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

[F1 VI. GLYCYRRHIZIC ACID. DETERMINATION OF GLYCYRRHIZIC ACID USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY U.K.

1. Scope U.K.

This method is suitable for the determination of glycyrrhizic acid in aniseed-flavoured spirit drinks using high performance liquid chromatography (HPLC). [F2 Regulation 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

F2 Words 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)

2. Normative references U.K.

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

3. Principle U.K.

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.

4. Reagents and materials U.K.

During the analysis, use only reagents of HPLC grade, absolute ethanol and water of grade 3 as defined by ISO 3696.

4.1. Ethanol 96 % vol. (CAS 64-17-5). U.K.

4.2. Ammonium glycyrrhizinate, C ₄₂ H ₆₂ O ₁₆ .NH ₃ (Glycyrrhizic acid ammonium salt) U.K.

(Mol. Wt.: 839,98)(CAS 53956-04-0): purity at least 90 %

(Mol. Wt.: glycyrrhizic acid 822,94).

4.3. Glacial acetic acid, CH ₃ COOH, (CAS 64-19-7). U.K.

4.4. Methanol, CH ₃ OH (CAS 67-56-1). U.K.

4.5. Ethanol 50 % vol. U.K.

For 1 000 ml at 20 ^o C:

• 96 % vol. ethanol (4.1): 521 ml

• Water (2.0): 511 ml.

4.6. Preparation of the HPLC elution solutions U.K.

4.6.1. Elution solvent A (example) U.K.

80 parts (by volume) of water (2.0)

20 parts (by volume) of acetic acid (4.3).

Degas the elution solvent for five minutes.

Note: U.K.

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.

4.6.2. Elution solvent B U.K.

Methanol (4.4).

4.7. Preparation of standard solutions U.K.

All standard solutions must be freshly prepared after two months.

4.7.1. Reference solution C U.K.

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.

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

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.

5. Apparatus and equipment U.K.

5.1. Separation system U.K.

5.1.1. High-performance liquid chromatograph. U.K.

5.1.2. Pumping system enabling one to achieve and maintain a constant or programmed rate of flow with great precision. U.K.

5.1.3. UV spectrophotometric detection system: to be set at 254 nm. U.K.

5.1.4. Solvent degassing system. U.K.

5.2. Computational integrator or recorder, the performance of which is compatible with the rest of the system. U.K.

5.3. Column (example): U.K.

• 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.

5.4. Laboratory equipment U.K.

5.4.1. Analytical balance with a precision of 0,1 mg U.K.

5.4.2. A-grade volumetric glassware U.K.

5.4.3. Micromembrane filtration arrangement for small volumes. U.K.

6. Chromatography conditions U.K.

6.1. Elution characteristics: (example) U.K.

• flow rate: 1 ml/minute,

• solvent A = 30 %,

• solvent B = 70 %.

6.2. Detection: U.K.

• UV = 254 nm

7. Procedure U.K.

7.1. Preparation of the spirit sample U.K.

Filter, if necessary, through a filter for organic solvents (pore diameter: 0,45 μm).

7.2. Determination U.K.

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:

  c

  is the concentration in grams per litre of glycyrrhizic acid in the spirit being analysed

  C

  is the concentration in grams per litre of ammonium glycyrrhizinate in the reference solution

  h

  is the area (or height) of the glycyrrhizic acid peak of the spirit being analysed

  H

  is the area (or height) of the glycyrrhizic acid peak of the reference solution

  P

  is the purity of the reference ammonium glycyrrhizinate (in %)

  823

  is the mass of one mole of glycyrrhizic acid

  840

  is the mass of one mole of ammonium glycyrrhizinate.

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

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:

  A

  pastis, blind duplicates

  B

  pastis, split levels (*)

  C

  pastis, blind duplicates

  D

  pastis, blind duplicates

  E

  pastis, blind duplicates]

Textual Amendments

F1 Inserted by Commission Regulation (EC) No 2091/2002 of 26 November 2002 amending Regulation (EC) No 2870/2000 laying down Community reference methods for the analysis of spirits drinks.