OIV-MA-F1-02 Hydroxymethylfurfural (HMF) by High-Performance Liquid Chromatography

Type IV method

 

1. Principle of the Method

High-performance liquid chromatography (HPLC)

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

2. Reagents

2.1.  Purified water for laboratory use and of quality standard EN ISO 3696

2.2.  Methanol, OH, distilled or HPLC quality. – CAS Number 67-59-1

2.3.  Acetic acid, CH3COOH, (ρ₂₀ = 1.05 g/ml). – CAS Number 64-19-7

2.4.  Mobile phase: water (2.1) -methanol (2.2)-acetic acid (2.3) previously filtered through a membrane filter (0.45 μm), (40:9:1 v/v).

This mobile phase must be prepared daily and degassed before use.

2.5.  Reference solution of hydroxymethylfurfural, 25 mg/l (m/v).

Into a 100 ml volumetric flask, place 25 mg of hydroxymethylfurfural, C₆H₃O₆, accurately weighed, and make up to the mark with methanol ( 2.2). Dilute this solution 1/10 with methanol (2.2) and filter through a membrane filter (0.45 μm).

If kept in a hermetically sealed brown glass bottle in a refrigerator, this solution will keep for two to three months.

(The concentration of the reference solution is given for guidance)

3. Equipment

3.1.  Apparatus

High-performance liquid chromatograph equipped with:

• a loop injector, 5 or 10 μl, (as an example),
• spectrophotometric detector for making measurements at 280 nm,
• column of octadecyl-bonded silica (e.g.: Bondapak C18 — Corasil, Waters Ass.),
• a recorder and, if required, an integrator,

Flow rate of mobile phase: 1.5 ml/minute (as an example).

3.2.  Membrane filtration apparatus, pore diameter 0.45 μm.

4. Procedure
   1.   Preparation of sample

Use the solution obtained by diluting the rectified concentrated must to 40% (m/v) (introduce 200 g of accurately weighed rectified concentrated must into a 500 ml volumetric flask. Make up to the mark with water and homogenise) and filter it through a membrane filter (0.45μm).

4.2.  Chromatographic determination

Inject 5 (or 10) μl of the sample prepared as described in paragraph 4.1. and 5 (or 10) μl of the reference hydroxymethylfurfural solution (2.5) into the chromatograph. Record the chromatogram.

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

The volume injected and the sequence are given for guidance. The chromatographic determination can also be done with a calibration curve

5. Expression of results

The hydroxymethylfurfural concentration in rectified concentrated musts is expressed in milligrams per kilogram of total sugars.

5.1.  Method of calculation

Let the hydroxymethylfurfural concentration in the 40% (m/v) solution of the rectified concentrated must be C mg/l.

The hydroxymethylfurfural concentration in milligrams per kilogram of total sugars is given by:

P = percentage (m/m) concentration of total sugars in the rectified concentrated must.

6. Characteristics of the method

 

Repeatability (r)

• r = 0.5 mg/kg total sugars

Reproducibility (R)

• R = 3.0 mg/kg total sugars

OIV-MA-F1-03 Determination of the acquired alcoholic strength by volume (ASV) of concentrated musts (CM) and grape sugar (or rectified concentrated musts, RCM)

Type IV method

 

1. Introduction

Concentrated musts (CM) and grape sugar (RCM) are viscous products with low alcohol contents; to determine their acquired ASV, a method must be used, the characteristics of which (linearity, repeatability, reproducibility, specificity, and detection and quantification limits) must be such that it is possible to measure alcohol contents of less than 1% vol.

2. Field of application

The method applies to concentrated musts and grape sugar.

3. Principle

A known mass of concentrated must (CM) or grape sugar is made alkaline by a suspension of calcium hydroxide and then distilled. The alcoholic strength by volume of the distillate is determined by electronic densitometry or by densitometry using a hydrostatic balance.

4. Reagents

• Suspension of 2M calcium hydroxide of analytical quality obtained by carefully pouring one litre of hot water (60°C to 70°C) on to about 120 g of unslaked lime (CaO).
• Antifoam solution obtained by dilution of 2 ml of concentrated silicone antifoam in 100 ml of water.
• Purified water for laboratory use and of quality EN ISO 3696.

5. Equipment

• Standard laboratory equipment including volumetric flasks 
• Analytic balance capable of weighing to within 0.1 g.
• Any type of distillation or steam distillation apparatus may be used provided that it satisfies the following test:
• Distil an ethanol‑water mixture with an alcoholic strength of 10% vol. five times in succession.  The distillate should have an alcoholic strength of at least 9.9% vol. after the fifth distillation; i.e. the loss of alcohol during each distillation should not be more than 0.02% vol.
• Electronic density meter or hydrostatic balance.

6. Procedure

• Homogenise the test sample by inverting the flask several times.
• In a 500 ml volumetric flask, weigh about 200 g of concentrated must or rectified concentrated must (to within 0.1 g). Note the weight (TS) of this test sample. Fill up to the mark with deionised water. This solution is about 40% m/v in must.

Obtaining the distillate

• Transfer 250 ml of the 40% solution to the distillation flask, add to the flask about 10 ml of calcium hydroxide in suspension, about 5 ml of antifoam solution and, where applicable, a boiling regulator (e.g. pieces of porcelain).
• Gently bring to the boil.
• Recover the distillate in a 100 ml volumetric flask (about 90 ml).
• Leave the distillate to return to ambient temperature, then fill up to the mark with deionised water.

Measurement of ASV

This is performed by electronic densitometry or by hydrostatic balance.

7. Calculation

• ASV measured = alcohol content given by the density meter, as % vol.
• TS = test sample of concentrated must or grape sugar, in weight.
• MV= density of concentrated must or rectified concentrated must, in g/ml

The results are expressed to 2 decimal places and rounded to within 0.05 %vol.

