ANNEX
F1X.DETERMINATION OF THE FOLLOWING WOOD COMPOUNDS IN SPIRIT DRINKS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): FURFURAL, 5-HYDROXYMETHYLFURFURAL, 5-METHYLFURFURAL, VANILLIN, SYRINGALDEHYDE, CONIFERALDEHYDE, SINAPALDEHYDE, GALLIC ACID, ELLAGIC ACID, VANILLIC ACID, SYRINGIC ACID AND SCOPOLETIN
1.Scope
The method pertains to the determination of furfural, 5-hydroxymethylfurfural, 5-methylfurfural, vanillin, syringaldehyde, coniferaldehyde, sinapaldehyde, gallic acid, ellagic acid, vanillic acid, syringic acid and scopoletin, by high-performance liquid chromatography.
2.Normative reference
Analytical method recognised by the General Assembly of the International Organisation of Vine and Wine (OIV) and published by OIV under the reference OIV-MA-BS-16: R2009.
3.Principle
Determination by high-performance liquid chromatography (HPLC), with detection by ultraviolet spectrophotometry at several wavelengths and by spectrofluorimetry.
4.Reagents
The reagents must be of analytical quality. The water used must be distilled water or water of at least equivalent purity. It is preferable to use microfiltered water with a resistivity of 18,2 M Ω.cm.
4.1.
96 % vol. alcohol.
4.2.
HPLC-quality methanol (Solvent B).
4.3.
Acetic acid diluted to 0,5 % vol. (Solvent A).
4.4.
Mobile phases: (given as an example only).
Solvent A (0,5 % acetic acid) and solvent B (pure methanol). Filter through a membrane (porosity 0,45 μm). Degas in an ultrasonic bath, if necessary.
4.5.
Reference standards of 99 % minimum purity: furfural, 5-hydroxymethyl furfural, 5-methylfurfural, vanillin, syringaldehyde, coniferaldehyde, sinapaldehyde, gallic acid, ellagic acid, vanillic acid, syringic acid and scopoletin.
4.6.
Reference solution: the standard substances are dissolved in a 50 % vol. aqueous-alcoholic solution. The final concentrations in the reference solution should be of the order of:
furfural: 5 mg/l; 5-hydroxymethyl furfural: 10 mg/l; 5-methylfurfural 2 mg/l; vanillin: 5 mg/l; syringaldehyde: 10 mg/l; coniferaldehyde: 5 mg/l; sinapaldehyde: 5 mg/l; gallic acid: 10 mg/l; ellagic acid: 10 mg/l; vanillic acid: 5 mg/l; syringic acid: 5 mg/l; scopoletin: 0,5 mg/l.
5.ApparatusStandard laboratory apparatus
5.1.
A high-performance liquid chromatograph capable of functioning in binary gradient mode and equipped with:
- 5.1.1.
A spectrophotometric detector capable of measuring at wavelengths from 260 to 340 nm. It is however preferable to work with a multiple wavelength detector with a diode array or similar, in order to confirm the purity of the peaks.
- 5.1.2.
A spectrofluorimetric detector — excitation wavelength: 354 nm, emission wavelength: 446 nm (for the trace determination of scopoletin; which is also detectable at 313 nm by spectrophotometry).
- 5.1.3.
An injection device capable of introducing 10 or 20 μl (for example) of the test sample.
- 5.1.4.
A high-performance liquid chromatography column, RP C18 type, 5 μm maximum particle size.
5.2.
Syringes for HPLC.
5.3.
Device for membrane-filtration of small volumes.
5.4.
Integrator-computer or recorder with performance compatible with the entire apparatus, and in particular, it must have several acquisition channels.
6.Procedure
6.1.Preparation of the solution to be injected
The reference solution and the spirit drink are filtered, if necessary, through a membrane with a maximum pore diameter of 0,45 μm.
6.2.
