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

[F1 VII. CHALCONES. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR VERIFYING THE PRESENCE OF CHALCONES IN PASTIS U.K.

1. Scope U.K.

This method is suitable for determining whether chalcones are present in aniseed-flavoured drinks or not. Chalcones are natural colorants of the flavonoid family that are present in liquorice root ( Glycyrrhiza glabra ).

For an aniseed-flavoured spirit to be called ‘ pastis ’ , it must contain chalcones ([F2 Regulation 110/2008]).

Textual Amendments

F2 Words in Annex Ch. 7 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)(c)

2. Normative references U.K.

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

3. Principle U.K.

A reference liquorice extract solution is prepared. The presence or absence of chalcones is determined using high-performance liquid chromatography (HPLC) with UV detection.

4. Reagents and materials U.K.

During the analysis, use only reagents of HPLC grade. The ethanol should be 96 % vol. Only water of grade 3 as defined by ISO 3696 should be used.

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

4.2. Acetonitrile, CH ₃ CN, (CAS 75-05-8) U.K.

4.3. Reference substance: Glycyrrhiza glabra : liquorice, ‘sweet root’ U.K.

Coarsely ground liquorice roots ( Glycyrrhiza glabra ). Average dimensions of the rodlike particles: length: 10 to 15 mm, thickness: 1 to 3 mm.

4.4. Sodium acetate, CH ₃ COONa, (CAS 127-09-3) U.K.

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

4.6. Preparation of solutions U.K.

4.6.1. Ethanol 50 % volume U.K.

For 1 000 ml at 20 °C:

• 96 % vol. ethanol (4.1): 521 ml,

• Water (2.0): 511 ml.

4.6.2. Solvent A: acetonitrile U.K.

Acetonitrile (4.2) of HPLC analytical purity.

Degas

4.6.3. Solvent B: 0,1 M sodium acetate buffer solution, pH 4,66. U.K.

Weigh 8,203 g of sodium acetate (4.4), add 6,005 g of glacial acetic acid (4.5) and make up to 1 000 ml with water (2) in a volumetric flask.

5. Preparation of the reference extract from Glycyrrhiza glabra (4.3) U.K.

5.1. Weigh 10 g of ground liquorice root ( Glycyrrhiza glabra ) (4.3) and place in a round-bottomed distillation flask U.K.

• add 100 ml of 50 % vol. ethanol (4.6.1),

• boil under reflux for one hour,

• filter,

• set the filtrate aside for later use.

5.2. Recover the liquorice extract from the filter U.K.

• place in a round-bottomed distillation flask,

• add 100 ml of 50 % vol. ethanol (4.6.1),

• boil under reflux for one hour,

• filter. Set aside the filtrate for later use.

5.3. The liquorice root extraction must be performed three times in succession. U.K.

5.4. Combine the three filtrates. U.K.

5.5. Evaporate the solvent phase (of 5.4) on a rotary evaporator. U.K.

5.6. Take up the residual extract (of 5.5) with 100 ml 50 % vol. ethanol (4.6.1). U.K.

6. Apparatus and equipment U.K.

6.1. Separation system. U.K.

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

6.1.2. Pumping system capable of achieving and maintaining a constant or programmed rate of flow at high pressure. U.K.

6.1.3. UV/visible spectrophotometric detection system that can be set at 254 and 370 nm. U.K.

6.1.4. Solvent degassing system: U.K.

6.1.5. Column oven that can be set at a temperature of 40 ± 0,1 °C. U.K.

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

6.3. Column U.K.

Material: stainless steel or glass

Internal diameter: 4 to 5 mm

Stationary phase: cross-linked silica with an octadecyl derived functional group (C18), particle size: 5 μm at most (cross-linked phase).

