(3)

The following Chapter VIII is inserted: ‘VIII. TOTAL SUGARS 1. Scope The HPLC–RI method is applicable for the determination of total sugars (expressed as invert sugar) in spirit drinks, with the exclusion of liqueurs containing egg and milk products. The method has been validated in an interlaboratory study for pastis, distilled anis, cherry liqueur, crème de (followed by the name of a fruit or the raw material used) and crème de cassis, at levels ranging from 10,86 g/l to 509,7 g/l. However, linearity of the instrument response was proven for the concentration range 2,5 g/l to 20,0 g/l. This method is not intended for determining low levels of sugars. 2. Normative references ISO 3696:1987 Waters for analytical use — Specifications and test methods. 3. Principle High-performance liquid chromatography assays of sugar solutions, in order to determine their glucose, fructose, sucrose, maltose and lactose concentrations. This method uses an alkylamine stationary phase and differential refractometry detection and is given as an example. The use of anion exchange resins as stationary phase would also be possible. 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) The elution solvent is composed of: 75 parts by volume of acetonitrile (4.6), 25 parts by volume of distilled water (4.7). Pass helium through at a slow rate for 5-10 minutes prior to use to degas. If the water being used has not been microfiltered, the solvent should be filtered with a filter for organic solvents with a pore size less than or equal to 0,45 μm. 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) Weigh 2 g each of the sugars to be analysed (4.1 to 4.5), transfer them without loss to a 100 ml volumetric flask. (NB 2,11 g of maltose monohydrate is equivalent to 2 g of maltose). Adjust to 100 ml with a 5 % vol. alcohol solution (4.10), shake and store at around + 4 °C. Prepare a new stock solution once a week. 4.12. Preparation of working standard solutions (2,5, 5,0, 7,5, 10,0 and 20,0 g/L) Dilute the stock solution, 20 g/l (4.11) appropriately with a 5 % vol. alcohol solution (4.10) to give five working standards of 2,5, 5,0, 7,5, 10,0 and 20,0 g/l. Filter with a filter of a pore size less than or equal to 0,45 μm (5.3). 5. Apparatus and Equipment 5.1. HPLC system capable of achieving baseline resolution of all of the sugars. 5.1.1. High-performance liquid chromatograph with a six-way injection valve fitted with a 10 μl loop or any other device, whether automatic or manual, for the reliable injection of microvolumes. 5.1.2. Pumping system enabling one to achieve and maintain a constant or programmed rate of flow with great precision. 5.1.3. Differential refractometer. 5.1.4. Computational integrator or recorder, the performance of which is compatible with the rest of the set-up. 5.1.5. Pre-column: It is recommended that a suitable pre-column is attached to the analytical column. 5.1.6. Column (example): Material: stainless steel or glass. Internal diameter: 2-5 mm. Length: 100-250 mm (depending on the packing particle size), for example, 250 mm if the particles are 5 μm in diameter. Stationary phase: alkylamine functional groups bonded to silica, maximum particle size 5 μm. 5.1.7. Chromatography conditions (example): Elution solvent (4.8), flow rate: 1 ml/minute. Detection: Differential refractometry. To make certain that the detector is perfectly stable, it should be switched on a few hours before use. The reference cell must be filled with the elution solvent. 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 On receipt, samples are to be stored at room temperature prior to analysis. 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 less than or equal to 0,45 μm (5.3). 7.2. PART B: HPLC 7.2.1. Determination Inject 10 μl of the standard solutions (4.12) and samples (7.1.2). Perform the analysis under suitable chromatography conditions, for example those described above. 7.2.2. Should any peak of a sample have a greater area (or height) than the corresponding peak in the most concentrated standard, then the sample should be diluted with distilled water and reanalysed. 8. Calculation Compare the two chromatograms obtained for the standard solution and spirit. Identify the peaks by their retention times. Measure their areas (or heights) to calculate the concentrations by the external standard method. Take into account any dilutions made to the sample. The final result is the sum of sucrose, maltose, lactose, glucose and fructose, expressed as invert sugar in g/l. Invert sugar is calculated as the sum of all monosaccharides and reducing disaccharides present, plus the stoichiometric amount of glucose and fructose calculated from the sucrose present. Invert sugar (g/l) = glucose (g/l) + fructose (g/l) + maltose (g/l) + lactose (g/l) + (sucrose (g/l) × 1,05). 1,05 = (molecular weight of fructose + molecular weight of glucose)/molecular weight of sucrose. 9. Method performance characteristics (precision) 9.1. Statistical results of the interlaboratory test 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 24 Number of samples 8 (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. Table 1 Fructose, glucose, maltose Analyte Fructose Glucose Maltose Samples (× 2) Crème de Cassis Standard (50 g/l) Aniseed-flavoured spirit drink Crème de Cassis Standard (50 g/l) Aniseed-flavoured spirit drink Standard (10 g/l) Mean value (g/l) 92,78 50,61 15,62 93,16 50,06 15,81 9,32 No of labs without outliers 21 22 21 23 19 21 22 Repeatability standard deviation, sr, (g/l) 2,34 2,12 0,43 3,47 1,01 0,48 0,54 Repeatability relative standard deviation, RSDr (%) 2,53 4,2 2,76 3,72 2,03 3,02 5,77 Repeatability limit, r (g/l) (r = 2,8 × sr) 6,56 5,95 1,21 9,71 2,84 1,34 1,51 Reproducibility standard deviation, sR (g/l) 7,72 3,13 0,84 9,99 2,7 0,88 1,4 Reproducibility relative standard deviation, RSDR (%) 8,32 6,18 5,37 10,72 5,4 5,54 15,06 Reproducibility limit, R (g/l) (R = 2,8 × sR) 21,62 8,76 2,35 27,97 7,57 2,45 3,93

