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Commission Regulation (EC) No 2870/2000 of 19 December 2000 laying down Community reference methods for the analysis of spirits drinks
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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.
ISO 3696:1987 Waters for analytical use — Specifications and test methods.
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.
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.
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.
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).
It is recommended that a suitable pre-column is attached to the analytical column.
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. |
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.
On receipt, samples are to be stored at room temperature prior to analysis.
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.
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.
=
glucose (g/l) + fructose (g/l) + maltose (g/l) + lactose (g/l) + (sucrose (g/l) × 1,05).
=
(molecular weight of fructose + molecular weight of glucose)/molecular weight of sucrose.
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 |
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, s r , (g/l) | 2,34 | 2,12 | 0,43 | 3,47 | 1,01 | 0,48 | 0,54 |
Repeatability relative standard deviation, RSD r (%) | 2,53 | 4,2 | 2,76 | 3,72 | 2,03 | 3,02 | 5,77 |
Repeatability limit, r (g/l) (r = 2,8 × s r ) | 6,56 | 5,95 | 1,21 | 9,71 | 2,84 | 1,34 | 1,51 |
Reproducibility standard deviation, s R (g/l) | 7,72 | 3,13 | 0,84 | 9,99 | 2,7 | 0,88 | 1,4 |
Reproducibility relative standard deviation, RSD R (%) | 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 |
Sucrose
a split level. | ||||||
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 a | 103,33 | 349,96 | 319,84 | 99,83 |
No of labs without outliers | 19 | 19 | 20 | 18 | 18 | 18 |
Repeatability standard deviation, s r (g/l) | 0,09 | 0,75 | 2,17 | 5,99 | 4,31 | 1,25 |
Repeatability relative standard deviation, RSD r (%) | 0,81 | 3,07 | 2,1 | 1,71 | 1,35 | 1,25 |
Repeatability limit, r (g/l) (r = 2,8 × s r ) | 0,25 | 2,1 | 6,07 | 16,76 | 12,06 | 3,49 |
Reproducibility standard deviation, s R (g/l) | 0,79 | 0,92 | 4,18 | 9,94 | 16,11 | 4,63 |
Reproducibility relative standard deviation, RSD R (%) | 7,31 | 3,76 | 4,05 | 2,84 | 5,04 | 4,64 |
Reproducibility limit, R (g/l) (R = 2,8 × s R ) | 2,22 | 2,57 | 11,7 | 27,84 | 45,12 | 12,97 |
Total Sugars
( Note: this data was calculated for total sugars, not invert sugar as defined in Section 8 above.) | |||||||
a split level.] ] | |||||||
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 a | 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, s r (g/l) | 0,13 | 0,75 | 0,77 | 2,17 | 5,89 | 5,59 | 2,71 |
Repeatability relative standard deviation, RSD r (%) | 1,16 | 3,07 | 2,45 | 2,1 | 1,69 | 1,1 | 1,24 |
Repeatability limit, r (g/l) (r = 2,8 × s r ) | 0,35 | 2,1 | 2,17 | 6,07 | 16,5 | 15,65 | 7,59 |
Reproducibility standard deviation s R (g/l) | 0,79 | 0,92 | 1,51 | 4,18 | 9,98 | 14,81 | 8,53 |
Reproducibility relative standard deviation, RSD R (%) | 7,25 | 3,76 | 4,79 | 4,04 | 2,85 | 2,91 | 3,9 |
Reproducibility limit R (g/l) (R = 2,8 × s R ) | 2,21 | 2,57 | 4,24 | 11,7 | 27,94 | 41,48 | 23,89 |
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