Determination of the 13C/12C isotope ratios of glucose, fructose, glycerol, ethanol in production of vitivinicultural origin by high-performance liquid chromatography coupled to isotope ratio mass spectrometry (Type-II-and-III)
OIV-MA-AS311-09 Determination of the 
isotope ratios of glucose, fructose, glycerol, ethanol in production of vitivinicultural origin by high-performance liquid chromatography coupled to isotope ratio mass spectrometry
Type II and III method
- Scope of application
This method applies to products of vitivinicultural origin.
This method is:
- type II for glucose, fructose and glycerol,
- type III for ethanol.
- Principle
The samples are injected into the HPLC instrument after any necessary dilution and filtration. After oxidation in a liquid interface, the 13C/12C isotope ratio of the compounds is determined using isotope ratio mass spectrometry. This liquid interface, symbolised by the acronym “co”, permits the chemical oxidation of the organic matter into CO2. HPLC-co-IRMS coupling can therefore be used to determine the isotope ratio of the following compounds simultaneously: glucose, fructose, glycerol and ethanol.
- Reagents
3.1. Pure water - resistivity > 18 M cm, HPLC quality
3.2. Ammonium persulfate - analytical purity – [CAS No.: 7727-54-0]
3.3. Orthophosphoric acid (concentration 85%) – analytical purity - [CAS No.: 7664-38-2]
3.4. Analytical-grade helium, used as a carrier gas [CAS No.: 07440-59-7]
3.5. Reference gas: analytical-grade 
(carbon dioxide), used as a secondary reference gas [CAS No.: 00124-38-9]
3.6. International standards
- Equipment
4.1. Everyday laboratory equipment
4.2. High-performance liquid chromatography instrument
4.3. Liquid interface for the oxidation of eluted compounds
4.4. Isotope ratio mass spectrometer
- Analysis of the samples
5.1. Preparation of the samples
Depending on the sugar, glycerol and ethanol contents, the samples should be diluted with the water (3.1) beforehand in order to obtain a concentration which is observable under the experimental conditions. Depending on the concentrations of the compounds, two measurements are needed with different dilutions.
5.2. Example of analytical conditions
Total analysis duration: 20 minutes
As an indication, the dilution of grape juices and wines is around 1:200, while that of concentrated musts is approximately 1:500.
HPLC:
Column: carbohydrate-type column (e.g. 700-CH Carbohydrate column, HyperRez XP Carbohydrate H+)
Injection volume: 25 μl
Mobile phase: water (3.1)
Flowrate: 0.4 mL/min
Column T°: 80 °C
Liquid Interface:
Solution of ammonium persulfate (3.2) (15% in mass) and orthophosphoric acid (2.5% in volume)
Peristaltic pump flow: 0.6 mL/min
Heater temperature: 93 °C
Flow of the helium carrier gas: 15 mL/min
Helium flow for drying: 50 mL/min
IRMS:
Trap current: 300 μA
5.3. Example chromatogram
Chromatogram of a sweet wine analysed using HPLC-co-IRMS
- Determination of isotope ratios
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The reference gas, CO2, is calibrated from international commercial standards. The isotope ratios are expressed in ‰ in relation to the Pee Dee Belemnite (PDB) and are defined as:
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*
Where: Sam = sample; St = standard; R = 13C/12C isotope ratio
- Method characteristics
The characteristics of the method for the measurement of the 
C isotope ratios of glucose, fructose, glycerol and ethanol by HPLC-co-IRMS have been determined from the results obtained from an inter-laboratory analysis of four samples (dry wine, sweet wine, grape juice and rectified concentrated must). The results obtained for each compound analysed and each type of matrix are annexed.
- Bibliography
- Cabanero, AI.; Recio, JL.; Rupérez, M. (2008) Isotope ratio mass spectrometry coupled to liquid and gas chromatography for wine ethanol characterization. Rapid Commun. Mass Spectrom. 22: 3111-3118.
- Cabanero, AI.; Recio, JL.; Rupérez, M. (2010) Simultaneous stable carbon isotopic analysis of wine glycerol and ethanol by liquid chromatography coupled to isotope ratio mass spectrometry. J. Agric. Food Chem. 58: 722-728.