Table 2 Sucrose Analyte Sucrose Samples Pastis Ouzo Cherry liqueur Crème de Menthe Crème de Cassis Standard (100 g/l) Mean value (g/l) 10,83 29,2 19,7 (*) 103,33 349,96 319,84 99,83 No of labs without outliers 19 19 20 18 18 18 Repeatability standard deviation, sr (g/l) 0,09 0,75 2,17 5,99 4,31 1,25 Repeatability relative standard deviation, RSDr (%) 0,81 3,07 2,1 1,71 1,35 1,25 Repeatability limit, r (g/l) (r = 2,8 × sr) 0,25 2,1 6,07 16,76 12,06 3,49 Reproducibility standard deviation, sR (g/l) 0,79 0,92 4,18 9,94 16,11 4,63 Reproducibility relative standard deviation, RSDR (%) 7,31 3,76 4,05 2,84 5,04 4,64 Reproducibility limit, R (g/l) (R = 2,8 × sR) 2,22 2,57 11,7 27,84 45,12 12,97

Table 3 Total Sugars (Note: this data was calculated for total sugars, not invert sugar as defined in Section 8 above.)

Samples Pastis Ouzo Aniseed-flavoured spirit drink Cherry liqueur Crème de Menthe Crème de Cassis Standard (220 g/l) Mean value (g/l) 10,86 29,2 19,7 (**) 31,59 103,33 349,73 509,69 218,78 No of Labs without outliers 20 19 20 20 18 18 19 Repeatability standard deviation, sr (g/l) 0,13 0,75 0,77 2,17 5,89 5,59 2,71 Repeatability relative standard deviation, RSDr (%) 1,16 3,07 2,45 2,1 1,69 1,1 1,24 Repeatability limit, r (g/l) (r = 2,8 × sr) 0,35 2,1 2,17 6,07 16,5 15,65 7,59 Reproducibility standard deviation sR (g/l) 0,79 0,92 1,51 4,18 9,98 14,81 8,53 Reproducibility relative standard deviation, RSDR (%) 7,25 3,76 4,79 4,04 2,85 2,91 3,9 Reproducibility limit R (g/l) (R = 2,8 × sR) 2,21 2,57 4,24 11,7 27,94 41,48 23,89