I.DETERMINATION OF ALCOHOLIC STRENGTH BY VOLUME OF SPIRIT DRINKS Introduction 1.Scope 2.Normative References 3.Terms and Definitions 3.1.Reference temperature: Note 1: 3.2.Density: 3.3.Specific gravity: Note 2: 3.4.Real alcoholic strength by volume: Note 3: 4.Principle APPENDIX I: PREPARATION OF DISTILLATE 1.Scope 2.Principle 3.Reagents and Materials 3.1.Anti-bumping granules. 3.2.Concentrated antifoam emulsion (for crème liqueurs). 4.Apparatus and equipment Note: 5.Storage of test samples 6.Procedure 6.1.Distillation apparatus verification. 6.2.Spirit drinks with alcoholic strength below 50 % vol. Note: 6.3.Spirit drinks with alcoholic strength above 50 % vol. Note: APPENDIX II: MEASUREMENT OF DENSITY OF DISTILLATE METHOD A:DETERMINATION OF REAL ALCOHOLIC STRENGTH BY VOLUME OF SPIRIT DRINKS...A.1.Principle A.2.Reagents and materials A.2.1.Sodium chloride solution (2 % w/v) A.3.Apparatus and Equipment Note 1: A.4.Procedure A.4.1.Calibration of pycnometer A.4.1.1.Calibration using a single-pan balance: A.4.1.1.1.Weigh the clean, dry pycnometer (P). A.4.1.1.2.Fill the pycnometer carefully with distilled water at ambient temperature...A.4.1.1.3.Weigh the tare bottle (T0). A.4.1.1.4.Calculation A.4.1.2.Calibration method using a twin-pan balance: A.4.2.Determination of alcoholic strength of test sample A.5.Method performance characteristics (precision) A.5.1.Statistical results of the interlaboratory test METHOD B:DETERMINATION OF REAL ALCOHOLIC STRENGTH BY VOLUME OF SPIRIT DRINKS...B.1.Principle B.2.Reagents and materials B.3.Apparatus and equipment B.4.Procedure B.5.Method performance characteristics (precision) B.5.1.Statistical results of the interlaboratory test METHOD C:DETERMINATION OF REAL ALCOHOLIC STRENGTH BY VOLUME OF SPIRIT DRINKS...C.1.Principle C.2.Reagents and materials C.2.1.Float cleaning solution (sodium hydroxide, 30 % w/v) C.3.Apparatus and Equipment Note 1: C.4.Procedure C.5.Method performance characteristics (precision) C.5.1.Statistical results of the interlaboratory test II.DETERMINATION OF TOTAL DRY EXTRACT OF SPIRIT DRINKS BY GRAVIMETRY...1.Scope 2.Normative References 3.Definition 4.Principle 5.Apparatus and Equipment 6.Sampling and samples 7.Procedure 7.1.Pipette 25 ml of the spirit containing less than 15...7.2.Complete the drying by placing the evaporating dish in a...8.Calculation 9.Method performance characteristics (precision) 9.1.Statistical results of the interlaboratory test III.DETERMINATION OF VOLATILE SUBSTANCES AND METHANOL OF SPIRIT DRINKS III.1.GENERAL REMARKS 1.Definitions 2.Gas chromatographic analysis of volatile compounds III.2.GAS CHROMATOGRAPHIC DETERMINATION OF VOLATILE CONGENERS: ALDEHYDES, HIGHER ALCOHOLS, ETHYL...1.Scope 2.Normative References 3.Definition 4.Principle 5.Reagents and materials 6.Apparatus and equipment 7.Sampling and samples. 8.Procedure (used for the validated method) 9.Calculation 10.Quality assurance and control (used for the validated method) 11.Method performance characteristics (precision) III.3.DETERMINATION OF VOLATILE ACIDITY OF SPIRIT DRINKS 1.Scope 2.Normative references 3.Definitions 3.1.Volatile acidity is calculated by deducting the fixed acidity from...3.2.Total acidity is the sum of titratable acidities. 3.3.Fixed acidity is the acidity of the residue left after...4.Principle 5.Reagents and materials 5.1.0,01 M sodium hydroxide solution (NaOH) 5.2.Mixed indicator solution: 6.Apparatus and equipment 7.Sampling and samples 8.Procedure 8.1.Total acidity 8.1.1.Preparation of sample 8.1.2.Titration 8.1.3.Calculation 8.2.Fixed acidity 8.2.1.Preparation of sample 8.2.2.Titration 8.2.3.Calculation 9.Calculation of volatile acidity 9.1.Expression in milliequivalents per l: 9.2.Expression in mg of acetic acid per l: 9.3.Expression in g of acetic acid per hl of pure...10.Method performance characteristics (Precision) 10.1.Statistical results of the interlaboratory test (1)‘Protocol for the design, conduct and interpretation of method-performance studies’,...