Commission Regulation (EC) No 2870/2000Show full title

III. DETERMINATION OF VOLATILE SUBSTANCES AND METHANOL OF SPIRIT DRINKS U.K.

III.1. GENERAL REMARKS U.K.

1. Definitions U.K.

[F1 Regulation 110/2008] sets minimum levels of volatile compounds other than ethanol and methanol for a series of spirit drinks (rum, spirits of viticultural origin, fruit spirits, etc.). For this series of drinks only, these levels are conventionally considered to be equivalent to the sum of the concentrations of:

Textual Amendments

F1 Words in Annex Ch. 3 Pt. 3.1 para. 1 substituted (31.12.2020) by The Agricultural Products, Food and Drink (Amendment etc.) (EU Exit) Regulations 2020 (S.I. 2020/1637), regs. 1(7), 16(6)(a)(i)

1.

volatile acids expressed as acetic acid;

2.

aldehydes expressed as ethanal by the sum of ethanal (acetaldehyde) and the ethanal fraction contained in 1,1-diethoxyethane (acetal);

3.

the following higher alcohols: propan-1-ol, butan-1-ol, butan-2-ol, 2-methylpropan-1-ol, assayed by individual alcohol and 2-methylbutan-1-ol, and 3-methylbutan-1-ol assayed as individual alcohol or the sum of the two;

4.

ethyl acetate.

The following are the conventional methods for measuring volatile compounds:

• the volatile acids by means of volatile acidity,

• the aldehydes (ethanal and acetal), ethyl acetate and the alcohols by means of gas chromatography (GPC).

2. Gas chromatographic analysis of volatile compounds U.K.

Gas chromatographic assays of volatile compounds other than those set out above may prove particularly interesting as a means of determining both the origin of the raw material used in the distillation and the actual conditions of distillation.

Some spirits contain other volatile components, such as aromatic compounds, which are characteristic of the raw materials used to obtain the alcohol, of the aroma of the spirit drink and of the special features of the preparation of the spirit. These compounds are important for evaluating the requirements set out in [F2 Regulation 110/2008].

Textual Amendments

F2 Words in Annex Ch. 3 Pt. 3.1 para. 2 substituted (31.12.2020) by The Agricultural Products, Food and Drink (Amendment etc.) (EU Exit) Regulations 2020 (S.I. 2020/1637), regs. 1(7), 16(6)(a)(ii)

III.2. GAS CHROMATOGRAPHIC DETERMINATION OF VOLATILE CONGENERS: ALDEHYDES, HIGHER ALCOHOLS, ETHYL ACETATE AND METHANOL U.K.

1. Scope U.K.

This method is suitable for use for the determination of 1,1-diethoxyethane (acetal), 2-methylbutan-1-ol (active amyl alcohol), 3-methylbutan-1-ol (isoamyl alcohol), methanol (methyl alcohol), ethyl ethanoate (ethyl acetate), butan-1-ol (n-butanol), butan-2-ol (sec-butanol), 2-methylpropan-1-ol (isobutyl alcohol), propan-1-ol (n-propanol) and ethanal (acetaldehyde) in spirit drinks using gas chromatography. The method uses an internal standard, for example pentan-3-ol. The concentrations of the analytes are expressed as grams per 100 litres of absolute alcohol; the alcoholic strength of the product must be determined prior to analysis. The spirit drinks that can be analysed using this method include whisky, brandy, rum, wine spirit, fruit spirit and grape marc spirit.

2. Normative References U.K.

ISO 3696:1987: Water for analytical laboratory use — Specifications and test methods.

3. Definition U.K.

Congeners are volatile substances formed along with ethanol during fermentation, distillation and maturation of spirit drinks.

4. Principle U.K.

Congeners in spirit drinks are determined by direct injection of the spirit drink, or appropriately diluted spirit drink, into a gas chromatography (GC) system. A suitable internal standard is added to the spirit drink prior to injection. The congeners are separated by temperature programming on a suitable column and are detected using a flame ionisation detector (FID). The concentration of each congener is determined with respect to the internal standard from response factors, which are obtained during calibration under the same chromatographic conditions as those of the spirit drink analysis.

