OIV-MA-VI-23 Method for ¹⁸O/¹⁶O isotope ratio determination of water in wine vinegar using isotopic mass spectrometry

Type II method

1. Object/Scope of application

The method enables the determination of the ¹⁸O/¹⁶O isotope ratio of water in wine vinegar after equilibration with CO₂, using isotope ratio mass spectrometry (IRMS).

2. Definitions

 

¹⁸O/¹⁶O:Isotope ratio of oxygen 18 to oxygen 16 in a given sample.

δ¹⁸O_V-SMOW:Relative scale for the expression of the isotope ratio of oxygen 18 to oxygen 16 in a given sample. δ¹⁸O_V-SMOW is calculated according to the following equation:

using the V-SMOW standard as a reference for the relative δ scale.

• BCR: Community Bureau of Reference
• IAEA: International Atomic Energy Agency (Vienna, Austria)
• IRMM: Institute for Reference Materials and Measurements
• IRMS: Isotope Ratio Mass Spectrometry
• m/z: Mass to charge ratio
• NIST: U.S. National Institute of Standards & Technology
• RM: Reference Material
• V-SMOW: Vienna Standard Mean Ocean Water (¹⁸O/¹⁶O = R_V-SMOW = 0.0020052)
• GISP: Greenland Ice Sheet Precipitation
• SLAP: Standard Light Antarctic Precipitation

3. Principle

The technique described below is based on the isotopic equilibration of water in samples of wine vinegar with a CO₂ gas standard, according to the following isotopic exchange reaction:

After equilibration, the carbon dioxide in the gaseous phase is used to analyse the ¹⁸O/¹⁶O isotopic ratio by means of Isotopic Ratio Mass Spectrometry (IRMS).

4. Reagents and materials

The materials and consumables depend on the method used (see point 6).

The following reference materials, working solutions and consumables can be used:

4.1. Reference materials

Name	Issuing organisation	δ18O according to V-SMOW
V-SMOW, RM 8535	IAEA / NIST	0 ‰
BCR-659	IRMM	-7.18 ‰
GISP, RM 8536	IAEA / NIST	-24.78 ‰
SLAP, RM 8537	IAEA / NIST	-55.5 ‰

4.2. Working solutions

4.2.1.    Carbon dioxide (or carbonic anhydride) as a secondary reference gas for measurement (CAS N° 00124-38-9).

4.2.2.    Carbon dioxide used for equilibration (depending on the instrument this gas could be the same as that which is listed in 5.2.1 or, in the case of cylinders with continuous flow systems, a helium-carbon dioxide mixture can also be used).

4.2.3.    Working solutions with δ¹⁸O_V-SMOW values calibrated according to international reference materials.

4.3. Consumables

Helium for analysis (CAS N° 07440-59-7)

 

5. Apparatus

5.1. Isotope ratio mass spectrometer

The Isotope ratio mass spectrometer (IRMS) enables the determination of the relative contents of ¹⁸0 and ¹⁶O in CO₂ gas with an internal precision of 0.05‰. Internal precision is defined here as the difference between two measurements of the same sample of CO₂.

The mass spectrometer designed for the determination of the isotopic composition of CO₂ gas is generally equipped with a triple collector capable of simultaneously measuring the following ion currents:

m/z =	44 (12C16O16O)
m/z =	45 (13C16O16O and 12C17O16O)
m/z =	46 (12C16O18O, 12C17O17O and 13C17O16O)

When measuring the corresponding intensities, the ¹⁸O/¹⁶O isotopic ratio is determined from the m/z = 46 and m/z = 44 intensity ratios after conducting a series of corrections for the ¹²C¹⁷O¹⁷O and ¹³C¹⁷O¹⁶O isobaric species, whose contributions can be calculated from the actual intensity observed for m/z= 45 and from the usual isotopic abundances for ¹³C and ¹⁷O in nature.

The isotope ratio mass spectrometer must either be equipped with:

• a double entry system (dual inlet system) to alternately measure the sample and a reference sample,
• or a continuous flow system that transfers quantitatively the CO₂ from the sample vials after equilibration and the CO₂ standard gas into the mass spectrometer.

5.2. Equipment and materials

All equipment and materials used must satisfy the stated requirements of the method or of the apparatus used (as specified by the manufacturer). However, other equipment and materials of equivalent performance can also be used.

5.2.1.    Vials with septa appropriate for the system used

5.2.2.    Volumetric pipettes with appropriate tips

5.2.3.    Temperature control system to carry out the isotopic exchange reaction at a constant temperature (typically within 1°C)

5.2.4.    Vacuum pump (if needed for the system used)

5.2.5.    Autosampler (if needed for the system used)

5.2.6.    Syringes for sampling (if needed for the system used)

5.2.7.    GC Column to separate CO₂ from other elementary gases (if needed for the system used)

5.2.8.    Water removal device (e.g. cryo-trap or selective permeable membranes)

6. Preparation of the samples

Wine vinegar samples as well as reference materials are used for analysis without any pre-treatment. If the sample contains impurities, it should be filtered with a 0.22 μm pore diameter filter.