(2)Horwitz, W. (1982) Analytical Chemistry , 54, 67A-76A. V.ANETHOLE. GAS CHROMATOGRAPHIC DETERMINATION OF TRANS-ANETHOLE IN SPIRIT DRINKS 1.Scope 2.Normative references 3.Principle 4.Reagents and materials 4.1.Ethanol 96 % vol. (CAS 64-17-5) 4.2.1-methoxy-4-(1-propenyl) benzene; (trans-anethole) (CAS 4180-23-8) 4.3.4-allylanisole, (estragole) (CAS 140-67-0), suggested internal standard (IS) 4.4.Ethanol 45 % vol. 4.5.Preparation of standard solutions 4.5.1.Standard solution A 4.5.2.Internal standard solution B 4.5.3.Solutions used to check the linearity response of the flame...4.5.4.Standard solution C 5.Apparatus and equipment 5.1.A capillary gas chromatograph fitted with a flame ionisation detector...5.2.Split/splitless injector 5.3.Capillary column, for example: 5.4.Common laboratory equipment: A grade volumetric glassware, analytical balance (precision:...6.Chromatography conditions 7.Samples 8.Procedure 8.1.Sample screening for estragole 8.2.Preparation of unknown samples 8.3.Blank 8.4.Linearity test 8.5.Determination 9.Calculation of response factor 9.1.Response factor (RF i ) calculation 9.2.Analysis of the linearity response test solutions 9.3.Analysis of the sample 10.Calculation of results 11.Quality assurance and control 12.Treatment of spirits sample containing large amount of sugar and...12.1.Principle 12.2.Reagents and materials 12.2.1.Ammonium sulphate, anhydrous, (CAS 7783-20-2). 12.2.2.Sodium phosphate, dibasic, dodecahydrate, (CAS 10039-32-4). 12.3.Apparatus and equipment 12.4.Procedure 12.4.1.Sample screening for estragole 12.4.2.Extraction 12.4.3.Preparation of the extracted sample to be analysed 12.5.Determination 12.6.Calculation of results 12.7.Quality control and assurance 13.Method performance characteristics (precision) VI.GLYCYRRHIZIC ACID. DETERMINATION OF GLYCYRRHIZIC ACID USING HIGH PERFORMANCE LIQUID...1.Scope 2.Normative references 3.Principle 4.Reagents and materials 4.1.Ethanol 96 % vol. (CAS 64-17-5). 4.2.Ammonium glycyrrhizinate, C 42 H 62 O 16 .NH 3...4.3.Glacial acetic acid, CH 3 COOH, (CAS 64-19-7). 4.4.Methanol, CH 3 OH (CAS 67-56-1). 4.5.Ethanol 50 % vol. 4.6.Preparation of the HPLC elution solutions 4.6.1.Elution solvent A (example) Note: 4.6.2.Elution solvent B 4.7.Preparation of standard solutions 4.7.1.Reference solution C 4.7.2.Standard solutions used to check the linearity of the response...5.Apparatus and equipment 5.1.Separation system 5.1.1.High-performance liquid chromatograph. 5.1.2.Pumping system enabling one to achieve and maintain a constant...5.1.3.UV spectrophotometric detection system: to be set at 254 nm....5.1.4.Solvent degassing system. 5.2.Computational integrator or recorder, the performance of which is compatible...5.3.Column (example): 5.4.Laboratory equipment 5.4.1.Analytical balance with a precision of 0,1 mg 5.4.2.A-grade volumetric glassware 5.4.3.Micromembrane filtration arrangement for small volumes. 6.Chromatography conditions 6.1.Elution characteristics: (example) 6.2.Detection: 7.Procedure 7.1.Preparation of the spirit sample 7.2.Determination 8.Method performance characteristics (precision) VII.CHALCONES. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR VERIFYING THE PRESENCE OF...1.Scope 2.Normative references 3.Principle 4.Reagents and materials 4.1.Ethanol 96 % vol. (CAS 64-17-5) 4.2.Acetonitrile, CH 3 CN, (CAS 75-05-8) 4.3.Reference substance: Glycyrrhiza glabra : liquorice, ‘sweet root’ 4.4.Sodium acetate, CH 3 COONa, (CAS 127-09-3) 4.5.Glacial acetic acid, CH 3 COOH, (CAS 64-19-7) 4.6.Preparation of solutions 4.6.1.Ethanol 50 % volume 4.6.2.Solvent A: acetonitrile 4.6.3.Solvent B: 0,1 M sodium acetate buffer solution, pH 4,66....5.Preparation of the reference extract from Glycyrrhiza glabra (4.3) 5.1.Weigh 10 g of ground liquorice root ( Glycyrrhiza glabra...5.2.Recover the liquorice extract from the filter 5.3.The liquorice root extraction must be performed three times in...5.4.Combine the three filtrates. 5.5.Evaporate the solvent phase (of 5.4) on a rotary evaporator....5.6.Take up the residual extract (of 5.5) with 100 ml...6.Apparatus and equipment 6.1.Separation system. 6.1.1.High-performance liquid chromatograph. 