Chromatographic operating conditions: carry out the analysis at ambient temperature by means of the equipment described in (5.1) and using the mobile phases (4.4) with a flow of approximately 0,6 ml per minute following the gradient below (given as an example only)
Time: 0 min 50 min 70 min 90 min
solvent A (water-acid): 100 % 60 % 100 % 100 %
solvent B (methanol): 0 % 40 % 0 % 0 %
Note that in certain cases this gradient should be modified to avoid co-elutions.
6.3.Determination
6.3.1.
Inject the reference standards separately, then mixed.
Adapt the operating conditions so that the resolution factors of the peaks of all the compounds are equal to at least 1.
6.3.2.
Inject the sample as prepared in 6.1.
6.3.3.
Measure the area of the peaks in the reference solution and the spirit drink and calculate the concentrations.
7.Expression of results
Express the concentration of each constituent in mg/l.
8.Performance characteristics of the method (precision)
The following data were obtained in 2009 from an international method-performance study on a variety of spirit drinks, carried out following internationally-agreed procedures (1) (2).
8.1.Furfural
Analyte | Furfural | |||||
---|---|---|---|---|---|---|
Samples | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 |
No of laboratories participating | 15 | 15 | 15 | 15 | 15 | 15 |
No of results accepted (laboratories) | 14 | 12 | 13 | 14 | 13 | 13 |
Mean value (mg/l) | 2,9 | 1,2 | 1,7 | 10,6 | 15,3 | 13,9 |
Repeatability standard deviation, sr (mg/l) | 0,04 | 0,05 | 0,04 | 0,18 | 0,23 | 0,20 |
Repeatability relative standard deviation, RSDr (%) | 1,4 | 4,5 | 2,3 | 1,7 | 1,5 | 1,5 |
Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,1 | 0,2 | 0,1 | 0,5 | 0,6 | 0,6 |
Reproducibility standard deviation, sR (mg/l) | 0,24 | 0,18 | 0,09 | 1,4 | 0,49 | 0,69 |
Reproducibility relative standard deviation, RSDR (%) | 8 | 15 | 5 | 13 | 3 | 5 |
Reproducibility limit, R (g/l) (R = 2,8 × sR) | 0,7 | 0,5 | 0,3 | 3,8 | 1,4 | 1,9 |
8.2.5-Hydroxymethylfurfural
Analyte | 5-Hydroxymethylfurfural | |||||
---|---|---|---|---|---|---|
Samples | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 |
No of laboratories participating | 16 | 16 | 16 | 16 | 16 | 16 |
No of results accepted (laboratories) | 14 | 14 | 14 | 14 | 14 | 14 |
Mean value (mg/l) | 5,0 | 11,1 | 9,4 | 33,7 | 5,8 | 17,5 |
Repeatability standard deviation, sr (mg/l) | 0,09 | 0,09 | 0,09 | 0,42 | 0,07 | 0,13 |
Repeatability relative standard deviation, RSDr (%) | 1,7 | 0,8 | 1,0 | 1,3 | 1,2 | 0,8 |
Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,2 | 0,3 | 0,3 | 1,2 | 0,2 | 0,4 |
Reproducibility standard deviation, sR (mg/l) | 0,39 | 1,01 | 0,50 | 4,5 | 0,4 | 1,6 |
Reproducibility relative standard deviation, RSDR (%) | 8 | 9 | 5 | 13 | 7 | 9 |
Reproducibility limit, R (g/l) (R = 2,8 × sR) | 1,1 | 2,8 | 1,4 | 12,5 | 1,1 | 4,6 |
8.3.5-Methylfurfural
Analyte | 5-Methylfurfural | |||||