Preferably, the reference materials used for calibration and data correction should be placed at the beginning and at the end of each series and inserted after every ten samples.

7. Procedure

The procedure generally used for the determination of the ¹⁸O/¹⁶O isotope ratio by means of equilibration of water with a CO₂ working solution and subsequent analysis by IRMS is presented below. These procedures can be altered according to the apparatus and equipment used, given that various kinds of equilibration devices with different functions are available. The two main technical procedures for introduction of CO₂ into the IRMS provide a dual inlet system and a continuous flow system respectively.

Note: all values given for volumes, temperatures, pressures and time periods are only indicative. Appropriate values must be obtained from specifications provided by the manufacturer or determined experimentally.

7.1. Manual equilibration

A defined volume of the sample/working solution is transferred into a flask using a pipette. The flask is then attached tightly to the manifold.

Each manifold is cooled down to below –80°C to deep-freeze the samples (manifolds equipped with capillary opening tubes do not require this freezing phase). Subsequently, the whole system is evacuated.

After reaching a stable vacuum, the CO₂ resulting from the working solution in the round bottom flasks is distributed. For the equilibration process, each manifold is placed in a water-bath at 25°C ( 1°C) for 12 hours (overnight). It is crucial that the temperature of the water-bath is kept constant and homogeneous.

After the equilibration process is complete, the resulting CO₂ is transferred from the flasks to the sample side bellow of the dual inlet system. The measurements are performed by comparing the isotope ratios of the CO₂ contained in the sample side with those of the CO₂ contained in the working solution side (gas

reference standard: CO₂) of the dual inlet. This approach is repeated till the last sample of the sequence has been measured.

7.2. Use of automatic equilibration apparatus

A defined volume of the sample/working solution is transferred into a round bottom flask using a pipette. The flasks containing the sample are attached to the equilibration apparatus and cooled down to below –80°C to deep-freeze the samples (systems equipped with capillary opening tubes do not require this freezing step). Subsequently, the whole system is evacuated.

After reaching a stable vacuum, the CO₂ resulting from the working solution in the round bottom flasks is distributed. Equilibrium is reached at a temperature of 22 1°C after a minimum period of 5 hours with moderate stirring (if possible). Since the equilibration duration depends on various parameters (e.g. the flask geometry, temperature, amount of stirring), the minimum equilibrium time required should be determined experimentally.

After the equilibration process is complete, the resulting CO₂ is transferred from the flasks to the sample -side bellows of the dual inlet system. Analysis is carried out by comparing the isotope ratios of the CO₂ contained in the sample side with those of the CO₂ contained in the working-solution side of the dual inlet. This approach is repeated till the last sample of the sequence has been measured.

7.3. Manual preparation and automatic equilibration and analysis using a dual inlet system coupled to a continuous flow IRMS

A defined volume of sample/working solution (e.g. 200 μL) is transferred to a vial using a pipette. The open vials are then placed in a closed chamber filled with the CO₂ used for equilibration (5.2.2). After several purges to eliminate any trace of air, the vials are closed and then placed on the thermal tray of the autosampler. The equilibration is reached after at least 8 hours at 40°C. Once the process of equilibration is complete, the CO₂ obtained is dried and then transferred into the sample side of the dual inlet system. The measurements are performed by comparing the isotope ratios of the CO₂ contained in the sample side and the working solution side (CO₂ reference standard gas) of the dual inlet several times. This process is repeated until the last sample of the sequence has been measured.

7.4. Use of automatic equilibration apparatus by means of a continuous flow system

A defined volume of the sample/working solution is transferred into a vial using a pipette. The sample vials are placed into a temperature controlled tray.

A mixture of He and CO₂ is added to the vials using a gas syringe. The CO₂ remains in the top of the vials for equilibration.

Equilibrium is reached at a typical temperature of 30 1°C after a minimum period of 18 hours.

After the equilibration process is complete, the resulting CO₂ is transferred by means of the continuous flow system into the mass spectrometer. The CO₂ gas standard is also introduced into the IRMS by means of the continuous flow system. The measurement is carried out according to the specific protocol for each kind of equipment.