6.1.2.Pumping system capable of achieving and maintaining a constant or...6.1.3.UV/visible spectrophotometric detection system that can be set at 254...6.1.4.Solvent degassing system: 6.1.5.Column oven that can be set at a temperature of...6.2.Computational integrator or recorder, the performance of which is compatible...6.3.Column 6.4.Common laboratory equipment, including: 6.5.Chromatography conditions (example). 6.5.1.Elution characteristics of solvents A (4.6.2) and B (4.6.3): 6.5.2.Flow rate: 1 ml/minute. 6.5.3.UV detector settings: 7.Procedure 7.1.Preparation of the spirit sample 7.2.Preparation of the residual liquorice extract (5.6) 7.3.Determination 7.3.1.Inject 20 μl of the prepared liquorice extract (7.2). Perform...7.3.2.Inject 20 μl of the sample (7.1) (aniseed-flavoured spirit sample)....7.3.3.Compare the two chromatograms. There must be a great similarity...8.Characteristic chromatogram for a pastis 9.Method performance characteristics (precision) VIII.TOTAL SUGARS 1.Scope 2.Normative references 3.Principle 4.Reagents and materials 4.1.Glucose (CAS 50-99-7), at least 99 % pure. 4.2.Fructose (CAS 57-48-7), at least 99 % pure. 4.3.Sucrose (CAS 57-50-1), at least 99 % pure. 4.4.Lactose (CAS 5965-66-2), at least 99 % pure. 4.5.Maltose monohydrate (CAS 6363-53-7), at least 99 % pure. 4.6.Pure acetonitrile (CAS 75-05-8) for HPLC analysis. 4.7.Distilled or demineralised water, preferably microfiltered. 4.8.Solvents (example) 4.9.Ethanol absolute (CAS 64-17-5). 4.10.Ethanol solution (5 %, v/v). 4.11.Preparation of stock standard solution (20 g/l) 4.12.Preparation of working standard solutions (2,5, 5,0, 7,5, 10,0 and...5.Apparatus and Equipment 5.1.HPLC system capable of achieving baseline resolution of all of...5.1.1.High-performance liquid chromatograph with a six-way injection valve fitted with...5.1.2.Pumping system enabling one to achieve and maintain a constant...5.1.3.Differential refractometer. 5.1.4.Computational integrator or recorder, the performance of which is compatible...5.1.5.Pre-column: 5.1.6.Column (example): 5.1.7.Chromatography conditions (example): 5.2.Analytical balance accurate to 0,1 mg. 5.3.Filtration set-up for small volumes using a 0,45 μm micromembrane....6.Sample storage 7.Procedure 7.1.PART A: Sample preparation 7.1.1.Shake the sample. 7.1.2.Filter the sample through a filter with a pore size...7.2.PART B: HPLC 7.2.1.Determination 7.2.2.Should any peak of a sample have a greater area...8.Calculation 9.Method performance characteristics (precision) 9.1.Statistical results of the interlaboratory test (1)‘ Protocol for the design, conduct and interpretation of method-performance...(2)Horwitz, W. (1982) Analytical Chemistry , 54, 67A-76A. IX.EGG YOLK. DETERMINATION OF EGG YOLK CONCENTRATION IN SPIRIT DRINKS...1.Scope 2.Normative references 3.Principle 4.Reagents and materials 4.1.Double-distilled water 4.2.Diatomaceous earth 4.3.Ethanol 96 % vol. (CAS 64-17-5) 4.4.15 % magnesium acetate (CAS 16674-78-5) solution 4.5.10 % sulphuric acid (CAS 7664-93-9) 4.6.1 N sulphuric acid. 4.7.0,16 g/l potassium dihydrogen phosphate (CAS 778-77-0), KH 2 PO...4.8.Reagent for phosphate determination: 5.Apparatus and equipment 5.1.100 ml conical flask 5.2.Ultrasonic bath (or magnetic stirrer) 5.3.100 ml volumetric flask 5.4.20 o C water bath 5.5.Filter (Whatman No 4 or equivalent) 5.6.Porcelain (or platinum) crucible 5.7.Boiling water bath 5.8.Hot plate 5.9.Muffle furnace 5.10.50 ml volumetric flask 5.11.20 ml volumetric flask 5.12.Spectrophotometer set at 420 nm 5.13.1 cm cuvette. 6.Samples 7.Procedure 7.1.Sample preparation 7.1.1.Weigh 10 g of the sample into a 100 ml...7.1.2.Add gradually 70 ml of ethanol (4.3) in small portions,...7.1.3.Transfer the contents of the flask to a 100 ml...7.1.4.Add a small amount of diatomaceous earth (4.2) and filter...7.1.5.Transfer 25 ml of the filtrate to a porcelain (or...7.1.6.Place the crucibles on a hot plate (5.8) and heat...7.1.7.Ash the residue by heating to incandescence at 600 o...7.1.8.Take up the ash with 10 ml of 10 %...7.2.Photometric phosphate assay 7.2.1.Comparative solution 7.2.1.1.Place 10 ml of 10 % sulphuric acid (4.5) in...7.2.1.2.Add to a 5 ml aliquot of this solution (7.2.1.1),...7.2.1.3.Stopper with a loosely inserted stopper, shake, and heat