BS EN 15911:2010 pdf free download Foodstuffs — Simultaneous determination of nine sweeteners by high performance liquid chromatography and evaporative light scattering detection
1 scope
This European Standard specifies a method for the simultaneous determination of nine sweeteners inbeverages and canned or bottled fruits by high performance liquid chromatography (HPLC) with evaporativelight scattering detection (HPLC-ELSD). This method has been validated in an interlaboratory study via theanalysis of spiked samples on the following matrices:
acesulfame-K (ACS-K) (from 38,3 mg/l to 383,5 mgll) in beverages and (from 38,4 mg/kg to 391,3 mg/kg)in canned fruits;
alitame (ALl) (from 31,1 mg/l to 114,5 mg/l) in beverages and (from 36 mg/kg to 175,2 mg/kg) in cannedfruits;
aspartame (ASP)(from 38,1 mg/l to 702 mg/l) in beverages and (from 37,2 mgkg to 1 120,2 mg/kg) incanned fruits;
cyclamic acid (CYC)(from 28,3 mg/l to 307,2 mg/) in beverages and (from 27,5 mg/kg to 1 100,6 mg/kg)in canned fruits;
dulcin (DUL) (from 55,0 mg/l to 115,1 mg/l) in beverages and (from 49,8 mg/kg to 172,6 mg/kg) in cannedfruits;
neotame(NEO)(from 37,6 mg/l to 115,3 mg/l) in beverages and (from 37,3 mg/kg to 173,7 mg/kg) incanned fruits;
neohesperidine dihydrochalcone (NHDC)(from 31,4 mg/l to 59,3 mgl) in beverages and (from35,3 mg/kg to 59,3 mg/kg) in canned fruits;
saccharin(SAC)(from 36,2 mg/l to 87,6 mg/l) in beverages and (from 44,3 mg/kg to 235,3 mg/kg) incanned fruits;
sucralose (SCL)(from 36,8 mg/ to 346,8 mg/l) in beverages and (from 35,3 mg/kg to 462,4 mg/kg) incanned fruits.
For further information on the validation see Clause 8 and Annex C.
NOTE The method has been fully validated [1] through collaborative trial, according to the luPAC HarmonisedProtocol [2], on analyte-matrix combinations of all nine sweeteners in beverages and canned or bottled fruits.
2Normative references
The following referenced documents are indispensable for the application of this document. For datedreferences,only the edition cited applies. For undated references,the latest edition of the referenceddocument (including any amendments) applies.
EN ISO 3696:1995,Water for analytical laboratory use— Specification and test methods (ISO 3696:1987)
3Principle
The procedure involves extraction of the nine sweeteners with a buffer solution, sample clean-up using solid-phase extraction cartridges followed by HPLC-ELSD analysis.
4 Reagents
During the analysis, unless otherwise stated, use only reagents of recognised analytical grade for HPLC analysis and water of at least grade 1 as defined in EN ISO 3696:1 995. When preparing solutions, the purity of the substances shall be taken into account.
4.1 Acesulfame-K, with a mass fraction w of at least 99,0 %.
4.2 Alitame, w ≥ 99,0 %.
4.3 Aspartame, w ≥ 99,0 %.
4.4 Dulcin, for HPLC.
4.5 Neotame, w ≥ 99,0 %.
4.6 Neohesperidine dihydrochalcone, w ≥ 95,0 %.
4.7 Saccharin, sodium salt dihydrate, w ≥ 98,0 %.
4.8 Sodium cyclamate, w ≥ 99,0 %.
4.9 Sucralose, w ≥ 99,0 %.
4.10 Formic acid, HCOOH for HPLC.
4.11 Triethylamine, (C 2 H 5 ) 3 N, w ≥ 99,5 %.
4.12 Methanol, for HPLC.
4.13 Acetone, for HPLC.
4.14 Buffer solution (pH = 4,5).
Dissolve 4 ml of formic acid (4.1 0) in 5 l of water. Adjust to pH 4,5 with approximately 1 2,5 ml triethylamine (4.1 1 ).
4.15 HPLC mobile phase A, methanol/buffer solution/acetone 69:24:7 (v/v/v).
Mix 690 ml of methanol (4.1 2) with 240 ml of buffer solution (4.1 4) and with 70 ml of acetone (4.1 3). Degas by sonication for 1 0 min.
4.16 HPLC Mobile phase B, methanol/buffer solution/acetone 1 1 :82:7 (v/v/v).
Mix 1 1 0 ml of methanol (4.1 2) with 820 ml of buffer solution (4.1 4) and with 70 ml of acetone (4.1 3). Degas by sonication for 1 0 min.
4.17 Mixed stock solution, containing ACS-K, ALI, ASP, CYC-Na, DUL, NEO, NHDC, SAC-Na and SCL;
mass concentration ρ(sweetener i) = 30 µg/ml to 250 µg/ml.
Prepare a mixed stock solution of all nine sweeteners by weighing the given masses of the individual sweetener standards (Table 1) first into a 1 00 ml beaker and dissolving them in 50 ml of methanol/water (1 :1 ) until complete dissolution. Then transfer the obtained solution quantitatively into a 500 ml volumetric flask and make up to the mark with the buffer solution (4.1 4). Mix thoroughly by sonication until complete dissolution.
4.18 Standard solutions. From the mixed stock solution (4.17) prepare a series of standard solutions containing the sweeteners at levels fitting appropriate limits, e.g. the highest concentration of the calibration shall be at least equivalent to 125 % of the given limits, such as those in Commission Directives [3], [4], [5] (see Table D.1), whilst taking the dilution steps within the procedure into account (see Table 2). For sweeteners not authorised by the current EU legislation (ALI, DUL and NEO) fictitious maximum usable dosages (MUD) are assumed at approximately 200 mg/l or 200 mg/kg.