- Guyon, F.; Gaillard, L.; Salagoïty, MH.; Médina, B. (2011) Intrinsic Ratios of Glucose, Fructose, Glycerol and Ethanol 13C/12C Isotopic Ratio Determined by HPLC-co-IRMS: Toward Determining Constants for Wine Authentication. Anal. Bioanal. Chem. 401:1551-1558
Annex Statistical treatment of the HPLC-co-IRMS inter-laboratory analysis for the determination of the precision of the method (repeatability and reproducibility)
List of laboratories in alphabetical order of country of origin.
|
Country |
Laboratory |
|
Belgium |
IRMM |
|
China |
CNRIFFI |
|
Czech Republic |
SZPI |
|
France |
SCL-33 |
|
Germany |
INTERTEK |
|
Germany |
UNI DUE |
|
Germany |
ELEMENTAR |
|
Germany |
QSI |
|
Germany |
LVI |
|
Italy |
FLORAMO |
|
Japan |
AKITA Univ. |
|
Spain |
MAGRAMA |
Responses:
12 laboratories / 14 responses
Treatment of the results of inter-laboratory analyses according to ISO 5725-2
Samples:
- 1 dry wine (Wine A)
- 1 sweet wine (Wine B)
- 1 rectified concentrated must (RCM)
- 1 grape juice
Analytical conditions:
Each sample was analysed in duplicate (repeatability) and double blind (reproducibility)
Expression of results in % vs. PDB
Precision of the glucose measurement
Repeatability and reproducibility
|
Wine B |
RCM |
Grape juice |
|
|
Number of laboratories |
12 |
12 |
12 |
|
Number of responses |
14 |
13 |
14 |
|
Number of responses retained (elimination of outliers) |
13 |
13 |
12 |
|
Minimum value |
-26.33 |
-25.04 |
-25.78 |
|
Maximum value |
-23.72 |
-23.74 |
-24.62 |
|
Mean value |
-25.10 |
-24.24 |
-25.19 |
|
Repeatability variance |
0.02 |
0.01 |
0.01 |
|
Repeatability standard deviation (Sr) |
0.14 |
0.10 |
0.09 |
|
Repeatability limit (r ‰) |
0.40 |
0.29 |
0.24 |
|
Reproducibility variance |
0.39 |
0.14 |
0.11 |
|
Reproducibility standard deviation (SR) |
0.62 |
0.38 |
0.33 |
|
Reproducibility limit (R ‰) |
1.77 |
1.06 |
0.94 |
Precision of the fructose measurement
Repeatability and reproducibility
|
Wine B |
RCM |
Grape juice |
|
|
Number of laboratories |
12 |
11 |
12 |
|
Number of responses |
14 |
13 |
14 |
|
Number of responses retained (elimination of outliers) |
13 |
13 |
13 |
|
Minimum value |
-25.56 |
-24.19 |
-25.33 |
|
Maximum value |
-24.12 |
-23.19 |
-23.98 |
|
Mean value |
-24.87 |
-23.65 |
-24.56 |
|
Repeatability variance |
0.02 |
0.03 |
0.02 |
|
Repeatability standard deviation (Sr) |
0.14 |
0.16 |
0.14 |
|
Repeatability limit (r ‰) |
0.40 |
0.46 |
0.39 |
|
Reproducibility variance |
0.15 |
0.10 |
0.18 |
|
Reproducibility standard deviation (SR) |
0.39 |
0.32 |
0.42 |
|
Reproducibility limit (R ‰) |
1.10 |
0.90 |
1.19 |
Precision of the glycerol measurement
Repeatability and reproducibility
|
Wine A |
Wine B |
|
|
Number of laboratories |
12 |
12 |
|
Number of responses |
12 |
12 |
|
Number of responses retained (elimination of outliers) |
11 |
11 |
|
Minimum value |
-32.91 |
-30.74 |
|
Maximum value |
-30.17 |
-28.27 |
|
Mean value |
-31.75 |
-29.54 |
|
Repeatability variance |
0.13 |
0.04 |
|
Repeatability standard deviation (Sr) |
0.36 |
0.19 |
|
Repeatability limit (r ‰) |
1.03 |
0.55 |
|
Reproducibility variance |
0.57 |
0.37 |
|
Reproducibility standard deviation (SR) |
0.76 |
0.61 |
|
Reproducibility limit (R ‰) |
2.14 |
1.72 |
Precision of the ethanol measurement
Repeatability and reproducibility
|
Wine A |
Wine B |
|
|
Number of laboratories |
12 |
12 |
|
Number of responses |
11 |
12 |
|
Number of responses retained (elimination of outliers) |
10 |
12 |
|
Minimum value |
-27.85 |
-27.60 |
|
Maximum value |
-26.50 |
-26.06 |
|
Mean value |
-27.21 |
-26.82 |
|
Repeatability variance |
0.03 |
0.03 |
|
Repeatability standard deviation (Sr) |
0.16 |
0.17 |
|
Repeatability limit (r ‰) |
0.47 |
0.47 |
|
Reproducibility variance |
0.16 |
0.23 |
|
Reproducibility standard deviation (SR) |
0.40 |
0.47 |
|
Reproducibility limit (R ‰) |
1.14 |
1.34 |