in...7.2.1.4.Fill a 1 cm cuvette (5.13) with this comparative solution....7.2.2.Sample solution 7.2.2.1.Add to a 5 ml aliquot of the ash solution...7.2.2.2.Stopper with a loosely inserted stopper, shake, and heat in...7.2.2.3.The yellow solution that develops is immediately analysed spectrophotometrically (5.12)...7.2.3.Calibration curve 7.2.3.1.To construct the calibration curve, add 2 ml aliquots of...7.2.3.2.Stopper with a loosely inserted stopper, shake, and heat in...7.2.3.3.Construction of the calibration curve: 8.Expression of results 9.Method performance characteristics (precision) X.DETERMINATION OF THE FOLLOWING WOOD COMPOUNDS IN SPIRIT DRINKS BY...1.Scope 2.Normative reference 3.Principle 4.Reagents 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). 4.5.Reference standards of 99 % minimum purity: furfural, 5-hydroxymethyl furfural,...4.6.Reference solution: the standard substances are dissolved in a 50...5.Apparatus 5.1.A high-performance liquid chromatograph capable of functioning in binary gradient...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,...6.Procedure 6.1.Preparation of the solution to be injected 6.2.Chromatographic operating conditions: carry out the analysis at ambient temperature...6.3.Determination 6.3.1.Inject the reference standards separately, then mixed. 6.3.2.Inject the sample as prepared in 6.1. 6.3.3.Measure the area of the peaks in the reference solution...7.Expression of results 8.Performance characteristics of the method (precision) 8.1.Furfural 8.2.5-Hydroxymethylfurfural 8.3.5-Methylfurfural 8.4.Vanillin 8.5.Syringaldehyde 8.6.Coniferaldehyde 8.7.Sinapaldehyde 8.8.Gallic acid 8.9.Ellagic acid 8.10.Vanillic acid 8.11.Syringic acid 8.12.Scopoletin (1)‘Protocol for the design, conduct and interpretation of method-performance studies’,...(2)Horwitz, W. (1982) Analytical Chemistry , 54, 67A-76A.
Commission Regulation (EC) No 2870/2000
of 19 December 2000
laying down Community reference methods for the analysis of spirits drinks
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Community,
Having regard to Council Regulation (EEC) No 1576/89 of 29 May 1989 laying down general rules on the definition, description and presentation of spirit drinks1, as amended by the Act of Accession of Austria, Finland and Sweden, and in particular Article 4(8) thereof,
Whereas:
(1)
Article 4(8) of Regulation (EEC) No 1576/89 provides for the adoption of methods to be used for analysing spirit drinks. Reference methods should be used to ensure compliance with Regulation (EEC) No 1576/89 and Commission Regulation (EEC) No 1014/90 of 24 April 1990 laying down detailed implementing rules on the definition, description and presentation of spirit drinks2, as last amended by Regulation (EC) No 2140/983, when any official control takes place or in the event of a dispute.
(2)
As far as possible, it would be useful to adopt and describe as Community analytical reference methods generally recognised methods.
(3)
In order to take account of scientific advances and of differences in the equipment of official laboratories, the use of methods based on principles of measurement other than the reference methods described in the Annex hereto should be permitted under the responsibility of the laboratory director, where those methods offer adequate guarantees as regards results and in particular meet the criteria set in the Annex to Council Directive 85/591/EEC of 20 December 1985 concerning the introduction of Community methods of sampling and analysis for the monitoring of foodstuffs intended for human consumption4 and if it can be shown that the variation in the accuracy, repeatability and reproducibility of the results obtained is within the limits of those obtained using the reference methods described in this Regulation. If that condition is met, the use of other analytical methods should be allowed. However, it is important to specify that in cases of dispute other methods may not replace reference methods.
(4)
The measures provided for in this Regulation are in accordance with the opinion of the Implementation Committee for Spirit Drinks,
HAS ADOPTED THIS REGULATION: