bottle containing the sample is placed in the heating system (4.3) set to 35 °C 2 °C, with stirring (4.4). The extraction of the headspace is carried out using the SPME system (4.2) for at least 15 minutes.

6.2.   Analysis

The fiber is then desorbed at 260 °C for at least 2 minutes in the injector of a gas chromatograph, in splitless mode (4.5). The separation is carried out using a capillary column with a non-polar stationary phase (4.8). The carrier gas is helium with a constant flow of 1 ml/min. A temperature program from 35 °C (for 3 min) to 265 °C (at 15 °C/min) is given as an example.

6.3.   Detection and quantification

Detection and quantification are carried out by mass spectrometry with a selection of specific ions. For example, the following ion ratio is suggested:

Analysis in SIM mode

Analyte

Interesting ions for detection (m/z):

Ion Quantification (m/z) :

2,4,6-TCA

195, 210, 212

195

(2,4,6-TCA)-d5

199, 215, 217

215

2,3,6-TCA

195, 210, 212

212

Analysis in tandem mode (MS/MS)

Analyte

Parent ions (m/z):

Daughter ion (m/z) :

2,4,6-TCA

212

169, 197

196

167, 169

(2,4,6-TCA)-d5

217

171, 199

For the determination of ECD, identify the analyte and internal standard (2,6-dibromoanisole or 2,3,6 trichloroanisole) in the chromatogram, by comparing the retention time of the sample peak corresponding to that of the standard solution peak.

  1. Calculations

The area of the chromatographic peak obtained for the 2,4,6-trichloroanisole is corrected by the area obtained for the chromatographic peak of the internal standard. The content in 2,4,6-trichloroanisole of each sample is obtained using a calibration curve. The points on this curve are obtained by tracing the relative responses of the 2,4,6-trichloroanisole/internal standard, obtained for aqueous-alcoholic solutions (3.7) containing known concentrations of 2,4,6-trichloroanisole, as a function of the concentrations of these solutions (3.14).

The results are given in ng/L of TCA present in the maceration, rounded off to the nearest 0.1 ng/L.

 

  1. Characteristics of the method

As an indication, the detection limit of the analysis of the macerations must be lower than 0.5 ng/L, and the quantification limit close to 1 ng/L. The coefficient of variation is lower than 5% for 5 ng/L, when the selected internal standard is the deuterated analogue TCA-.

An interlaboratory trial was carried out in order to validate the method. This interlaboratory trial was not carried out according to the OIV protocol and the validation parameters mentioned in the FV 1224.

  1. Bibliography
  • HERVÉ E., PRICE S., BURNS G., Chemical analysis of TCA as a quality control tool for natural corks. ASEV Annual Meeting. 1999.
  • ISO standard 20752:2007 Cork stoppers — Determination of releasable 2, 4, 6-trichloroanisol (TCA).
  • FV 1224 - Résultats de l’analyse collaborative Ring test 3-TCA SPME.

Determining the presence and content of polychlorophenols and polychloroanisols in wines, cork stoppers, wood and bentonites used as atmospheric traps (Type-IV)

OIV-MA-AS315-17 Determining the presence and content of polychlorophenols and polychloroanisols in wines, cork stoppers, wood and bentonites used as atmospheric traps

Type IV method

 

  1. Scope

All wines, cork stoppers, bentonites (absorption traps) and wood.

  1. Principle

Determination of 2,4,6-trichloroanisol, 2,4,6-trichlorophenol, 2,3,4,6-tetrachloroanisol, 2,3,4,6-tetrachlorophenol, pentachloroanisol and pentachlorophenol by gas chromatography, by injecting a hexane extract of the wine and an ether/hexane extract of the solid samples to be analyzed and internal calibration.

  1. Reagents

Preliminary remark: all the reagents and solvents must be free of the compounds to be determined listed in 2 at the detection limit.

3.1.  Purity of hexane > 99 %

3.2.  Purity of ethylic ether > 99 %

3.3.  Ether/hexane mixture (50/50; v/v)

3.4.  or 2,5-dibromophenol purity 99 %

3.5.  Pure ethanol

3.6.  Pure deionized water, TCA free, type II in accordance with ISO standard EN 3696

3.7.  50 % vol.  aqueous-alcoholic solution. Place 100 ml of absolute ethanol (3.<5) in a graduated 200-ml flask (4.9.9), add 200 ml of deionized water (3.6), and homogenize.