8. Calculations

The intensities for m/z = 44, 45, 46 are recorded for each sample and reference material analysed in a series of measurements. The ¹⁸O/¹⁶O isotope ratios are then calculated by a computer, using the IRMS apparatus’ software, according to the principles explained in point 6.1. In practice, the ¹⁸O/¹⁶O isotope ratios are measured against a working solution previously calibrated against the V-SMOW. Changes in the instrumental conditions while measuring on line may entail small variations. If this occurs, the δ ¹⁸O of the samples must be corrected according to the difference in the δ ¹⁸O value from the working solution and its assigned value, which was calibrated beforehand against V-SMOW. The correction that is applied to the samples varies in a linear fashion with respect to the values of the two working solutions that precede and follow the samples. Indeed, the working solution must be measured at the beginning and at the end of all sample series. A correction can therefore be calculated for each sample using linear interpolation between two values (the difference between the assigned value and the measured value of the working solution).

The final results are presented as δ¹⁸O_V-SMOW values expressed in ‰.

δ¹⁸O_V-SMOW values are calculated according to the following equation:

The δ¹⁸O value normalized with respect to the V-SMOW/SLAP scale is calculated according to the following equation:

The δ¹⁸O_V-SMOW value accepted for SLAP is -55.5‰ (see point 5.1).

9. Validation of the method

An international collaborative study was carried out (Thomas and Jamin, 2009) for validation of the method.

The repeatability limit (r) is, on average, 0.15‰.

The reproducibility limit (R) is, on average, 0.54‰.

The data and results of the international collaborative study are presented in Annex A.

10. Bibliography

• OIV-MA-AS1-08: Reliability of analytical results (Resolution 5/99)
• OIV-MA-AS1-09: Protocol for the design, conducts and interpretation of collaborative studies (Resolution 6/2000)
• OIV-MA-AS2-12: Method for ¹⁸O/¹⁶O isotope ratio determination of water in wines and must
• IUPAC Protocol in W. Horwitz, Pure Appl. Chem. 67 (2) (1995) 331
• Thomas, F., Jamin, E. (2009). (2)H NMR and (13)C-IRMS analyses of acetic acid from vinegar, (18)O-IRMS analysis of water in vinegar: International collaborative study report. Analytica Chimica Acta, 649, 98-105

Annex A: Results of the international collaborative study

 

This document sets out the results of the study for the validation of the method of analysis of the ¹⁸O/¹⁶O ratio of water in wine vinegar (Thomas and Jamin 2009).

The study was carried out in accordance with documents OIV-MA-AS1-08 and OIV-MA-AS1-09.

1. Participating laboratories

14 laboratories participated in the study:

Bundesinstitut für Risikobewertung, Berlin	Germany
Central Science Laboratory, York	United Kingdom
Custom Technical Laboratory, Prague	Czech Republic
Chemical Institute of the Hungarian Customs and Finance Guard, Budapest	Hungary
Eurofins, Nantes	France
FEM-IASMA, San Michele all’Adige, Trento	Italy
Joint Research Center, Ispra	Italy
Arbitral Agroalimentario Del MAPA, Madrid	Spain
Landesuntersuchungsamt, Speyer	Germany
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Wurzburg	Germany
Service Commun des Laboratoires, Bordeaux	France
Service Commun des Laboratoires, Montpellier	France
Service Commun des Laboratoires, Paris	France
Unione Italiana Vini, Verona	Italy

2. Samples

The study was carried out on 5 double-blind samples: three wine vinegar samples (B, C, D), an alcohol vinegar sample (A) and a wine vinegar sample with 20% of A (Thomas and Jamin, 2009).

For each sample, the δ¹⁸C of the water was analysed. The results are indicated in the following table.

3. Statistical evaluation

Statistical calculations were performed according to the OIV-MA-AS1-09 protocol, which is based on Horwitz’ IUPAC protocol. Outliers were removed in the following way: Cochran tests for removal of the laboratories with the highest variance; single- and pair-value Grubbs tests for individual or paired individual outliers; then more Cochran tests, etc., keeping a proportion of outliers <2/9.  The standard deviations of repeatability (sr) and of reproducibility (sR) were then calculated for each material from valid results pairs from the blind duplicates.

For more information, refer to the publication by Thomas and Jamin, 2009.

Description of the sample	N° of valid results	N° of replicates	δ13C average (‰)	sr (‰)	Repeatability limit r (2.8×Sr) ‰	sR (‰)	Reproducibility limit R (2.8× SR) ‰
A	8	2	-6.96	0.06	0.17	0.22	0.62
B	8	2	-0.86	0.08	0.22	0.21	0.59
C	8	2	-1.38	0.02	0.06	0.2	0.56
B+20%A	8	2	-1.79	0.05	0.14	0.2	0.56
D	5	2	-1.23	0.05	0.14	0.14	0.39

The average repeatability limit (r) was 0.15‰, and the average reproducibility limit (R) was 0.54‰, comparable to the values observed for wine alcohol (OIV-MA-AS2-12).