8. Characteristics of the method

8.1.  Linearity of response

The linearity of the density meter for low ASV values is one of the critical parameters of this method. A standard range of 10 aqueous-alcoholic solutions of ASV ranging between 0 and 5%vol. was prepared. Each solution was analysed 3 times.

The response of the density meter is perfectly linear within this range as shown by the calibration line in Figure 1.

Figure 1: Linearity of determination of the ASV by electronic densitometry between 0 and 5% level

8.2.  Specificity of the method

The second critical point of this method is the distillation of viscous musts containing small quantities of alcohol. In order to verify the specificity, known quantities of ethanol (from 0.25% vol to 5% vol) were added to CMs and grape sugar. The supplemented test specimens were distilled in the conditions defined earlier, then the distillates were analysed by electronic densitometry or by hydrostatic balance.

The results are shown in Table 1. The recovery rate is satisfactory, ranging between 88% and 99%. As shown by the line in Figure 2, the method is specific (slope in the vicinity of 1, intercept point in the vicinity of 0).

Table 1: Recovery rate for determination of the acquired ASV of CMs and Grape Sugar

Test specimen	Initial content (%vol.)	Added content (%vol.)	Recovered content (%vol.)	Recovery rate (%)
CM 1	0.00	0.25	0.22	88
CM 1	0.00	1.00	0.98	98
Grape Sugar (RCM) 1	0.00	1.00	0.94	94
Grape Sugar (RCM) 1	0.00	2.00	1.97	99
CM 2	0.00	0.50	0.44	88
Grape Sugar (RCM)2	0.00	5.00	4.94	99

 

Figure 2 : Specificity of determination of the acquired ASV of CMs and Grape Sugar

8.3.  Repeatability

The repeatability of the method was determined using 20 test specimens of CM or grape sugar supplemented with alcohol or not. Each CM or RCM test specimen was analysed 3 times, in order to ensure identical conditions. The repeatability limits obtained are as follows:

Table 2: Repeatability of determination of the acquired ASV of CMs and Grape Sugar
Repeatability for electronic densitometry		Calculated value
	Standard deviation	0.009
	CV or RSD as %	0.9%
	r limit	0.024 %vol.
	r limit as %	3%
Repeatability for Hydrostatic balance		Calculated value
	Standard deviation	0.013
	CV or RSD as %	1.7%
	r limit	0.038 %vol.
	r limit as %	5,3%

8.4.  Reproducibility

The reproducibility of the results is determined by analysing the same must twice, at different dates during a given period of time. The results are given in Table 3.

Table 3 - Reproducibility of determination of the acquired ASV of CMs and grape sugar
Reproducibility for electronic densitometry		Calculated value
	Standard deviation	0.043
	CV or RSD as %	3%
	R limit	0.12%vol.
	R limit as %	9%
Reproducibility for Hydrostatic balance		Calculated value
	Standard deviation	0.026
	CV or RSD as %	3.4%
	R limit	0.076%vol.
	R limit as %	10.6%

8.5.  Detection and quantification limits

The limits of detection (LD) and quantification (LQ) estimated based on the linearity study are as follows:

• LD = 0.01%vol.
• LQ = 0.05%vol.

The quantification limit was verified by analysis of musts having an ASV at a concentration level of 0.05%vol.

8.6.  Uncertainty

Uncertainty, evaluated based on the reproducibility standard deviation, is 0.10%vol.

OIV-MA-F1-05 Total acidity

Type IV method

 

1. Definition

The total acidity of the rectified concentrated must is the sum of its titrable acidities when it is titrated to pH 7 against a standard alkaline solution.

Carbon dioxide is not included in the total acidity.

2. Principle of the method

2.1.  Potentiometric titration or titration with bromothymol blue as an indicator and comparison with an end-point colour standard.

3. Reagents

3.1.  Buffer solutions

3.1.1.      pH 7.0:

monopotassium phosphate, (	107.3 g
1 M sodium hydroxide (NaOH) solution	500 ml
Water to	1000 ml

3.1.2.      pH 4.0

Solution of potassium hydrogen phthalate, 0.05 M, containing 10.211 g of potassium hydrogen phthalate (C₈H₅KO₄) per litre at 20 °C.

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

For example:

• pH 1.679 0.01 at 25°C
• pH 4.005 0.01 at 25°C
• pH 7.000 0.01 at 25°C
  2.   0,1 M sodium hydroxide (NaOH) solution.
  3.   4 g/l bromothymol blue indicator solution:

Bromothymol blue (	4 g
Neutral ethanol 96 % vol	200 ml

Dissolve and add:

Water free of	200 ml
1 M sodium hydroxide solution sufficient to produce blue-green colour (pH 7) approximately	7.5 ml
Water to:	1000 ml

4. Apparatus

 

4.1.  Potentiometer with scale graduated in pH values, and electrodes.

As a reminder, the glass electrode must be kept in distilled water. The calomel/saturated potassium chloride electrode must be kept in a saturated potassium chloride solution. A combined electrode is most frequently used: it should be kept in distilled water.

4.2.  Conical flask 100 ml.

5. Procedure

5.1.  Preparation of sample:

Introduce 200 g of accurately weighed rectified concentrated must. Make up to the mark with 500 ml water. Homogenize.

5.2.  Potentiometric titration

5.2.1.      Zeroing of the apparatus

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

5.2.2.      Calibration of the pH meter

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

5.2.3.      Method of measurement

Into a conical flask (4.4), introduce a 50 ml of the sample, prepared as described in 5.1.

Add about 10 ml of distilled water and then add the 0.1 M sodium hydroxide solution (3.2) from the burette until the pH is equal to 7 at 20 °C. The sodium hydroxide must be added slowly and the solution stirred continuously.

Let n ml be the volume of 0.1 M NaOH added.

5.3.  Titration with indicator (bromothymol blue)

5.3.1.      Preliminary test: end-point colour determination.

Into a conical flask (4.4) place 25 ml of boiled distilled water, 1 ml of bromothymol blue solution (3.3) and 50 ml of the sample prepared as in (5.1).

Add the 0.1 M sodium hydroxide solution (3.2) until the colour changes to blue-green.

Then add 5 ml of the pH 7 buffer solution (3.1)

5.3.2.      Measurement

Into a conical flask (4.4) place 30 ml of boiled distilled water, 1 ml of bromothymol blue solution (3.3) and 50 ml of the sample, prepared as described in 5.1.

Add 0.1 M sodium hydroxide solution (3.2) until the same colour is obtained as in the preliminary test above (5.3.1).

Let n ml be the volume of 0.1 M sodium hydroxide added.

6. Expression of results

6.1.  Method of calculation

The total acidity expressed in milliequivalents per kilogram of rectified concentrated must is given by:

The total acidity expressed in milliequivalents per kilogram of total sugars is given by:

P = % concentration (m/m) of total sugars.

It is recorded to one decimal place.

7. Characteristics of the method

Repeatability (r)

• r = 0.4 meq /kg total sugars

Reproducibility (R)

• R = 2.4 meq /kg total sugars

OIV-MA-F1-06 pH

Type IV method

 

1. Principle

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

2. Reagents

2.1.  Buffer solutions

2.1.1.      Saturated solution of potassium hydrogen tartrate, containing at least 5.7 g of potassium hydrogen tartrate per litre () at 20 °C. (This solution may be kept for up to two months by adding 0.1 g of thymol per 200 ml.) pH/temperature

• 3.57 at 20 °C
• 3.56 at 25 °C
• 3.55 at 30 °C

2.1.2.      Solution of potassium hydrogen phthalate, 0.05 M, containing 10.211 g of potassium hydrogen phthalate () per litre at 20 °C.

(Maximum keeping period, two months.)

pH/temperature

• 3.999 at 15 °C
• 4.003 at 20 °C
• 4.008 at 25 °C
• 4.015 at 30 °C

2.1.3.      Solution containing:

monopotassium phosphate,	3.402 g
dipotassium phosphate,	4.354 g
Water to	1000 ml

(maximum keeping period, two months)

pH/temperature

• 6.90 at 15 °C
• 6.88 at 20 °C
• 6.86 at 25 °C
• 6.85 at 30 °C

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

For example:

• pH 1.679 0.01 at 25°C
• pH 4.005 0.01 at 25°C
• pH 7.000 0.01 at 25°C

3. Apparatus
   1.   pH meter with a scale calibrated in pH units and enabling measurements to be made to at least 0.01.
   2.   Electrodes:
      1.       Glass electrode.
      2.       Calomel-saturated potassium chloride reference electrode
      3.       Or a combined electrode.

4. Procedure
   1.   Preparation of the sample for analysis

Dilute the rectified concentrated must with water to produce a concentration of 25 0.5 % (m/m) of total sugars (25° Brix).

If P is the percentage concentration (m/m) of total sugars in the rectified concentrated must, weigh a mass of:

2500/P

and make up to 100 g with water.

The water used must have a conductivity below 2 microsiemens per cm.

4.2.  Zeroing of the apparatus

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

4.3.  Calibration of the pH meter

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

4.4.  Determination

Dip the electrode into the sample to be analysed, the temperature of which should be between 20 and 25 °C and as close as possible to 20 °C.

Read the pH value directly off the scale.

Carry out at least two determinations on the same sample.

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

5. Expression of results

The pH of the 25 % (m/m) (25 ° Brix) solution of rectified concentrated must is quoted to two decimal places.

6. Characteristics of the method

Repeatability (r)

• r = 0.07

Reproducibility (R)

• R = 0.07

OIV-MA-F1-07 Sulphur dioxide

Type IV method

 

1. Definitions

Free sulphur dioxide is defined as the sulphur dioxide present in the must in the following forms:

The equilibrium between these forms is a function of pH and temperature:

represents molecular sulphur dioxide.

Total sulphur dioxide is defined as the total of all the various forms of sulphur dioxide present in the must, either in the free state or combined with its constituents.

2. Materials

Total sulphur dioxide is extracted from the previously diluted rectified concentrated must by entrainment at high temperature (approximately 100 °C).

2.1.  Reagents

2.1.1.      Phosphoric acid, 85 % (H₃PO₄) (ρ₂₀ = 1.71 g/ml).

2.1.2.      Hydrogen peroxide solution, 9.1 g H₂O₂/litre (three volumes).

2.1.3.      Indicator reagent:

methyl red	100 mg
methylene blue	50 mg
alcohol 50 % vol	100 ml

2.1.4.      Sodium hydroxide solution (NaOH), 0.01 M.

2.2.  Apparatus

2.2.1.      The apparatus used should conform to the diagram shown below, particularly with regard to the condenser.

Fig. 1 The dimensions given are in millimetres. The internal diameters of the four concentric tubes forming the condenser are 45, 34, 27 and 10 mm.

The gas feed tube to the bubbler B ends in a small sphere of 1 cm diameter with 20 0.2-mm diameter holes around its largest horizontal circumference. Alternatively, this tube may end in a frit glass plate which produces a large number of very small bubbles and thus ensures good contact between the liquid and gaseous phases.

The gas flow through the apparatus should be approximately 40 litres per hour. The bottle on the right of the diagram is intended to restrict the pressure reduction produced by the water pump to 20 to 30 cm of water. To regulate the vacuum to its correct value, a flowmeter with a semi-capillary tube should be installed between the bubbler and the bottle.

2.2.2.      A microburette.

3. Procedure
   1.   For rectified concentrated musts, use the solution obtained by diluting the sample to be analysed to 40 % (m/v) as indicated in the chapter 'Total acidity', section 5.1. Introduce 50 ml of this solution and 5 ml of phosphoric acid (2.2.1) into the 250 ml flask A of the entrainment apparatus. Connect the flask to the apparatus.
   2.   Place 2 to 3 ml of hydrogen peroxide solution (2.2.2) in the bubbler B, neutralize with the 0.01 M sodium hydroxide solution (2.2.4) and bring the must in the flask A to the boil using a small flame of 4 to 5 cm height which should directly touch the bottom of the flask. Do not put the flask on a metal plate but on a disc with a hole of approximately 30 mm diameter in it. This is to avoid overheating substances extracted from the sample that are deposited on the walls of the flask.

Maintain boiling while passing a current of air (or nitrogen). Within 15 minutes the total sulphur dioxide has been carried over and oxidized. Determine the sulphuric acid which has formed by titration with the 0.01 M sodium hydroxide solution (2.2.4).

Let n ml be the volume used.

4. Calculation

Total sulphur dioxide in milligrams per kilogram of total sugars (50 ml prepared test sample (3.1):

(where P = percentage concentration (m/m) of total sugars

5. Expression of results

 

Total sulphur dioxide is expressed in mg/kg of total sugars.

6. Characteristics of the method

 

Repeatability (r)

• 50 ml test sample < 50 mg/l; r = 1 x 250/P mg/kg of total sugars

Reproducibility (R)

• 50 ml test sample < 50 mg/l; R = 9 x 250/P mg/kg of total sugars

OIV-MA-F1-09 Specific methods for the analysis of grape sugar

Type I method

ANNEX A: TOTAL CATIONS

1. Principle

The test sample is treated by a strongly acid cation exchanger. The cations are exchanged with H⁺. Total cations are expressed by the difference between the total acidity of the effluent and that of the test sample.

2. Apparatus

2.1.  Glass column of internal diameter 10 to 11 mm and length approximately 300 mm, fitted with a drain tap.

2.2.  pH meter with a scale graduated at least in 0.1 pH units.

2.3.  Electrodes:

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

3. Reagents

3.1.  Strongly acid cation exchange resin in H⁺ form pre-swollen by soaking in water overnight.

3.2.  Sodium hydroxide solution, 0.1 M.

3.3.  Paper pH indicator.

The water used must be purified water for laboratories, with specific conductivity below 2 μS cm-1 at 20°C, for example EN ISO 3696 type II water.

4. Procedure

The pH meter must be calibrated according to the method OIV MA AS313-15

4.1.  Preparation of sample

Use the solution obtained by diluting the rectified concentrated must to 40% (m/v). Introduce 200 g of accurately weighed rectified concentrated must. Make up to the mark with 500 ml water. Homogenize.

4.2.  Total acidity of the rectified concentrated must

Titrate the acidity of the concentrated must in 100 ml of sample prepared as in 4.1 with the 0.1M sodium hydroxide solution until the pH is equal to 7 at 20 °C. The alkaline solution should be added slowly and the solution continuously shaken. Let n1 ml be the volume of 0.1 M sodium hydroxide solution used.

4.3.  Preparation of the ion exchange column

Introduce into the column approximately 10 ml pre-swollen ion exchanger in H⁺ form. Rinse the column with distilled water until all acidity has been removed, using the paper indicator to monitor this.

4.4.  Ion exchange

Pass 100 ml of the rectified concentrated must solution prepared as in paragraph 4.1 through the column at the rate of one drop every second. Collect the effluent in a beaker. Rinse the column with 50 ml of distilled water. Titrate the acidity in the effluent (including the rinse water) with the 0.1 M sodium hydroxide solution until the pH is 7 at 20°C. The alkaline solution should be added slowly and the solution continuously shaken. Let n2 ml be the volume of 0.1 M sodium hydroxide solution used.

5. Expression of the results

The total cations are expressed in milliequivalents per kilogram of total sugar to one decimal place.

5.1.  Calculations

Total acidity of the rectified concentrated must in milliequivalents per kilogram:

• a=2.5 n1

Acidity of the effluent expressed in milliequivalents per kilogram of rectified concentrated must:

• E= 2.5 n2.

Total cations in milliequivalents per kilogram of total sugars:

• P=percentage concentration (m/m) of total sugars.

5.2.  Repeatability (r)

• r = 0.3

OIV-MA-F1-11 Specific methods for the analysis of grape sugar

Type IV method

 

ANNEX D.2: HEAVY METALS

 

Determination of lead content by Inductively Coupled Plasma Mass Spectrometry (ICP-MS).

The analysis of Pb in rectified concentrated grape musts can also be performed applying the method OIV/OENO 344/2010 (Multielemental Analysis Using ICP-MS), with the following modification added at the end of point N°5 (Sample preparation):

5.      Sample preparation

This method can also be applied to the analysis of Pb in rectified concentrated grape musts. For this purpose, a prior mineralisation of the sample is required. A digestion of the samples in a close vessel microwave system is recommended. The following procedure is given as a way of example:

 

Ad 1 g of must, 2 ml of nitric acid (3.4) and 8 ml of water (3.1) in a microwave vessel, and apply the following microwave digestion programme:

Stage	ramp	°C	Hold
1	20 min	200	20 min

Once the samples have been digested, transfer them to a 50 ml plastic tube (4.6), dilute with water (3.1) to 30g and homogenize.

OIV-MA-F1-12 Specific methods for the analysis of grape sugar

Type II method

Determination of meso-inositol, scyllo-inositol and sucrose

 

1. Principle

Rectified Concentrated Must (RCM) is mainly composed of sugars, polyalcohols and water contained in grapes. All the other organic and mineral components are removed during the rectification process.

Meso-inositol, scyllo-inositol and sucrose are determined through gas chromatography (GC) following silanisation.

The sucrose possibly found in small amounts in the RCM is stable for some months since hydrolysis is greatly slowed down due to the absence of organic and mineral acids, which are removed during the rectification process, to a very low water content, and to a high level of glucose and fructose.

2. Reagents

2.1.  Xylitol (CAS no. 87-99-0)

Internal standard: (aqueous solution of at a precisely known concentration of about 10 g/L prepared at the time).

2.2.  Meso-inositol () (CAS no. 87-89-8)

2.3.  Scyllo-inositol () (CAS no. 488-59-5)

2.4.  Glucose (), fructose (), sucrose ()

2.5.  Bis(trimethylsilyl)trifluoroacetamide - (BSTFA) – () (CAS no. 25561-30-2)

Warning: This is a dangerous and inflammable product. Inflammable liquids and vapours may provoke serious skin burns and serious eye lesions. Wear gloves/protective clothing. Protect your eyes/face.

2.6.  Trimethylchlorosilane () – TMCS – (CAS no. 75-77-4)

Warning: This is a dangerous and inflammable product. Harmful to the skin. Provokes serious skin burns and serious ocular lesions. Toxic when inhaled. May irritate the respiratory tract. Keep away from sources of heat/sparks/free flames /heated surfaces. Do not smoke. Avoid inhaling the vapours. Wear gloves/protective clothing. Protect your eyes/face.

2.7.  Pyridine p.a. () (CAS no. 110-86-1)

Warning: This is a dangerous and inflammable product. Noxious when inhaled, in contact with the skin and when swallowed.

2.8.  Absolute ethanol () (CAS no. 64-17-5)

Warning: This is an inflammable product. Liquid and vapours easily inflammable. Keep away from sources of heat/sparks/free flames /heated surfaces. Do not smoke.

2.9.  Type 1 Water in conformity with ISO 3696 standard or deionised water with resistivity 18M cm

2.10.        The silanising reagent is also available in ready to use kits (e.g. HMDS+TMSCl+Pyridine 3:1:9 Supelco cod. 33038)

2.11.        Technical gases: nitrogen, hydrogen, helium and air for gas chromatography and for the dehydration phases

3. Apparatus

3.1.  Gas chromatograph

3.2.  Capillary column able to guarantee a minimum efficiency of N=250,000 plates/column for sucrose at 1 g/L

For example, OV-1 (25 m x 0.30 mm x 0.15 µm) or DB-5 (60 m x 0.25 mm x 0.10 μm).

Operating conditions (only as an example):

carrier gas: pure hydrogen or helium, for gas chromatography,

Hydrogen: Warning: This is an extremely inflammable gas. Store the container in a well-ventilated place and far from flames and sparks. – Do not smoke. Avoid the piling up of electrostatic loads.

carrier gas flow rate: about 2 mL/min,

injector temperature: 250 °C,

temperature of flame ionisation detector (FID): 300 °C,

programming of temperature: 1 minute at 160 °C, 4 °C/minute up to 260 °C, constant temperature of 260 ° C for 15 minutes,

splitter ratio: about 1:20,

auxiliary gases: pure hydrogen and air for gas chromatography

3.3.  Integrator

3.4.  Microsyringe: 10 μL

3.5.  Micropipettes: 10, 100, 400 and 1000 μ

3.6.  2 mL flasks with Teflon stopper

3.7.  Oven

3.8.  Technical balance, analytical balance able to ensure an accuracy of ± 0.1 mg

3.9.  Flasks of 50 and 100 mL

3.10.        Dryer

4. Procedure

4.1.  Preparation of the sample

In a 50 mL flask, weigh a quantity “p” of rectified concentrated must ranging between 4.9 and 5.1 g, noting the weight of the substance with the precision of 0.1 mg.

Then add 1 mL of xylitol standard solution (2.1) and bring to volume with water (2.9).

4.2.  Dehydration of the sample

After mixing, 100 μL of solution is taken and placed in a flask (3.6) where it is dried under a gentle stream of nitrogen.

100 μL of absolute ethanol (2.8) may be added to facilitate evaporation.

Note 1: In case a precise dose of sucrose is desired, the diluted solution should be prepared just before the silanisation, in order to limit hydrolysis of the sucrose in the diluted acqueous solution.

Note 2: The repeated measurements of the sucrose content have to be performed on diluted solutions prepared every time before every silanisation.

4.3.  Derivatization

The residue is carefully dissolved in 100 μL of pyridine (2.7) and 100 μL of bis (trimethylsilyl)trifluoroacetamide (2.5) and 10 μL of trimethylchlorosilane (2.6) are added. The flask is closed with the Teflon stopper and placed in the oven at 70 °C for 70 minutes.

Take the sealed flasks out of the oven and leave to cool in the dryer in the dark at room temperature for an hour before injecting into the gas chromatograph.

The substance conserved in the sealed flask and kept in the dryer in the dark at room temperature is stable for three days.

Note 3: If working with a silanisation kit, 400 μL of reagent should be used per 100 μL of the dehydrated and diluted sample, as described in 4.1.

Note 4: The silanisation is considered successful if the solution, after a sole phase, has a clear appearance or leaves a slight, white residue. There should not be a dark residue since this would indicate an excess of non-derivatised sugar or an aged silanising substance.

Note 5: Should there be a white suspension, wait until the solid matter deposits on the bottom without centrifuging.

4.4.  Gas chromatographic analysis

1 μL is taken with a syringe (3.4) and injected into the chromatograph with the aforementioned splitter.

The chromatogramme should not show a confluence of peaks (sign of a badly performed silanisation), but characteristic peaks as shown in the attached figures. (Fig.9-Fig.12).

4.5.  Peak integration criteria

Integrate the gas chromatographic peaks with respect to the horizontal baseline. In case of peaks that are not perfectly resolved, trace the horizontal baseline starting from the deeper valleys that delimit the peak in question. Trace a vertical line downwards starting from the valleys of the peaks up to the baseline to identify the peak area.

Do not use the valley-valley integration method.

An example of the application of these criteria is given in Figure 10 for the internal standard, in Figure 11 for the inositols and in Figure 12 for the sucrose.

Note 6: In case a precise dose of sucrose is desired, it is important to respect the integration criteria explained in paragraph 4.4. and shown in Figure 4.

5. Calculations

5.1.  Calculation of response factors

 Note: instead of the calculation of response factors a calibration curve can be used

5.1.1.     A solution is prepared containing:

• 60 g/L of glucose,
• 60 g/L of fructose,
• 1 g/L of meso-inositol,
• 1 g/L of sucrose,

(weigh 60 g of glucose and 60 g of fructose with precision 1 g; then 1 g of meso-inositol and 1 g of sucrose with precision 0.1 mg) and lastly bring to volume of 1 litre with water.

5.1.2.     Silanisation of the reference solution

Carry out the operations described in paragraph 4.1 starting with  5 mL of said solution in place of the 5 g of RMC

Take 5 mL of the solution and proceed as in paragraph 4.

5.1.3.     Gaschromatographic response factors

The results for meso-inositol and sucrose with respect to xylitol are calculated from the chromatogram.

In the case of scyllo-inositol, which has a retention time lying between the last peak of the anomeric form of glucose and the peak for meso-inositol (see Figure 11), use the same response factors achieved for meso-inositol.

Where: = area of the meso-inositol peak; = area of the sucrose peak; = area of the internal standard peak; = concentration of meso-inositol in mg/L; = concentration of sucrose in mg/L; = concentration of internal standard in mg/L, the following formula is true:

The solution for the calculation of the response factors has to be prepared and analysed on the same day (see note 1 of paragraph 4.1).

5.2.  Formulation of the results

Meso-inositol, scyllo-inositol and sucrose are expressed in mg/kg of Total Sugars (mg/kg TS) without decimals.

5.2.1.     Concentrations expressed in mg/L for the 10% (w/v) solution of RCM (4.1):

5.2.2.     Concentrations expressed in mg/kg of Total Sugars (mg/kg TS) for the meso-inositol and the scyllo-inositol, and for the sucrose in the RCM.

Indicating with “i” any of the three compounds:

Where “w” is the weighed amount in g of the RCM and “G” is the percentage of sugar of the RCM expressed in °Brix [or % (m/m) of sucrose]. The sugar percentage of the RCM sample should be measured using the OIV-MA-AS2-02 method.

6. Characteristics of the method

6.1.  Critical points

The method regards the analysis of sugars and polyalcohols found in extremely small quantities in a matrix of glucose and fructose in very high concentrations. It is thus necessary to verify the method’s capacity to furnish linear responses in the range of concentrations proposed and that are sufficiently accurate compared with known values.

Furthermore, the method provides for gas chromatographic analysis of the silanised compounds obtained through derivatisation of the sugars. These compounds are sensitive to humidity and tend to deteriorate with time. It is therefore important to verify the adequacy of the instructions regarding conservation and handling of these compounds.

Lastly, sucrose is subject to hydrolysis due to the quantity of residual water in the RCM (from 30 to 45%). However, the low acidity of the matrix and the high concentration of glucose and fructose slow down the hydrolysis process and allow accurate measurements to be carried out. It is therefore important to check that the analysis timeframes are fast compared to the hydrolysis in order to allow repeatable measurements of the sucrose.

6.2.  Linearity

A series of six synthetic samples were prepared (including the blank) containing a matrix of glucose and fructose obtained by weighing equal quantities of the two sugars in relation to a 60% (w/w) content of total sugars in the initial RCM.

To five of these synthetic samples, precise amounts in increasing quantities of meso-inositol and sucrose were added so as to achieve the concentrations shown in the following table:

	concentration added
No.	meso-inositol	sucrose
	(mg/kg TS)	(mg/kg TS)
1	0 (blank)	0 (blank)
2	214.7	427.3
3	420.0	857.7
4	840.2	1675.8
5	1727.0	3338.0
6	2514.0	6719.0

The samples were then diluted (4.1), the diluted solution was dehydrated (4.2), silanised (4.3) and analysed with the GC (4.4-4.5).

The samples were then silanised and analysed using GC. The results were verified in order of linearity, showing on the graph the ratio between the peak areas of the meso-inositol peaks and that of the internal standard (), and the ratio between the concentration of meso-inositol and of the internal standard (in mg/L), indicated by The GC analysis was conducted twice and the following data refer to the mean of the two values.

The same treatment was applied to the sucrose as follows

Fig. 1 Linearity of meso-inositol

The linearity of the meso-inositol is highly satisfactory (R>0.998) in the entire range of concentration studied.

Fig. 2 Linearity of sucrose

In relation to sucrose, the linear relationship hypothesised over the entire range of concentrations studied did not lead to a satisfactory correlation (R=0.967) but, by narrowing down the linearity field to the synthetic sample no. 5, the correlation became comparable to that of meso-inositol (R>0.997).

Following the instructions in par. 5.1, the following response factors were obtained:

RF rel. Meso-inositol/Xylitol I.S.	RF rel. Sucrose/Xylitol I.S.
1.04 0.03 (mean σ; n=4)	0.36 0.06 (mean σ; n=4)

6.3.  Specificity

The relationship between the added meso-inositol and that determined by GC is linear in the entire measurement range studied, and the slope of the line is very close to one and intercept is very close to zero.

Fig. 3 Specificity of meso-inositol

Also, the recovery is satisfactory at between 95 and 105%, as seen in the following table:

added (mg/kg TS)	determined by GC ±σ (n=2) (mg/kg TS)	Recovery (%)
0	0	-
214.7	213 2	99%
420.0	419 3	100%
840.2	852 20	102%
1727.0	1668 214	100%
2514.0	2587 36	99%

As to the specificity of the sucrose, the concentrations obtained from the calculation of the added sucrose and that determined by GC conform and are in a linear relationship with each other, with a slope of one and intercept of almost zero, on the condition, however, that the concentration range is more restricted compared to that studied.

The following graph shows that the linear relationship does not extend up to the last concentration level of about 6700 mg/kg TS

Fig. 4 Specificity of sucrose

Also, the recovery is satisfactory, at between 90 and 110%, excluding the higher concentration levels, as seen in the following table:

added (mg/kg TS)	determined by GC σ (n=2) (mg/kg TS)	Recovery (%)
0	0	-
427.3	423 16	99%
857.7	913 37	107%
1675.8	1852 344	111%
3338.0	3297 284	99%
6719.0	9220 19	137%

6.4.  Stability of sucrose in rectified concentrated must

A sample of the RCM with added sucrose was kept at room temperature and analysed at regular time intervals in order to see the incidence of the hydrolysis phenomenon of sucrose in RCM.              The results are summarised in the following table:

	t = 0 days	t=9 days	t=53 days
Meso-inositol (mg/kg TS)	2227	2100	2052
Scyllo-inositol (mg/kg TS)	424	430	394
Sucrose (mg/kg TS)	4631	5108	4969

Both the meso-inositol and the scyllo-inositol, as well as the sucrose, do not show significant variations in concentration compared to the initial value up to 53 days after the preparation.

This fact appears to be evident in the following graph

Fig. 5 Stability over time

6.5.  Stability of the silanised sample

The silanised product obtained as described in point 4.2 was conserved as described in the same paragraph. At 24-hour intervals the silanised sample was analysed with the gas chromatograph following the procedure set out in point 4.3 and the succeeding points.

The results are described in the following table:

	t=0 mean ± SD (n=3)	t=24 h mean SD (n=3)	t=48 h mean SD (n=3)	t=72 h mean SD (n=3)	t=96 h mean SD (n=3)
Meso-inositol (mg/kg TS)	2424 109	2347 44	2358 17	2453 39	2478 15
Scyllo-inositol (mg/kg TS)	261 7	254 2	256 4	257 3	264 1
Sucrose (mg/kg TS)	6233 971	6500 200	6633 58	6733 321	6600 436

There are no significant differences between the results obtained from the same silanised sample up to 4 days after silanisation, adopting the measures for the conservation of the silanised sample described in point 4.2.

This fact is evident in the following graphs:

Fig. 6 Stability of Meso-inositol

Fig. 7 Stability of Scyllo-inosito

Fig. 8 Stability of Sucrose

6.6.  Precision

Precision parameters obtained in the interlaboratory test conducted in April 2014 between 8 Italian laboratories. The Ring Test was performed on a sample with added sucrose at a concentration of 1 g of sucrose / 1 kg RCM

	sucrose	meso-inositol	scyllo-inositol
Number of participating laboratories	8	8	8
Number of accepted test results	23	23	23
Mean values (mg/kg TS)	1665	954	145
Repeatability
Repeatability standard deviation (Sr)	78	52	11
Relative repeatability standard deviation (%RSDr)	4.7	5.4	7.3
Repeatability limit (r)	219	146	29
HORRAT r = / RSD(R) Horwitz	0.9	1.0	1.0
Reproducibility
Reproducibility standard deviation (SR)	122	76	19
Relative reproducibility standard deviation (%RSDR)	7.4	8.0	13
Reproducibility limit (R)	343	213	55
RSD(R) Horwitz %	5.2	5.7	7.6
HORRAT R = / RSD(R) Horwitz	1.4	1.4	1.8
/	0.64	0.68	0.58

7. Bibliography

• Versini G., Dalla Serra A. and Margheri G. (1984). Polialcooli e zuccheri minori nei mosti concentrati rettificati. Possibili parametri di genuinità? Vignevini, 11(3), 41-47
• Monetti A., Versini G., Dalpiaz G. and Raniero F. (1996). Sugar adulterations control in concentrated rectified grape musts by finite mixture distribution analysis of the myo-inositol and scyllo-insitol content and the D/H methyl ratio of fermentative ethanol. Journal of Agricultural and Food Chemistry, 44-8: 2194-2210.

Figures

 

Fig. 9 Chromatogram of the reference solution for the calculation of response factors

 

Fig. 10 Chromatogram of an RCM sample – Internal Standard ZONE

 

Fig. 11 Chromatogram of an RCM sample - “Inositol” ZONE

Fig. 12 Chromatogram of an RCM sample –with 0.5 g/kg sucrose added

 

OIV-MA-F1-13 Specific methods for the analysis of grape sugar- Folin-Ciocalteu Index

Type IV method

1. Definition

The Folin-Ciocalteu Index is (IFC) the result obtained from the application of the method described below.

This method applies to rectified concentrated must (RCM).

2. Principle of the method

All phenolic compounds contained in RCM are oxidized by the Folin-Ciocalteu reagent. In RCM other reducing substances can interfere such as, for example, sugars and sulfur dioxide. This interference can be evaluated by measuring a "blank" devoid of phenolic substances, prepared by treating the corresponding sample with activated charcoal.