---|---|---|---|---|---|---|
Samples | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 |
No of laboratories participating | 11 | 11 | 11 | 11 | 11 | 11 |
No of results accepted (laboratories) | 11 | 11 | 8 | 11 | 10 | 11 |
Mean value (mg/l) | 0,1 | 0,2 | 0,1 | 0,5 | 1,7 | 0,8 |
Repeatability standard deviation, sr (mg/l) | 0,01 | 0,01 | 0,02 | 0,02 | 0,03 | 0,07 |
Repeatability relative standard deviation, RSDr (%) | 10,7 | 6,1 | 13,6 | 4,7 | 2,0 | 10,0 |
Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,0 | 0,0 | 0,1 | 0,1 | 0,1 | 0,2 |
Reproducibility standard deviation, sR (mg/l) | 0,03 | 0,04 | 0,03 | 0,18 | 0,20 | 0,26 |
Reproducibility relative standard deviation, RSDR (%) | 35 | 18 | 22 | 39 | 12 | 35 |
Reproducibility limit, R (g/l) (R = 2,8 × sR) | 0,1 | 0,1 | 0,1 | 0,5 | 0,6 | 0,7 |
8.4.Vanillin
Analyte | Vanillin | |||||
---|---|---|---|---|---|---|
Samples | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 |
No of laboratories participating | 16 | 15 | 16 | 16 | 16 | 16 |
No of results accepted (laboratories) | 16 | 15 | 16 | 16 | 16 | 16 |
Mean value (mg/l) | 0,5 | 0,2 | 1,2 | 1,2 | 3,2 | 3,9 |
Repeatability standard deviation, sr (mg/l) | 0,03 | 0,02 | 0,06 | 0,11 | 0,11 | 0,09 |
Repeatability relative standard deviation, RSDr (%) | 6,8 | 9,6 | 4,6 | 8,9 | 3,5 | 2,3 |
Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,1 | 0,1 | 0,2 | 0,3 | 0,3 | 0,3 |
Reproducibility standard deviation, sR (mg/l) | 0,09 | 0,06 | 0,18 | 0,27 | 0,41 | 0,62 |
Reproducibility relative standard deviation, RSDR (%) | 19 | 25 | 15 | 22 | 13 | 16 |
Reproducibility limit, R (g/l) (R = 2,8 × sR) | 0,3 | 0,2 | 0,5 | 0,8 | 1,2 | 1,7 |
8.5.Syringaldehyde
Analyte | Syringaldehyde | |||||
---|---|---|---|---|---|---|
Samples | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 |
No of laboratories participating | 16 | 15 | 16 | 16 | 16 | 16 |
No of results accepted (laboratories) | 13 | 13 | 13 | 12 | 14 | 13 |
Mean value (mg/l) | 1,0 | 0,2 | 4,8 | 3,2 | 10,5 | 9,7 |
Repeatability standard deviation, sr (mg/l) | 0,03 | 0,02 | 0,04 | 0,08 | 0,10 | 0,09 |
Repeatability relative standard deviation, RSDr (%) | 2,6 | 8,1 | 0,8 | 2,6 | 0,9 | 0,9 |
Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,1 | 0,1 | 0,1 | 0,2 | 0,3 | 0,3 |
Reproducibility standard deviation, sR (mg/l) | 0,08 | 0,07 | 0,23 | 0,19 | 0,39 | 0,43 |
Reproducibility relative standard deviation, RSDR (%) | 8 | 33 | 5 | 6 | 4 | 4 |
Reproducibility limit, R (g/l) (R = 2,8 × sR) | 0,2 | 0,2 | 0,7 | 0,5 | 1,1 | 1,2 |
8.6.Coniferaldehyde
Analyte | Coniferaldehyde | |||||
---|---|---|---|---|---|---|
Samples | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 |
No of laboratories participating | 13 | 12 | 13 | 12 | 13 | 13 |
No of results accepted (laboratories) | 12 | 12 | 13 | 12 | 13 | 13 |
Mean value (mg/l) | 0,2 | 0,2 | 0,6 | 0,8 | 4,6 | 1,3 |
Repeatability standard deviation, sr (mg/l) | 0,02 | 0,02 | 0,03 | 0,03 | 0,09 | 0,06 |
Repeatability relative standard deviation, RSDr (%) | 9,2 | 9,8 | 4,6 | 4,3 | 1,9 | 4,5 |
Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,04 | 0,04 | 0,07 | 0,09 | 0,24 | 0,16 |
Reproducibility standard deviation, sR (mg/l) | 0,04 | 0,04 | 0,11 | 0,18 | 0,38 | 0,25 |
Reproducibility relative standard deviation, RSDR (%) | 23 | 27 | 21 | 23 | 8 | 19 |
Reproducibility limit, R (g/l) (R = 2,8 × sR) | 0,1 | 0,1 | 0,3 | 0,5 | 1,1 | 0,7 |
8.7.Sinapaldehyde
Analyte | Sinapaldehyde | |||||
---|---|---|---|---|---|---|
Samples | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 |
No of laboratories participating | 14 | 14 | 14 | 14 | 15 | 14 |
No of results accepted (laboratories) | 14 | 13 | 12 | 13 | 13 | 12 |
Mean value (mg/l) | 0,3 | 0,2 | 0,2 | 1,6 | 8,3 | 0,3 |
Repeatability standard deviation, sr (mg/l) | 0,02 | 0,01 | 0,02 | 0,06 | 0,14 | 0,03 |
Repeatability relative standard deviation, RSDr (%) | 7,5 | 4,6 | 11,2 | 3,7 | 1,6 | 11,4 |
Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,06 | 0,03 | 0,06 | 0,17 | 0,38 | 0,08 |
Reproducibility standard deviation, sR (mg/l) | 0,09 | 0,05 | 0,08 | 0,20 | 0,81 | 0,18 |
Reproducibility relative standard deviation, RSDR (%) | 31 | 27 | 46 | 13 | 10 | 73 |
Reproducibility limit, R (g/l) (R = 2,8 × sR) | 0,2 | 0,2 | 0,2 | 0,6 | 2,3 | 0,5 |
8.8.Gallic acid
Analyte | Gallic acid | |||||
---|---|---|---|---|---|---|
Sample | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 |
No of laboratories participating | 16 | 15 | 16 | 16 | 16 | 16 |
No of results accepted (laboratories) | 15 | 14 | 16 | 16 | 16 | 16 |
Mean value (mg/l) | 1,2 | 0,4 | 2,0 | 6,1 | 7,3 | 21,8 |
Repeatability standard deviation, sr (mg/l) | 0,07 | 0,04 | 0,06 | 0,18 | 0,18 | 0,60 |
Repeatability relative standard deviation, RSDr (%) | 6,1 | 8,1 | 2,9 | 3,0 | 2,4 | 2,8 |
Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,2 | 0,1 | 0,2 | 0,5 | 0,5 | 1,7 |
Reproducibility standard deviation, sR (mg/l) | 0,43 | 0,20 | 0,62 | 3,3 | 2,2 | 7,7 |
Reproducibility relative standard deviation, RSDR (%) | 36 | 47 | 31 | 53 | 30 | 35 |
Reproducibility limit, R (g/l) (R = 2,8 × sR) | 1,2 | 0,6 | 1,7 | 9,1 | 6,2 | 21,7 |
8.9.Ellagic acid
Analyte | Ellagic acid | |||||
---|---|---|---|---|---|---|
Samples | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 |
No of laboratories participating | 7 | 7 | 7 | 7 | 7 | 7 |
No of results accepted (laboratories) | 7 | 7 | 7 | 7 | 7 | 6 |
Mean value (mg/l) | 3,2 | 1,0 | 9,5 | 13 | 13 | 36 |
Repeatability standard deviation, sr (mg/l) | 0,20 | 0,16 | 0,30 | 0,41 | 0,95 | 0,34 |
Repeatability relative standard deviation, RSDr (%) | 6,3 | 16 | 3,2 | 3,2 | 7,4 | 1,0 |
Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,6 | 0,4 | 0,9 | 1,1 | 2,7 | 1,0 |
Reproducibility standard deviation, sR (mg/l) | 1,41 | 0,42 | 4,0 | 5,0 | 4,9 | 14 |
Reproducibility relative standard deviation, RSDR (%) | 44 | 43 | 42 | 39 | 39 | 40 |