5. Reagents and materials U.K.

Unless otherwise stated, use only reagents of a purity greater than 97 %, purchased from an ISO-accredited supplier with a certificate of purity, free from other congeners at test dilution (this may be confirmed by injection of individual congener standards at the test dilution using GC conditions as in 6.4) and only water of at least grade 3 as defined in ISO 3696. Acetal and acetaldehyde must be stored in the dark at < 5 °C, all other reagents may be stored at room temperature.

5.1.

Ethanol absolute (CAS 64-17-5).

5.2.

Methanol (CAS 67-56-1).

5.3.

Propan-1-ol (CAS 71-23-8).

5.4.

2-methylpropan-1-ol (CAS 78-33-1).

5.5.

Acceptable internal standards: pentan-3-ol (CAS 584-02-1), pentan-1-ol (CAS 71-41-0), 4-methylpentan-1-ol (CAS 626-89-1) or methyl nonanoate (CAS 1731-84-6).

5.6.

2-methylbutan-1-ol (CAS 137-32-6).

5.7.

3-methylbutan-1-ol (CAS 123-51-3).

5.8.

Ethyl acetate (CAS 141-78-6).

5.9.

Butan-1-ol (CAS 71-36-3).

5.10.

Butan-2-ol (CAS 78-92-2).

5.11.

Acetaldehyde (CAS 75-07-0).

5.12.

Acetal (CAS 105-57-7).

5.13.

40 % v/v ethanol solution

To prepare 400 ml/l ethanol solution pour 400 ml ethanol (5.1) into a 1-litre volumetric flask, make up to volume with distilled water and mix.

5.14.

Preparation and storage of standard solutions (procedure used for the validated method).

All standard solutions must be stored at < 5 °C and be prepared freshly on a monthly basis. Masses of components and solutions should be recorded to the nearest 0,1 mg.

5.14.1.

Standard solution — A U.K.

Pipette the following reagents into a 100-ml volumetric flask, containing approximately 60-ml ethanol solution (5.13) to minimise component evaporation, make up to volume with ethanol solution (5.13) and mix thoroughly. Record the weight of the flask, each component added and the total final weight of contents.

Component	Volume (ml)
Methanol (5.2)	3,0
Propan-1-ol (5.3)	3,0
2-methylpropan-1-ol (5.4)	3,0
2-methylbutan-1-ol (5.6)	3,0
3-methylbutan-1-ol (5.7)	3,0
Ethyl acetate (5.8)	3,0
Butan-1-ol (5.9)	3,0
Butan-2-ol (5.10)	3,0
Acetaldehyde (5.11)	3,0
Acetal (5.12)	3,0

Note 1: U.K.

It is preferable to add acetal and acetaldehyde last in order to minimise losses through evaporation. U.K.

5.14.2.

Standard solution — B U.K.

Pipette 3 ml of pentan-3-ol, or other suitable internal standard, (5.5) into a 100-ml volumetric flask, containing approximately 80 ml ethanol solution (5.13), make up to volume with ethanol solution (5.13) and mix thoroughly.

Record the weight of the flask, the weight of pentan-3-ol or other internal standard added and the total final weight of contents.

5.14.3.

Standard solution — C U.K.

Pipette 1 ml solution A (5.14.1) and 1 ml solution B (5.14.2) into a 100-ml volumetric flask containing approximately 80 ml ethanol solution (5.13), make up to volume with ethanol solution (5.13) and mix thoroughly.

Record the weight of the flask, each component added and the total final weight of contents.

5.14.4.

Standard solution — D U.K.

In order to maintain analytical continuity, prepare a quality control standard using the previously prepared standard A (5.14.1). Pipette 1 ml solution A (5.14.1) into a 100-ml volumetric flask containing approximately 80 ml ethanol solution (5.13), make up to volume with ethanol solution (5.13) and mix thoroughly.

Record the weight of the flask, each component added and the total final weight of contents.

5.14.5.

Standard solution — E U.K.

Pipette 10 ml solution B (5.14.2) into a 100-ml volumetric flask containing approximately 80 ml ethanol solution (5.13), make up to volume with ethanol solution (5.13) and mix thoroughly.

Record the weight of the flask, each component added and the total final weight of contents.

5.14.6.

Standard solutions used to check the linearity of response of FID U.K.

Into separate 100-ml volumetric flasks, containing approximately 80 ml ethanol (5.13), pipette 0, 0,1, 0,5, 1,0, 2,0 ml solution A (5.14.1) and 1 ml solution B (5.14.2), make up to volume with ethanol solution (5.13) and mix thoroughly.