6.4. Common laboratory equipment, including: U.K.

6.4.1.

analytical balance. (precision: ± 0,1 mg);

6.4.2.

distillation apparatus with a reflux condenser, comprising, for example:

• a 250 ml round-bottomed flask with a standardised ground-glass joint,

• a 30 cm long reflux condenser, and

• a heat source (any pyrogenic reaction involving the extractive matter must be avoided by using an appropriate arrangement).

6.4.3.

Rotary evaporation apparatus.

6.4.4.

Filtration set-up (i.e. Buchner funnel).

6.5. Chromatography conditions (example). U.K.

6.5.1. Elution characteristics of solvents A (4.6.2) and B (4.6.3): U.K.

• shift from 20/80 (v/v) to 50/50 (v/v) gradient in 15 minutes,

• shift from 50/50 (v/v) to 75/25 (v/v) gradient in five minutes,

• equal strength at 75/25 (v/v) for five minutes,

• stabilisation of the column between injections,

• equal strength at 20/80 (v/v) for five minutes.

6.5.2. Flow rate: 1 ml/minute. U.K.

6.5.3. UV detector settings: U.K.

• the detector must be set at 370 nm to detect the presence of chalcones and then at 254 nm to detect glycyrrhizic acid.

  Note: U.K.

  the change of wavelength (from 370 nm to 254 nm) must be carried out 30 seconds before the beginning of the peak of elution of glycyrrhizic acid. U.K.

7. Procedure U.K.

7.1. Preparation of the spirit sample U.K.

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

7.2. Preparation of the residual liquorice extract (5.6) U.K.

Make a one in ten dilution with 50 % vol. ethanol (4.6.1) before analysis.

7.3. Determination U.K.

7.3.1. Inject 20 μl of the prepared liquorice extract (7.2). Perform the analysis using the chromatography conditions described above (6.5). U.K.

7.3.2. Inject 20 μl of the sample (7.1) (aniseed-flavoured spirit sample). Perform the analysis using the chromatography conditions described above (6.5). U.K.

7.3.3. Compare the two chromatograms. There must be a great similarity between the two chromatograms in the chalcone exit zone (during the detection at 370 nm under the analysis conditions described above) (see Figure 1). U.K.

8. Characteristic chromatogram for a pastis U.K.

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

Results of the interlaboratory test:

• the following table gives the performance for recognition of presence or absence of chalcones in pastis and aniseed-flavoured spirits.

• 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	14
  Number of samples	11
  Analyte	chalcones

  a Inconsistent results between the two duplicates, attributed to a sampling error
  Samples	A	B	C	D	E	F
  Number of laboratories retained after eliminating outliers	14	14	14	14	14	13
  Number of outliers (laboratories)	—	—	—	—	—	1 a
  Number of accepted results	28	14	14	28	28	26
  Number of results for presence of chalcones	28	14	14	0	28	0
  Number of results for absence of chalcones	0	0	0	28	0	26
  Percentage of correct results (%)	100	100	100	100	100	100

  Samples	G	H	I	J	K
  Number of laboratories retained after eliminating outliers	14	14	14	14	14
  Number of outliers (laboratories)	—	—	—	—	—
  Number of accepted results	28	14	14	28	28
  Number of results for presence of chalcones	0	0	0	0	0
  Number of results for absence of chalcone	28	14	14	28	28
  Percentage of correct results (%)	100	100	100	100	100

  Sample types:

  A

  pastis, blind duplicates

  B

  pastis, single sample

  C

  pastis, single sample

  D

  ‘ pastis ’ (not containing chalcones), blind duplicates

  E

  ‘ pastis ’ (not containing chalcones), blind duplicates

  F

  aniseed-flavoured liqueur (not containing chalcones), blind duplicates

  G

  aniseed-flavoured liqueur (not containing chalcones), blind duplicates

  H

  ouzo (not containing chalcones), single sample

  I

  ouzo (not containing chalcones), single sample

  J

  anis (not containing chalcones), blind duplicates

  K

  ‘ pastis ’ (not containing chalcones), 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.