Commission Regulation (EC) No 2870/2000 of 19 December 2000 laying down Community reference methods for the analysis of spirits drinks (c. 2870)

ANNEX U.K.

[F1 X. 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 U.K.

1. Scope U.K.

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 U.K.

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 U.K.

Determination by high-performance liquid chromatography (HPLC), with detection by ultraviolet spectrophotometry at several wavelengths and by spectrofluorimetry.

4. Reagents U.K.

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. U.K.

4.2. HPLC-quality methanol (Solvent B). U.K.

4.3. Acetic acid diluted to 0,5 % vol. (Solvent A). U.K.

4.4. Mobile phases: (given as an example only). U.K.

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. U.K.

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: U.K.

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. Apparatus U.K.

Standard laboratory apparatus

5.1. A high-performance liquid chromatograph capable of functioning in binary gradient mode and equipped with: U.K.

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. U.K.

5.3. Device for membrane-filtration of small volumes. U.K.

5.4. Integrator-computer or recorder with performance compatible with the entire apparatus, and in particular, it must have several acquisition channels. U.K.

6. Procedure U.K.

6.1. Preparation of the solution to be injected U.K.

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) U.K.

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 U.K.

6.3.1. Inject the reference standards separately, then mixed. U.K.

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. U.K.

6.3.3. Measure the area of the peaks in the reference solution and the spirit drink and calculate the concentrations. U.K.

7. Expression of results U.K.

Express the concentration of each constituent in mg/l.

8. Performance characteristics of the method (precision) U.K.

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 U.K.

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, s r (mg/l)	0,04	0,05	0,04	0,18	0,23	0,20
Repeatability relative standard deviation, RSD r (%)	1,4	4,5	2,3	1,7	1,5	1,5
Repeatability limit, r (mg/l) (r = 2,8 × s r )	0,1	0,2	0,1	0,5	0,6	0,6
Reproducibility standard deviation, s R (mg/l)	0,24	0,18	0,09	1,4	0,49	0,69
Reproducibility relative standard deviation, RSD R (%)	8	15	5	13	3	5
Reproducibility limit, R (g/l) (R = 2,8 × s R )	0,7	0,5	0,3	3,8	1,4	1,9

8.2. 5-Hydroxymethylfurfural U.K.

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, s r (mg/l)	0,09	0,09	0,09	0,42	0,07	0,13
Repeatability relative standard deviation, RSD r (%)	1,7	0,8	1,0	1,3	1,2	0,8
Repeatability limit, r (mg/l) (r = 2,8 × s r )	0,2	0,3	0,3	1,2	0,2	0,4
Reproducibility standard deviation, s R (mg/l)	0,39	1,01	0,50	4,5	0,4	1,6
Reproducibility relative standard deviation, RSD R (%)	8	9	5	13	7	9
Reproducibility limit, R (g/l) (R = 2,8 × s R )	1,1	2,8	1,4	12,5	1,1	4,6

8.3. 5-Methylfurfural U.K.

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, s r (mg/l)	0,01	0,01	0,02	0,02	0,03	0,07
Repeatability relative standard deviation, RSD r (%)	10,7	6,1	13,6	4,7	2,0	10,0
Repeatability limit, r (mg/l) (r = 2,8 × s r )	0,0	0,0	0,1	0,1	0,1	0,2
Reproducibility standard deviation, s R (mg/l)	0,03	0,04	0,03	0,18	0,20	0,26
Reproducibility relative standard deviation, RSD R (%)	35	18	22	39	12	35
Reproducibility limit, R (g/l) (R = 2,8 × s R )	0,1	0,1	0,1	0,5	0,6	0,7

8.4. Vanillin U.K.

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, s r (mg/l)	0,03	0,02	0,06	0,11	0,11	0,09
Repeatability relative standard deviation, RSD r (%)	6,8	9,6	4,6	8,9	3,5	2,3
Repeatability limit, r (mg/l) (r = 2,8 × s r )	0,1	0,1	0,2	0,3	0,3	0,3
Reproducibility standard deviation, s R (mg/l)	0,09	0,06	0,18	0,27	0,41	0,62
Reproducibility relative standard deviation, RSD R (%)	19	25	15	22	13	16
Reproducibility limit, R (g/l) (R = 2,8 × s R )	0,3	0,2	0,5	0,8	1,2	1,7

8.5. Syringaldehyde U.K.

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, s r (mg/l)	0,03	0,02	0,04	0,08	0,10	0,09
Repeatability relative standard deviation, RSD r (%)	2,6	8,1	0,8	2,6	0,9	0,9
Repeatability limit, r (mg/l) (r = 2,8 × s r )	0,1	0,1	0,1	0,2	0,3	0,3
Reproducibility standard deviation, s R (mg/l)	0,08	0,07	0,23	0,19	0,39	0,43
Reproducibility relative standard deviation, RSD R (%)	8	33	5	6	4	4
Reproducibility limit, R (g/l) (R = 2,8 × s R )	0,2	0,2	0,7	0,5	1,1	1,2

8.6. Coniferaldehyde U.K.

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, s r (mg/l)	0,02	0,02	0,03	0,03	0,09	0,06
Repeatability relative standard deviation, RSD r (%)	9,2	9,8	4,6	4,3	1,9	4,5
Repeatability limit, r (mg/l) (r = 2,8 × s r )	0,04	0,04	0,07	0,09	0,24	0,16
Reproducibility standard deviation, s R (mg/l)	0,04	0,04	0,11	0,18	0,38	0,25
Reproducibility relative standard deviation, RSD R (%)	23	27	21	23	8	19
Reproducibility limit, R (g/l) (R = 2,8 × s R )	0,1	0,1	0,3	0,5	1,1	0,7

8.7. Sinapaldehyde U.K.

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, s r (mg/l)	0,02	0,01	0,02	0,06	0,14	0,03
Repeatability relative standard deviation, RSD r (%)	7,5	4,6	11,2	3,7	1,6	11,4
Repeatability limit, r (mg/l) (r = 2,8 × s r )	0,06	0,03	0,06	0,17	0,38	0,08
Reproducibility standard deviation, s R (mg/l)	0,09	0,05	0,08	0,20	0,81	0,18
Reproducibility relative standard deviation, RSD R (%)	31	27	46	13	10	73
Reproducibility limit, R (g/l) (R = 2,8 × s R )	0,2	0,2	0,2	0,6	2,3	0,5

8.8. Gallic acid U.K.

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, s r (mg/l)	0,07	0,04	0,06	0,18	0,18	0,60
Repeatability relative standard deviation, RSD r (%)	6,1	8,1	2,9	3,0	2,4	2,8
Repeatability limit, r (mg/l) (r = 2,8 × s r )	0,2	0,1	0,2	0,5	0,5	1,7
Reproducibility standard deviation, s R (mg/l)	0,43	0,20	0,62	3,3	2,2	7,7
Reproducibility relative standard deviation, RSD R (%)	36	47	31	53	30	35
Reproducibility limit, R (g/l) (R = 2,8 × s R )	1,2	0,6	1,7	9,1	6,2	21,7

8.9. Ellagic acid U.K.

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, s r (mg/l)	0,20	0,16	0,30	0,41	0,95	0,34
Repeatability relative standard deviation, RSD r (%)	6,3	16	3,2	3,2	7,4	1,0
Repeatability limit, r (mg/l) (r = 2,8 × s r )	0,6	0,4	0,9	1,1	2,7	1,0
Reproducibility standard deviation, s R (mg/l)	1,41	0,42	4,0	5,0	4,9	14
Reproducibility relative standard deviation, RSD R (%)	44	43	42	39	39	40
Reproducibility limit, R (g/l) (R = 2,8 × s R )	4,0	1,2	11	14	14	40

8.10. Vanillic acid U.K.

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, s r (mg/l)	0,03	0,04	0,03	0,10	0,13	0,21
Repeatability relative standard deviation, RSD r (%)	14,2	16,5	2,3	12,6	5,3	7,7
Repeatability limit, r (mg/l) (r = 2,8 × s r )	0,1	0,1	0,1	0,3	0,4	0,6
Reproducibility standard deviation, s R (mg/l)	0,06	0,05	0,51	0,2	1,22	0,70
Reproducibility relative standard deviation, RSD R (%)	28	20	35	31	51	26
Reproducibility limit, R (g/l) (R = 2,8 × s R )	0,2	0,1	1,4	0,7	3,4	2,0

8.11. Syringic acid U.K.

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, s r (mg/l)	0,03	0,02	0,06	0,13	0,08	0,11
Repeatability relative standard deviation, RSD r (%)	6,7	12,6	2,3	9,0	2,3	2,3
Repeatability limit, r (mg/l) (r = 2,8 × s r )	0,1	0,1	0,2	0,4	0,2	0,3
Reproducibility standard deviation, s R (mg/l)	0,08	0,05	0,29	0,26	0,43	0,67
Reproducibility relative standard deviation, RSD R (%)	19	29	11	18	13	14
Reproducibility limit, R (g/l) (R = 2,8 × s R )	0,2	0,1	0,8	0,7	1,2	1,9

8.12. Scopoletin U.K.

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, s r (mg/l)	0,0024	0,0008	0,0018	0,0014	0,0054	0,0040
Repeatability relative standard deviation, RSD r (%)	2,6	2,2	1,6	3,3	0,8	2,7
Repeatability limit, r (mg/l) (r = 2,8 × s r )	0,007	0,002	0,005	0,004	0,015	0,011
Reproducibility standard deviation, s R (mg/l)	0,01	0,01	0,03	0,01	0,09	0,02
Reproducibility relative standard deviation, RSD R (%)	15	16	23	17	15	15
Reproducibility limit, R (g/l) (R = 2,8 × s R )	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. U.K.

(2) Horwitz, W. (1982) Analytical Chemistry , 54, 67A-76A.] U.K.

Textual Amendments

F1 Inserted by Commission Implementing Regulation (EU) 2016/635 of 22 April 2016 amending the Annex to Regulation (EC) No 2870/2000 as regards certain reference methods for the analysis of spirit drinks.