Record the weight of the flask, each component added and the total final weight of contents.

5.14.7.

QC standard solution U.K.

Pipette 9 ml standard solution D (5.14.4) and 1 ml of standard solution E (5.14.5) into a weighing vessel and mix thoroughly.

Record the weight of the flask, each component added and the total final weight of contents.

6. Apparatus and equipment U.K.

6.1.

Apparatus capable of measuring the density and alcoholic strength.

6.2.

Analytical balance, capable of measuring to four decimal places.

6.3.

A temperature programmed gas chromatograph fitted with a flame ionisation detector and integrator or other data handling system capable of measuring peak areas or peak heights.

6.4.

Gas chromatographic column(s), capable of separating the analytes such that the minimum resolution between the individual components (other than 2-methylbutan-1-ol and 3-methylbutan-1-ol) is at least 1.3.

Note 2: U.K.

The following columns and GC conditions are suitable examples: U.K.

1.

A retention gap 1 m × 0,32 mm i.d. connected to a CP-WAX 57 CB column 50 m × 0,32 mm i.d. 0,2 μm film thickness (stabilised polyethylene glycol) followed by a Carbowax 400 column 50 m × 0,32 mm i.d. 0,2 μm film thickness. (Columns are connected using press-fit connectors.)

Carrier gas and pressure:	Helium (135 kPa)
Column temperature:	35 °C for 17 min., 35 to 70 °C at 12 °C/min., hold at 70 °C for 25 min.
Injector temperature:	150 °C
Detector temperature:	250 °C
Injection volume:	1 μl, split 20 to 100:1

2.

A retention gap 1 m × 0,32 mm i.d. connected to a CP-WAX 57 CB column 50 m × 0,32 mm i.d. 0,2 μm film thickness (stabilised polyethylene glycol). (Retention gap is connected using a press-fit connector.)

Carrier gas and pressure:	Helium (65 kPa)
Column temperature:	35 °C for 10 min., 35 to 110 °C at 5 °C/min., 110 to 190 °C at 30 °C/min., hold at 190 °C for 2 min.
Injector temperature:	260 °C
Detector temperature:	300 °C
Injection volume:	1 μl, split 55:1

3.

A packed column (5 % CW 20M, Carbopak B), 2 m × 2 mm i.d.

Column temperature:	65 °C for 4 min., 65 to 140 °C at 10 °C/min., hold at 140 °C for 5 min., 140 to 150 °C at 5 °C/min., hold at 150 °C for 3 min.
Injector temperature:	65 °C
Detector temperature:	200 °C
Injection volume:	1 μl

7. Sampling and samples. U.K.

7.1.

Laboratory sample

On receipt, the alcoholic strength of each sample is measured (6.1).

8. Procedure (used for the validated method) U.K.

8.1.

Test portion

8.1.1.

Weigh an appropriate sealed weighing vessel and record the weight.

8.1.2.

Pipette 9 ml laboratory sample into the vessel and record the weight (M_SAMPLE).

8.1.3.

Add 1 ml of standard solution E (5.14.5) and record the weight (M_IS).

8.1.4.

Shake the test material vigorously (at least 20 inversions). Samples must be stored at less than 5 °C prior to analysis in order to minimise any volatile losses.

8.2.

Blank test U.K.

8.2.1.

Using a four decimal place balance (6.2), weigh an appropriate sealed weighing vessel and record the weight.

8.2.2.

Pipette 9 ml 400 ml/l ethanol solution (5.13) into the vessel and record the weight.

8.2.3.

Add 1 ml of standard solution E (5.14.5) and record the weight.

8.2.4.

Shake the test material vigorously (at least 20 inversions). Samples must be stored at less than 5 °C prior to analysis in order to minimise any volatile losses.

8.3.

Preliminary test U.K.

Inject standard solution C (5.14.3) to ensure that all of the analytes are separated with a minimum resolution of 1.3 (except 2-methylbutan-1-ol and 3-methylbutan-1-ol).

8.4.

Calibration U.K.

The calibration should be checked using the following procedure. Ensure that the response is linear by successively analysing in triplicate each of the linearity standard solutions (5.14.6) containing internal standard (IS). From the integrator peak areas or peak heights for each injection calculate the ratio R for each congener and plot a graph of R versus the concentration ratio of congener to internal standard (IS), C. A linear plot should be obtained, with a correlation coefficient of at least 0,99.