3.8.  Internal standard:

3.8.1.      200 mg/l stock solution. Place 20 mg of internal standard (3.4) in a graduated 100-ml flask (4.9.8), add the 50 % volume aqueous-alcoholic solution (3.7) and homogenize.

3.8.2.      Internal standard solution (2 mg/l). Place 1 ml of the stock solution of internal standard (3.8.1) in a graduated 100-ml flask (4.9.8), add the 50% vol aqueous-alcoholic solution (3.7) and homogenize.

3.8.3.      Internal standard solution (20 µg/l). Place 1 ml of stock solution of internal standard (3.8.2) in a 100 ml graduated flask (4.9.8), add with 50 % vol aqueous-alcoholic solution

3.9.  Pure products

  • 2,4,6-trichloroanisole: 99 %, case: 87-40-1
  • 2, 4, 6-trichlorophenol: 99.8 %, case: 88-06-2
  • 2,3,5,6-tetrachloroanisole: 99 %, case: 6936-40-9 (note: the product sought in the samples is 2,3,4,6-tetrachloroanisole but is does not exist on the market)
  • 2, 3, 4, 6-tetrachlorophenol: 99 %, case: 58-90-2
  • pentachloroanisole: 99 %, case: 1825-21-1
  • pentachlorophenol: 99 %, case: 87-86-5
    1.         Reagents for derivatisation - Piridine: acetic anydride (1:0,4) vol.
      1. Piridine: 99 %
      2. Acetic anydride: 98 %
    2.         Calibration stock solution at 200 mg/l

In a graduated 100-ml flask (4.9.8), place approximately 20 mg of the pure reference products (3.9.1 to 3.9.6) but whose exactly weight is known (4.7), add absolute ethanol (3.5). Homogenize.

3.12.        Intermediate calibration solution at 200 μg/l

In a graduated 100-ml flask (4.9.8) filled with absolute ethanol (3.5), add 100 μl of the calibration stock solution at 200 mg/l (3.11) using the 100- μl micro-syringe (4.9.1) and homogenize.

3.13.        Calibration surrogate solution at 4 μg/l

In a graduated 50-ml flask (4.9.7) containing 50 % vol aqueous-alcoholic solution (3.7) add 1 ml of the intermediate calibration solution at 200 μg/l (3.11) using a 1-ml pipette (4.9.6). Add to volume 50 ml with pure ethanol (3.5) and homogenize.

3.14.        Calibration solutions. It is possible to prepare various standard solutions with various concentrations by adding, using the 100- μl micro-syringe of (4.9.1), for example 50 μl of the surrogate calibration solution at 4 μg/l (3.12) to 50 ml of wine to enrich it with 4 ng/l of the substances to be determined.

The same reasoning can be used to prepare calibration solutions of various concentrations, either using aqueous-alcoholic solutions, or wine, or to enrich an extraction medium with a known quantity of pure products.

3.15.        Commercially available Bentonite.

  1. Apparatus

4.1.  Gas phase chromatograph with Split-splitless injector coupled to an electron capture detector. (It is likewise possible to use a mass spectrometer)

4.2.  Capillary tube of non-polar steady-state phénylmethylpolysiloxane type: (0.32 mm x 50 m, thickness of film 0.12 μm or the equivalent

4.3.  Chromatographic conditions, as an example:

4.3.1.      Injection in "split-splitless" mode (valve closing time 30 seconds)

4.3.2.      Carrier gas flow rate: 30 ml/min including 1 ml in the column Hydrogen U ®2  (It is likewise possible to use helium)  

4.3.3.      Auxiliary gas flow rate: 60 ml/min – Nitrogen with chromatographic purity ( 99,9990 %). It is also possible to use argon methane.

4.3.4.      Furnace gradient temperature for information purposes:

  • from 40 °C to 160 °C at a rate of 2 °C/min
  • from 160 °C to 200 °C at a rate of 5 °C/min
  • step at 220 °C for 10 min
    1.       Injector temperature: 250 °C
    2.       Detector temperature: 250 °C 
  1.   Acquisition and integration: acquisition is by computer. The peaks of the various compounds identified by comparison with the reference are then integrated.
  2.   Magnetic agitator.
  3.   Vortex with adaptation for 30-ml flask (4.9.3)
  4.   Precision balance to within 0.1 mg
  5.   Manual or electric household grate
  6.   Laboratory equipment:
    1.       100- μl micro-syringe
    2.       10- μl micro-syringe
    3.       30-ml flask closing with a screwed plug and cover with one side Teflon-coated
    4.       10-ml stick pipette graduated 1/10 ml
    5.       5-ml stick pipette graduated 1/10 ml
    6.       1-ml precision pipette
    7.       Graduated 50-ml flask
    8.       Graduated 100-ml flask
    9.       Graduated 200-ml flask
    10. 10 100-ml separating funnel
    11. Pasteur pipettes and suitable propipette pear
    12. Household aluminum foil, roll-form.
    13. Centrifuge
  1. Sample preparation

5.1.  The stopper is grated (4.8) or cut into pieces (dimension < 3 mm)

5.2.  Wood is cut with a clipper to obtain pieces (dimension < 3 mm)

5.3.  The bentonite (3.15) (30 g for example) is spread out over a strip of aluminum foil (4.9.12) of approximately 30 cm x 20 cm and is exposed to the atmosphere to be analyzed for at least 5 days.

  1. Operating method

6.1.  Extraction process for solid samples:

6.1.1.      Stopper: in a 30-ml flask (4.9.3), place approximately 1 g of grated stopper (5.1) but of a precisely known weight (4.7)

6.1.2.      Wood: in a 30-ml flask (4.9.3), place approximately 2 g of wood chips (5.2) but of a precisely known weight (4.7)

6.1.3.      Control Bentonite: in a 30-ml flask (4.9.3), place approximately 5 g of bentonite (3.15) but of a precisely known weight (4.7)

6.1.4.      Sample bentonite: in a 30-ml flask (4.9.3), place approximately 5 g of bentonite (5.3) of a precisely known weight (4.7)

6.1.5.      Add 10 ml (4.9.4) of ether/hexane mixture (3.3)

6.1.6.      Add with the micro-syringe (4.9.1) 50 μl of the internal standard solution (3.8.2)

6.1.7.      Agitate with the vortex (4.6) for 3 min

6.1.8.      Recover the ether/hexane liquid phase in a 30-ml flask (4.9.3)

6.1.9.      Repeat the extraction operation on the sample with 2 times 5 ml of ether/hexane mixture (3.3)

6.1.10. Final extract: mix the 3 phases of ether/hexane.

6.2.  Extraction of the wine and calibration solution

6.2.1.      Sample 50 ml of wine or calibration solution (using the graduated flask (4.9.7)

6.2.2.      Place them in the 100-ml graduated flask (4.9.8)

6.2.3.      Add with the microsyringe (4.9.1) 50 μl of internal standard (3.8.3)

6.2.4.      Add 4 ml (4.9.5) of hexane (3.1)

6.2.5.      Carry out the extraction using the magnetic stirrer (4.5) for 5 min.

6.2.6.      Elutriate into the funnel (4.9.10)

6.2.7.      Recover the organic phase with the emulsion in a 30-ml flask (4.9.3) and aqueous phase  in the 100-ml graduated flask (4.9.8)

6.2.8.      Repeat the extraction of the wine or calibration solution using 2 ml of hexane (3.1)

6.2.9.      Carry out the extraction using the magnetic stirrer (4.5) for 5 min.

6.2.10. Elutriate into the funnel (4.9.10)

6.2.11. Recover the organic phase with the emulsion in the same 30-ml flask mentioned in 6.2.7 (containing the organic phase obtained upon the first extraction)

6.2.12. Break the emulsion of the organic phase by centrifugation (4.9.13) by eliminating the lower aqueous phase using a Pasteur pipette (4.9.11) fitted with a propipette pear.

6.2.13. Final wine extract and calibration solutions: the residual organic extract

6.3.  Analyze:

6.3.1.      Add final extract (6.1.11 or 6.2.13) 100 μl (4.9.1) of the pyridine acetic anydride reagent mixture (3.10) for the derivatisation.

6.3.2.      Mix using a magnetic stirrer (4.5) for 10 min.

6.3.3.      Inject 2 μl of derivatised final extract (6.3.2) into the chromatograph

  1. Calculation

Response factor = concentration of calibration solution (3.13) * (Peak area of the internal standard / *(Peak area of the pure product in the calibration solution).

Check the calibration by ensuring the response factors +/- 10 %.

  1. Results

The results are expressed in ng/l for the wine and ng/g for the cork stoppers, bentonites and wood.

  1. Characteristics of the method

9.1.  Coverage rate

The coverage rate calculated in relation to the quantities added in terms of wood chips, polychloroanisols and polychlorophenols of 115 ng/g is:

  • 2,4,6-trichloroanisol: 96 %
  • 2,4,6-trichlorophenol: 96 %
  • 2,3,4,6-tetrachloroanisol: 96 %
  • 2,3,4,6-tetrachlorophenol: 97 %
  • pentachloroanisol: 96 %
  • pentachlorophenol: 97 %

9.2.  Measurement repeatability

Calculated for each product, the uncertainties are as follows:

In a stopper ng/g

Mean

Standard deviation

Repeatability

2,4,6-trichloroanisol

1.2

0.1

0.28

2,4,6-trichlorophenol

26

3.3

9.24

2,3,4,6-tetrachloroanisol

1.77

0.44

1.23

2,3,4,6-tetrachlorophenol

2.59

  0.33

0.92

pentachloroanisol

23.3

2.9

8.12

pentachlorophenol

7.39

  1.91

5.35

In wood with 23 ng/g

Standard deviation

Repeatability

2,4,6-trichloroanisol

1.9

5.3

2,4,6-trichlorophenol

1.9

5.3

2,3,4,6-tetrachloroanisol

2.6

7.4

2,3,4,6-tetrachlorophenol

3.3

9.3

pentachloroanisol

2.7

7.5

pentachlorophenol

3.6

10.1

In wine with 10 ng/l

Standard deviation

Repeatability

2,4,6-trichloroanisol

0,4

1,1

2,4,6-trichlorophenol

2,1

5,9

2,3,4,6-tetrachloroanisol

0,6

1,7

2,3,4,6-tetrachlorophenol

4

11,2

pentachloroanisol

1,2

3,4

pentachlorophenol

6,5

18,2

In bentonite with15ng/g

Standard deviation

Repeatability

2,4,6-trichloroanisol

0,9

2,5

2,4,6-trichlorophenol

4

11,2

2,3,4,6-tetrachloroanisol

1,2

3,4

2,3,4,6-tetrachlorophenol

5,2

14,6

pentachloroanisol

4,3

12,0

pentachlorophenol

12,1

33,9

9.3.  Detection limits (DL) and quantification limits (QL) calculated according to the OIV method:

9.3.1.      Wood

DL in ng/g

QL in ng/g

2,4,6-trichloroanisol

0.72

2.4

2,4,6-trichlorophenol

0.62

2.0

2,3,4,6-tetrachloroanisol

0.59

2.0

2,3,4,6-tetrachlorophenol

1.12

3.74

pentachloroanisol

0.41

1.4

pentachlorophenol

0.91

3.1

9.3.2.      Bentonite

DL in ng/g

QL in ng/g

2,4,6-trichloroanisol

0.5

1

2,4,6-trichlorophenol

1

3

2,3,4,6-tetrachloroanisol

0.5

1

2,3,4,6-tetrachlorophenol

1

3

pentachloroanisol

0.5

1

pentachlorophenol

Not det.

Not det.

9.3.3.      Stopper

DL in ng/g

QL in ng/g

2,4,6-trichloroanisol

0.5

1.5

2,4,6-trichlorophenol

1

2

2,3,4,6-tetrachloroanisol

0.5

1.5

2,3,4,6-tetrachlorophenol

1

2

pentachloroanisol

0.5

1.5

pentachlorophenol

1

2

9.3.4.      Wine

DL in ng/l

QL in ng/l

2,4,6-trichloroanisol

0.3

1

2,4,6-trichlorophenol

1

3

2,3,4,6-tetrachloroanisol

0.3

1

2,3,4,6-tetrachlorophenol

0.3

1

pentachloroanisol

0.5

3

pentachlorophenol

1

3

®2 Air Liquide

Analysis of biogenic amines in musts and wines HPLC (Type-II)

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
  1. 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]
  1. Principle

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

  1. 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.