The Folin-Ciocalteu reagent is formed from a mixture of phosphotungstic acid (H₃PW₁₂O₄₀) and phosphomolybdic acid () which, after oxidation of the reducing substances present in the RCM, is reduced to a mixture of the blue oxides of tungsten () and molybdenum (). The blue coloration produced has a maximum absorption in the region of 750 nm.

3. Reagents

These must be of analytical reagent quality.

The water used must be distilled or water of equivalent purity.

3.1.  Chemicals

3.1.1.      Sodium tungstate dihydrate, (CAS Number: 10213-10-2);

3.1.2.      3.1.2 Sodium molybdate dihydrate, (CAS Number: 10102-40-6);

3.1.3.      Phosphoric acid solution 85 wt. % in , , ρ ₂₀ = 1.71 g/ml (CAS Number 7664-38-2);

3.1.4.      Hydrochloric acid, HCl, ρ ₂₀ = 1.2 g/ml (CAS Number 7647-01-0);

3.1.5.      Lithium sulfate monohydrate, (CAS Number: 10102-25-7);

3.1.6.      Bromine, (CAS Number: 7726-95-6);

3.1.7.      Sodium carbonate anhydrous, (CAS Number: 497-19-8);

3.1.8.      Activated charcoal decolorizing (CAS Number 7440-44-0).

3.2.  Folin-Ciocalteu reagent

This reagent is available commercially in a form ready for use.

It may be prepared as follows: dissolve 100 g of sodium tungstate (· 2) and 25 g of sodium molybdate (· 2) in 700 ml of distilled water. Add 50 ml of 85 % phosphoric acid (ρ ₂₀ = 1.71 g/ml) and 100 ml of concentrated hydrochloric acid (ρ ₂₀ = 1.19 g/ml). Bring to the boil and boil for 10 hours under reflux conditions. Then add 150 g of  lithium sulphate () and a few drops of bromine and boil once more for 15 minutes. Allow to cool and make up to one liter with distilled water.

3.3.  Anhydrous sodium carbonate, Na₂CO₃, made up into a 20 % w/v solution.

4. Apparatus

Normal laboratory apparatus, particularly:

4.1.  100 ml volumetric flasks.

4.2.  Spectrophotometer capable of operating at 750 nm.

4.3.  Technical balance ( 0.01 g)

4.4.  50 ml graduated cylinder;

4.5.  50 ml beaker;

4.6.  Magnetic stirrer;

4.7.  Rapid-filtration filter papers;

4.8.  5 ml calibrated pipette;

4.9.  Optical-glass cuvette;

4.10.        Ultrasonic bath

5. Procedure

Good repeatability of results is achieved by using scrupulously clean apparatus (volumetric flasks and spectrophotometer cells).

5.1.  Preparation of sample

Let P = percentage (m/m) of total sugars in the rectified concentrated must.

Dilute the RCM with distilled water until a total sugar concentration of  the solution equal to 25 0.5 % (m/m) (25° Brix): weigh a mass in g equal to 2500/P into a volumetric flask (4.1) and make up to 100 g with water.

5.2.  Preparation of a blank sample

Take 10 ml from solution 5.1 using the graduated cylinder (4.4), put into a 50 ml beaker (4.5) and add 0,5g of activated carbon (3.1.8).

Leave in contact for 15 minutes with the aid of a magnetic stirrer (4.6), and then filter the solution thought a rapid-filtration paper (4.7).

5.3.  Color reaction

5.3.1.      Color development in the sample

Introduce the following reagents or solutions into a 100 ml volumetric flask (4.1) strictly in the order given below:

• 5 ml of the sample (5.1) using a pipette (4.8),
• 50 ml of distilled water by means of a graduated cylinder (4.4) ,
• 5 ml of Folin-Ciocalteu reagent (3.2) using a pipette (4.8),
• 20 ml of sodium carbonate solution (3.2) by means of a graduated cylinder (4.4).

Make up to 100 ml with distilled water. Stir to homogenize. Wait 30 minutes for the reaction to stabilize.

During this period the flasks should be put in an ultrasonic bath (4.10) for 15 minutes to remove any bubbles that may interfere in the spectrophotometric measurement.

5.3.2.      Color development in blank

Proceed as described in (5.3.1) by replacing the sample (5.1) with a blank (5.2).

5.4.  Measurement

Determine the absorbance at 750 nm through an optical-glass cuvette with a path length of 1 cm, with respect to a blank (5.2).

If the absorbance is not around 0.3 an appropriate dilution should be made until the measured absorbance falls to around 0.3.

6. Expression of results

 

6.1.  Method of calculation

The result is expressed in the form of an index obtained by multiplying the absorbance by 16, with one decimal point.

The FCI value calculated should be corrected for the blank according to the following formula:

FCI measured at 25°Brix = 16 d (A – )

where:

• A = absorbance at 750 nm measured on the sample at 25 ± 0.5 % (m/m) (5.1)
• = absorbance at 750 nm measured on the blank (5.2)
• d = an appropriate factor if a dilution has been done (5.4)

7. Method performance

The method was checked in the ICQRF laboratory in Conegliano (Italy) analyzing two RCM samples. Each sample was further enriched in SO₂ at a concentration 5 mg/Kg TS (mg per Kg of total sugar). The samples were analyzed three times:

Samples	FC index at 25°Brix			Media	SD	r=2.8xSD
RCM 1	5.4	4.9	5.3	5.2	0.3	0.8
RCM 2	2.2	2.4	1.9	2.2	0.2	0.7

Further data was provided from the ASAE laboratory (Portugal):

Number of samples	Concentration range	Sr	r	SR	R
21	From 1 to 5.3	0.25	0.7
21	From 1 to 5.0			0.91	2.5