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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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VI.
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This method is suitable for the determination of glycyrrhizic acid in aniseed-flavoured spirit drinks using high performance liquid chromatography (HPLC). [F2Regulation 110/2008] specifies that any aniseed-flavoured spirit called ‘ pastis ’ must contain between 0,05 and 0,5 g of glycyrrhizic acid per litre.
Textual Amendments
F2Words in Annex Ch. 6 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)(b)
ISO 3696: 1987 Water for analytical laboratory use — Specifications and test methods.
The glycyrrhizic acid concentration is determined using high-performance liquid chromatography (HPLC) with UV detection. A standard solution and the test sample are filtered and they are separately injected directly into the HPLC system.
During the analysis, use only reagents of HPLC grade, absolute ethanol and water of grade 3 as defined by ISO 3696.
(Mol. Wt.: 839,98)(CAS 53956-04-0): purity at least 90 %
(Mol. Wt.: glycyrrhizic acid 822,94).
For 1 000 ml at 20 o C:
96 % vol. ethanol (4.1): 521 ml
Water (2.0): 511 ml.
80 parts (by volume) of water (2.0)
20 parts (by volume) of acetic acid (4.3).
Degas the elution solvent for five minutes.
If the water being used has not been microfiltered, it is advisable to filter the prepared elution solvent on a filter for organic solvents with a pore size less than or equal to 0,45 μm. U.K.
Methanol (4.4).
All standard solutions must be freshly prepared after two months.
Weigh to the nearest 0,1 mg, 25 mg of ammonium glycyrrhizinate (4.2) in a 100 ml volumetric flask. Add some 50 % vol. ethanol (4.5) and dissolve the ammonium glycyrrhizinate. When it has dissolved make up to the mark with 50 % vol. ethanol (4.5).
Filter through a filter for organic solvents.
A 1,0 g/l stock solution is prepared by weighing, to the nearest 0,1 mg, 100 mg of ammonium glycyrrhizinate in a 100 ml volumetric flask. Add some 50 % vol. ethanol (4.5) and dissolve the ammonium glycyrrhizinate. When it has dissolved make up to the mark with 50 % vol. ethanol (4.5).
At least four other solutions corresponding to 0,05, 0,1, 0,25 and 0,5 g/l of ammonium glycyrrhizinate are prepared by pipetting respectively 5 ml, 10 ml, 25 ml and 50 ml of the 1,0 g/l stock solution in separate 100 ml volumetric flasks. Then make up to the mark with 50 % vol. ethanol (4.5) and mix up thoroughly.
Filter all solutions though a filter for organic solvents.
Material: stainless steel or glass
Internal diameter: 4 to 5 mm
Length: 100 to 250 mm
Stationary phase: cross-linked silica with a (preferably spherical) octadecyl functional group (C18), maximum particle size: 5 μm.
flow rate: 1 ml/minute,
solvent A = 30 %,
solvent B = 70 %.
UV = 254 nm
Filter, if necessary, through a filter for organic solvents (pore diameter: 0,45 μm).
Once the chromatography conditions have stabilised,
inject 20 μl of the reference solution C (4.7.1),
inject 20 μl of the sample solution,
compare the two chromatograms. Identify the glycyrrhizic acid peaks from their retention times. Measure their areas (or heights) and calculate the concentration in g/l to two decimal figures using the following equation:
where:
is the concentration in grams per litre of glycyrrhizic acid in the spirit being analysed
is the concentration in grams per litre of ammonium glycyrrhizinate in the reference solution
is the area (or height) of the glycyrrhizic acid peak of the spirit being analysed
is the area (or height) of the glycyrrhizic acid peak of the reference solution
is the purity of the reference ammonium glycyrrhizinate (in %)
is the mass of one mole of glycyrrhizic acid
is the mass of one mole of ammonium glycyrrhizinate.
Statistical results of the interlaboratory test:
the following table give the values for glycyrrhizic acid.
The following data were obtained from an international method performance study carried out to internationally agreed procedures.
Year of interlaboratory test | 1998 |
Number of laboratories | 16 |
Number of samples | 5 |
Analyte | glycyrrhizic acid |
Samples | A | B | C | D | F |
---|---|---|---|---|---|
Number of laboratories retained after eliminating outliers | 13 | 14 | 15 | 16 | 16 |
Number of outliers (laboratories) | 3 | 2 | 1 | — | — |
Number of accepted results | 26 | 28 | 30 | 32 | 32 |
Mean value g/l | 0,046 | 0,092 (*) 0,099 | 0,089 | 0,249 | 0,493 |
Repeatability standard deviation (S r ) g/l | 0,001 | 0,001 | 0,001 | 0,002 | 0,003 |
Repeatability relative standard deviation (RSD r ) (%) | 1,5 | 1,3 | 0,7 | 1,0 | 0,6 |
Repeatability limit (r) g/l | 0,002 | 0,004 | 0,002 | 0,007 | 0,009 |
Reproducibility standard deviation (S R ) g/l | 0,004 | 0,007 | 0,004 | 0,006 | 0,013 |
Reproducibility relative standard deviation (RSD R ) (%) | 8,6 | 7,2 | 4,0 | 2,5 | 2,7 |
Reproducibility limit (R) g/l | 0,011 | 0,019 | 0,01 | 0,018 | 0,037 |
Sample types:
pastis, blind duplicates
pastis, split levels (*)
pastis, blind duplicates
pastis, blind duplicates
pastis, blind duplicates]
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