Reproducibility limit, R (g/l) (R = 2,8 × sR) | 4,0 | 1,2 | 11 | 14 | 14 | 40 |
8.10.Vanillic acid
Analyte | Vanillic acid | |||||
---|---|---|---|---|---|---|
Samples | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 |
No of laboratories participating | 15 | 15 | 15 | 15 | 15 | 15 |
No of results accepted (laboratories) | 12 | 11 | 14 | 14 | 15 | 14 |
Mean value (mg/l) | 0,2 | 0,2 | 1,5 | 0,8 | 2,4 | 2,7 |
Repeatability standard deviation, sr (mg/l) | 0,03 | 0,04 | 0,03 | 0,10 | 0,13 | 0,21 |
Repeatability relative standard deviation, RSDr (%) | 14,2 | 16,5 | 2,3 | 12,6 | 5,3 | 7,7 |
Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,1 | 0,1 | 0,1 | 0,3 | 0,4 | 0,6 |
Reproducibility standard deviation, sR (mg/l) | 0,06 | 0,05 | 0,51 | 0,2 | 1,22 | 0,70 |
Reproducibility relative standard deviation, RSDR (%) | 28 | 20 | 35 | 31 | 51 | 26 |
Reproducibility limit, R (g/l) (R = 2,8 × sR) | 0,2 | 0,1 | 1,4 | 0,7 | 3,4 | 2,0 |
8.11.Syringic acid
Analyte | Syringic acid | |||||
---|---|---|---|---|---|---|
Samples | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 |
No of laboratories participating | 16 | 15 | 16 | 16 | 16 | 16 |
No of results accepted (laboratories) | 16 | 15 | 15 | 15 | 16 | 15 |
Mean value (mg/l) | 0,4 | 0,2 | 2,5 | 1,4 | 3,4 | 4,8 |
Repeatability standard deviation, sr (mg/l) | 0,03 | 0,02 | 0,06 | 0,13 | 0,08 | 0,11 |
Repeatability relative standard deviation, RSDr (%) | 6,7 | 12,6 | 2,3 | 9,0 | 2,3 | 2,3 |
Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,1 | 0,1 | 0,2 | 0,4 | 0,2 | 0,3 |
Reproducibility standard deviation, sR (mg/l) | 0,08 | 0,05 | 0,29 | 0,26 | 0,43 | 0,67 |
Reproducibility relative standard deviation, RSDR (%) | 19 | 29 | 11 | 18 | 13 | 14 |
Reproducibility limit, R (g/l) (R = 2,8 × sR) | 0,2 | 0,1 | 0,8 | 0,7 | 1,2 | 1,9 |
8.12.Scopoletin
Analyte | Scopoletin | |||||
---|---|---|---|---|---|---|
Samples | Whisky | Brandy | Rum | Cognac 1 | Bourbon | Cognac 2 |
No of laboratories participating | 10 | 10 | 10 | 10 | 10 | 10 |
No of results accepted (laboratories) | 9 | 8 | 9 | 8 | 8 | 8 |
Mean value (mg/l) | 0,09 | 0,04 | 0,11 | 0,04 | 0,65 | 0,15 |
Repeatability standard deviation, sr (mg/l) | 0,0024 | 0,0008 | 0,0018 | 0,0014 | 0,0054 | 0,0040 |
Repeatability relative standard deviation, RSDr (%) | 2,6 | 2,2 | 1,6 | 3,3 | 0,8 | 2,7 |
Repeatability limit, r (mg/l) (r = 2,8 × sr) | 0,007 | 0,002 | 0,005 | 0,004 | 0,015 | 0,011 |
Reproducibility standard deviation, sR (mg/l) | 0,01 | 0,01 | 0,03 | 0,01 | 0,09 | 0,02 |
Reproducibility relative standard deviation, RSDR (%) | 15 | 16 | 23 | 17 | 15 | 15 |
Reproducibility limit, R (g/l) (R = 2,8 × sR) | 0,04 | 0,02 | 0,07 | 0,02 | 0,26 | 0,06 |
(1)
‘Protocol for the design, conduct and interpretation of method-performance studies’, Horwitz, W. (1995) Pure and Applied Chemistry, 67, 332-343.
(2)
Horwitz, W. (1982) Analytical Chemistry, 54, 67A-76A.