8.5.

Determination U.K.

Inject standard solution C (5.14.3) and 2 QC standard solutions (5.14.7). Follow with unknown samples (prepared according to 8.1 and 8.2) inserting one QC standard every 10 samples to ensure analytical stability. Inject one standard solution C (5.14.3) after every 5 samples.

9. Calculation U.K.

An automated system of data handling can be used, provided the data can be checked using the principles described in the method below.

Measure either peak areas or peak heights for congener and internal standard peaks.

9.1.

Response factor calculation. U.K.

From the chromatogram of the injection of standard solution C (5.14.3), calculate response factors for each congener using equation (1). where:

IS

=

Internal Standard

Conc. congener

=

concentration of congener in solution C (5.14.3)

Conc. IS

=

concentration of internal standard in solution C (5.14.3).

9.1.2.

Sample analysis U.K.

Using equation (2) below, calculate the concentration of each congener in the samples. where:

M_SAMPLE

=

weight of sample (8.1.2);

M_IS

=

weight of internal standard (8.1.3);

Conc. IS

=

concentration of internal standard in solution E (5.14.5);

RF

=

response factor calculated using equation 1.

9.1.3.

Quality control standard solution analysis U.K.

Using equation (3) below, calculate the percentage recovery of the target value for each congener in the quality control standards (5.14.7):

The concentration of the analyte in the QC standard is calculated using equations (1) and (2) above.

9.2.

Final presentation of results U.K.

Results are converted from μ/g to g per 100 litres absolute alcohol for samples using equation (4): where

ρ

=

density in kg/m³.

Results are quoted to 3 significant figures and a maximum of one decimal place e.g. 11,4 g per 100 l absolute alcohol.

10. Quality assurance and control (used for the validated method) U.K.

Using equation (2) above, calculate the concentration of each congener in the quality control standard solutions prepared by following the procedure as in 8.1.1 to 8.1.4. Using equation (3), calculate the percentage recovery of the target value. If the analysed results are within ± 10 % of their theoretical values for each congener, analysis may proceed. If not, an investigation should be made to find the cause of the inaccuracy and remedial action taken as appropriate.

11. Method performance characteristics (precision) U.K.

Statistical results of the interlaboratory test: the following tables give the values for the following compounds: ethanal, ethyl acetate, acetal, total ethanal, methanol, butan-2-ol, propan-1-ol, butan-1-ol, 2-methyl-propan-1-ol, 2 methyl-butan-1-ol, 3 methyl-butan-1-ol.

The following data were obtained from an international method performance study carried out to internationally agreed procedures.

Year of interlaboratory test	1 997
Number of laboratories	32
Number of samples	5
Analyte	ethanal

Sample types
A Brandy; blind duplicates. B Kirsch; blind duplicates. C Grappa; blind duplicates. D Whisky; split levels (*). E Rum; split levels (*).
Samples	A	B	C	D	E
Number of laboratories retained after eliminating outliers	28	26	27	27	28
Number of outliers (laboratories)	2	4	3	3	2
Number of accepted results	56	52	54	54	56
Mean value ( ) μg/g	63,4	71,67	130,4	38,4	28,6
				13,8 (*)	52,2 (*)
Repeatability standard deviation (Sr) μg/g	3,3	1,9	6,8	4,1	3,6
Repeatability relative standard deviation (RSDr) (%)	5,2	2,6	5,2	15,8	8,9
Repeatability limit (r) μg/g	9,3	5,3	19,1	11,6	10,1
Reproducibility standard deviation (SR) μg/g	12	14	22	6,8	8,9
Reproducibility relative standard deviation (RSDR) (%)	18,9	19,4	17,1	26,2	22,2
Reproducibility limit (R) μg/g	33,5	38,9	62,4	19,1	25,1

Year of interlaboratory test	1 997
Number of laboratories	32
Number of samples	5
Analyte	ethyl acetate

Sample types
A Brandy; blind duplicates. B Kirsch; blind duplicates. C Grappa; blind duplicates. D Whisky; split levels (*). E Rum; split levels (*).
Samples	A	B	C	D	E
Number of laboratories retained after eliminating outliers	24	24	25	24	24
Number of outliers (laboratories)	2	2	1	2	2
Number of accepted results	48	48	50	48	48
Mean value ( ) μg/g	96,8	1 046	120,3	112,5	99,1
				91,8 (*)	117,0 (*)
Repeatability standard deviation (Sr) μg/g	2,2	15	2,6	2,1	2,6
Repeatability relative standard deviation (RSDr) (%)	2,3	1,4	2,1	2,0	2,4
Repeatability limit (r) μg/g	6,2	40,7	7,2	5,8	7,3
Reproducibility standard deviation (SR) μg/g	6,4	79	8,2	6,2	7,1
Reproducibility relative standard deviation (RSDR) (%)	6,6	7,6	6,8	6,2	6,6
Reproducibility limit (R) μg/g	17,9	221,9	22,9	17,5	20,0

Year of interlaboratory test	1 997
Number of laboratories	32
Number of samples	5
Analyte	acetal

Sample types
A Brandy; blind duplicates. B Kirsch; blind duplicates. C Grappa; blind duplicates. D Whisky; split levels (*). E Rum; split levels (*).
Samples	A	B	C	D	E
Number of laboratories retained after eliminating outliers	20	21	22	17	21
Number of outliers (laboratories)	4	3	2	4	3
Number of accepted results	40	42	44	34	42
Mean value ( ) μg/g	35,04	36,46	68,5	20,36	15,1
				6,60 (*)	28,3 (*)
Repeatability standard deviation (Sr) μg/g	0,58	0,84	1,6	0,82	1,9
Repeatability relative standard deviation (RSDr) (%)	1,7	2,3	2,3	6,1	8,7
Repeatability limit (r) μg/g	1,6	2,4	4,4	2,3	5,3
Reproducibility standard deviation (SR) μg/g	4,2	4,4	8,9	1,4	3,1
Reproducibility relative standard deviation (RSDR) (%)	12,1	12,0	13,0	10,7	14,2
Reproducibility limit (R) μg/g	11,8	12,2	25,0	4,0	8,7

Year of interlaboratory test	1 997
Number of laboratories	32
Number of samples	5
Analyte	total ethanal

Sample types
A Brandy; blind duplicates. B Kirsch; blind duplicates. C Grappa; blind duplicates. D Whisky; split levels (*). E Rum; split levels (*).
Samples	A	B	C	D	E
Number of laboratories retained after eliminating outliers	23	19	22	21	22
Number of outliers (laboratories)	1	5	2	3	2
Number of accepted results	46	38	44	42	44
Mean value ( ) μg/g	76,5	85,3	156,5	45,4	32,7
				15,8 (*)	61,8 (*)
Repeatability standard deviation (Sr) μg/g	3,5	1,3	6,5	4,4	3,6
Repeatability relative standard deviation (RSDr) (%)	4,6	1,5	4,2	14,2	7,6
Repeatability limit (r) μg/g	9,8	3,5	18,3	12,2	10,0
Reproducibility standard deviation (SR) μg/g	13	15	24,1	7,3	9,0
Reproducibility relative standard deviation (RSDR) (%)	16,4	17,5	15,4	23,7	19,1
Reproducibility limit (R) μg/g	35,2	41,8	67,4	20,3	25,2

Year of interlaboratory test	1 997
Number of laboratories	32
Number of samples	5
Analyte	Methanol

Sample types
A Brandy; blind duplicates. B Kirsch; blind duplicates. C Grappa; blind duplicates. D Whisky; split levels (*). E Rum; split levels (*).
Samples	A	B	C	D	E
Number of laboratories retained after eliminating outliers	26	27	27	28	25
Number of outliers (laboratories)	4	3	3	1	4
Number of accepted results	52	54	54	56	50
Mean value ( ) μg/g	319,8	2 245	1 326	83,0	18,6
				61,5 (*)	28,9 (*)
Repeatability standard deviation (Sr) μg/g	4,4	27	22	1,5	1,3
Repeatability relative standard deviation (RSDr) (%)	1,4	1,2	1,7	2,1	5,6
Repeatability limit (r) μg/g	12,3	74,4	62,5	4,3	3,8
Reproducibility standard deviation (SR) μg/g	13	99	60	4,5	2,8
Reproducibility relative standard deviation (RSDR) (%)	3,9	4,4	4,6	6,2	11,8
Reproducibility limit (R) μg/g	35,2	278,3	169,1	12,5	7,9

Year of interlaboratory test	1 997
Number of laboratories	32
Number of samples	4
Analyte	butan-2-ol

Sample types
A Brandy; blind duplicates. B Kirsch; blind duplicates. C Grappa; blind duplicates. E Rum; split levels (*).
Samples	A	B	C	E
Number of laboratories retained after eliminating outliers	21	27	29	22
Number of outliers (laboratories)	4	3	1	3
Number of accepted results	42	54	58	44
Mean value ( ) μg/g	5,88	250,2	27,57	5,83
				14,12 (*)
Repeatability standard deviation (Sr) μg/g	0,4	2,2	0,87	0,64
Repeatability relative standard deviation (RSDr) (%)	6,8	0,9	3,2	6,4
Repeatability limit (r) μg/g	1,1	6,1	2,5	1,8
Reproducibility standard deviation (SR) μg/g	0,89	13	3,2	0,87
Reproducibility relative standard deviation (RSDR) (%)	15,2	5,1	11,5	8,7
Reproducibility limit (R) μg/g	2,5	35,5	8,9	2,4

Year of interlaboratory test	1 997
Number of laboratories	32
Number of samples	5
Analyte	propan-1-ol

Sample types
A Brandy; blind duplicates. B Kirsch; blind duplicates. C Grappa; blind duplicates. D Whisky; split levels (*). E Rum; split levels (*).
Samples	A	B	C	D	E
Number of laboratories retained after eliminating outliers	29	27	27	29	29
Number of outliers (laboratories)	2	4	3	2	2
Number of accepted results	58	54	54	58	58
Mean value ( ) μg/g	86,4	3 541	159,1	272,1	177,1
				229,3 (*)	222,1 (*)
Repeatability standard deviation (Sr) μg/g	3,0	24	3,6	2,3	3,3
Repeatability relative standard deviation (RSDr) (%)	3,4	0,7	2,3	0,9	1,6
Repeatability limit (r) μg/g	8,3	68,5	10,0	6,4	9,1
Reproducibility standard deviation (SR) μg/g	5,3	150	6,5	9,0	8,1
Reproducibility relative standard deviation (RSDR) (%)	6,1	4,1	4,1	3,6	4,1
Reproducibility limit (R) μg/g	14,8	407,2	18,2	25,2	22,7

Year of interlaboratory test	1 997
Number of laboratories	32
Number of samples	5
Analyte	propan-1-ol

Sample types
A Brandy; blind duplicates. B Kirsch; blind duplicates. C Grappa; blind duplicates. (*).
Samples	A	B	C
Number of laboratories retained after eliminating outliers	20	22	22
Number of outliers (laboratories)	4	4	6
Number of accepted results	40	44	44
Mean value ( ) μg/g	3,79	5,57	7,54
Repeatability standard deviation (Sr) μg/g	0,43	0,2	0,43
Repeatability relative standard deviation (RSDr) (%)	11,2	3,6	5,6
Repeatability limit (r) μg/g	1,1	0,6	1,2
Reproducibility standard deviation (SR) μg/g	0,59	0,55	0,82
Reproducibility relative standard deviation (RSDR) (%)	15,7	9,8	10,8
Reproducibility limit (R) μg/g	1,7	1,5	2,3

Year of interlaboratory test	1 997
Number of laboratories	32
Number of samples	5
Analyte	2-methylpropan-1-ol

Sample types
A Brandy; blind duplicates. B Kirsch; blind duplicates. C Grappa; blind duplicates. D Whisky; split levels (*). E Rum; split levels (*).
Samples	A	B	C	D	E
Number of laboratories retained after eliminating outliers	28	31	30	26	25
Number of outliers (laboratories)	3	0	1	5	6
Number of accepted results	56	62	60	52	50
Mean value ( ) μg/g	174,2	111,7	185,0	291,0	115,99
				246,8 (*)	133,87 (*)
Repeatability standard deviation (Sr) μg/g	2,3	1,6	2,5	1,8	0,74
Repeatability relative standard deviation (RSDr) (%)	1,3	1,4	1,3	0,7	0,6
Repeatability limit (r) μg/g	6,4	4,5	6,9	5,0	2,1
Reproducibility standard deviation (SR) μg/g	8,9	8,9	9,7	6,0	6,2
Reproducibility relative standard deviation (RSDR) (%)	5,1	8,0	5,2	2,2	5,0
Reproducibility limit (R) μg/g	24,9	24,9	27,2	16,9	17,4

Year of interlaboratory test	1 997
Number of laboratories	32
Number of samples	5
Analyte	2-methyl-butan-1-ol

Sample types
A Brandy; blind duplicates. B Kirsch; blind duplicates. C Grappa; blind duplicates. D Whisky; split levels (*). E Rum; split levels (*).
Samples	A	B	C	D	E
Number of laboratories retained after eliminating outliers	25	26	25	27	25
Number of outliers (laboratories)	3	2	3	1	2
Number of accepted results	50	52	50	54	50
Mean value ( ) μg/g	113,0	48,3	91,6	72,1	39,5
				45,2 (*)	61,5 (*)
Repeatability standard deviation (Sr) μg/g	2,1	1,5	1,7	2,3	2,3
Repeatability relative standard deviation (RSDr) (%)	1,9	3,1	1,8	3,9	4,5
Repeatability limit (r) μg/g	6,0	4,2	4,7	6,4	6,3
Reproducibility standard deviation (SR) μg/g	7,4	3,8	6,6	4,7	4,5
Reproducibility relative standard deviation (RSDR) (%)	6,6	7,9	7,2	8,1	8,8
Reproducibility limit (R) μg/g	20,8	10,7	18,4	13,3	12,5

Year of interlaboratory test	1 997
Number of laboratories	32
Number of samples	5
Analyte	3-methyl-butan-1-ol

Sample types
A Brandy; blind duplicates. B Kirsch; blind duplicates. C Grappa; blind duplicates. D Whisky; split levels (*). E Rum; split levels (*).
Samples	A	B	C	D	E
Number of laboratories retained after eliminating outliers	23	23	24	27	21
Number of outliers (laboratories)	5	5	4	1	6
Number of accepted results	46	46	48	54	42
Mean value ( ) μg/g	459,4	242,7	288,4	142,2	212,3
				120,4 (*)	245,6 (*)
Repeatability standard deviation (Sr) μg/g	5,0	2,4	3,4	2,4	3,2
Repeatability relative standard deviation (RSDr) (%)	1,1	1,0	1,2	1,8	1,4
Repeatability limit (r) μg/g	13,9	6,6	9,6	6,6	9,1
Reproducibility standard deviation (SR) μg/g	29,8	13	21	8,5	6,7
Reproducibility relative standard deviation (RSDR) (%)	6,5	5,2	7,3	6,5	2,9
Reproducibility limit (R) μg/g	83,4	35,4	58,8	23,8	18,7

[F3 III.3. DETERMINATION OF VOLATILE ACIDITY OF SPIRIT DRINKS U.K.

1. Scope U.K.

The method has been validated in an interlaboratory study for rum, brandy, marc and fruit spirits, at levels ranging from 30 mg/l to 641 mg/l.

2. Normative references U.K.

ISO 3696: 1987 Water for analytical use — Specifications and test methods.

3. Definitions U.K.

3.1. Volatile acidity is calculated by deducting the fixed acidity from the total acidity. U.K.

3.2. Total acidity is the sum of titratable acidities. U.K.

3.3. Fixed acidity is the acidity of the residue left after evaporating the spirit to dryness. U.K.

4. Principle U.K.

The total acidity and fixed acidity are determined by titration or by potentiometry.

5. Reagents and materials U.K.

During the analysis, unless otherwise stated, use only reagents of recognised analytical grade and water of at least grade 3 as defined in ISO 3696:1987.

5.1. 0,01 M sodium hydroxide solution (NaOH) U.K.

5.2. Mixed indicator solution: U.K.

• Weigh 0,1 g of indigo carmine and 0,1 g of phenol red.

• Dissolve in 40 ml water and make up to 100 ml with ethanol.

6. Apparatus and equipment U.K.

Indirect laboratory apparatus, grade A glassware and the following:

6.1.

Water pump

6.2.

Rotary evaporator or ultrasonic bath

6.3.

Equipment for potentiometric titration (optional).

7. Sampling and samples U.K.

Samples are stored at room temperature prior to analysis.

8. Procedure U.K.

8.1. Total acidity U.K.

8.1.1. Preparation of sample U.K.

The spirit is irradiated with ultrasonic (ultrasonication) or stirred two minutes under a vacuum to rid it of carbon dioxide if required.

8.1.2. Titration U.K.

Pipette 25 ml of the spirit into a 500 ml Erlenmeyer flask.

Add about 200 ml of cooled boiled distilled water (prepared fresh daily) and 2-6 drops of the mixed indicator solution (5.2).

Titrate with the 0,01 M sodium hydroxide solution (5.1) until the yellow-green colour changes to violet in the case of colourless spirits, the yellow-brown colour to red-brown in the case of brown-coloured spirits respectively.

The titration may also be carried out by potentiometry, to pH 7,5.

Let n ₁ ml be the volume of the 0,01 M sodium hydroxide solution added.

8.1.3. Calculation U.K.

The total acidity (TA) expressed in milliequivalents per l of spirit is equal to 0,4 × n ₁ .

The total acidity (TA′) expressed in mg of acetic acid per l of spirit is equal to 24 × n ₁ .

8.2. Fixed acidity U.K.

8.2.1. Preparation of sample U.K.

Evaporate 25 ml of the spirit to dryness:

• Pipette 25 ml of the spirit into a flat-bottomed cylindrical evaporating dish 55 mm in diameter. During the first hour of evaporation the evaporating dish is placed on the lid of a boiling water bath so that the liquid will not boil, as this could lead to losses through splattering.

• Complete the drying by placing the evaporating dish in a drying oven at 105 °C for two hours. Allow the evaporating dish to cool in a desiccator.

8.2.2. Titration U.K.

Dissolve the residue left after evaporating with cooled boiled distilled water (prepared fresh daily) and make up to a volume to circa 100 ml and add 2-6 drops of the mixed indicator solution (5.2).

Titrate with the 0,01 M sodium hydroxide solution (5.1).

The titration may also be carried out by potentiometry, to pH 7,5.

Let n ₂ ml be the volume of the 0,01 M sodium hydroxide solution added.

8.2.3. Calculation U.K.

The fixed acidity (FA) expressed in milliequivalents per l of spirit is equal to 0,4 × n ₂ .

The fixed acidity (FA) expressed in mg of acetic acid per l of spirit is equal to 24 × n ₂ .

9. Calculation of volatile acidity U.K.

9.1. Expression in milliequivalents per l: U.K.

Let:

TA

=

total acidity in milliequivalents per l

FA

=

fixed acidity in milliequivalents per l

Volatile acidity, VA, in milliequivalents per l is equal to:

TA – FA.

9.2. Expression in mg of acetic acid per l: U.K.

Let:

TA′

=

total acidity in mg of acetic acid per l

FA′

=

fixed acidity in mg of acetic acid per l

Volatile acidity, VA, in mg of acetic acid per l is equal to:

TA′ – FA′.

9.3. Expression in g of acetic acid per hl of pure 100 % vol. alcohol is equal to: U.K.

where A is the alcoholic strength by volume of the spirit drink.

10. Method performance characteristics (Precision) U.K.

10.1. Statistical results of the interlaboratory test U.K.

The following data were obtained from an international method performance study carried out to internationally agreed procedures (1) (2).

Year of interlaboratory test	2000
Number of laboratories	18
Number of samples	6

Samples	A	B	C	D	E	F
Number of laboratories retained after eliminating outliers	16	18	18	14	18	18
Number of outliers (laboratories)	2			4
Number of accepted results	32	36	36	28	36	36
Mean value (mg/L)	272* 241*	30	591* 641*	46	107	492
Repeatability standard deviation, s r (mg/l)	8,0	3,6	15,0	3,7	6,7	8,5
Repeatability relative standard deviation, RSDr (%)	3,1	11,8	2,4	8,0	6,2	1,7
Repeatability limit, r (mg/l)	23	10	42	10	19	24
Reproducibility standard deviation, s R (mg/l)	8,5	8,4	25,0	4,55	13,4	24,4
Reproducibility relative standard deviation, RSD R (%)	3,3	27,8	4,1	9,9	12,5	5,0
Reproducibility limit, R (mg/l)	24	23	70	13	38	68

Sample types:

A

Plum spirit; split level *

B

Rum I; blind duplicates

C

Rum II; split level *

D

Slivovitz; blind duplicates

E

Brandy; blind duplicates

F

Marc spirit; blind duplicates.

(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

F3 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.