1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 15911:2010Foodstuffs Simultaneousdetermination of ninesweeteners by highperformance liquidchromatography andevaporative light scatteringdetectionBS EN 15911:2010 BRITISH ST
2、ANDARDNational forewordThis British Standard is the UK implementation of EN 15911:2010.The UK participation in its preparation was entrusted to TechnicalCommittee AW/-/3, Food analysis - Horizontal methods.A list of organizations represented on this committee can beobtained on request to its secreta
3、ry.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2010ISBN 978 0 580 66428 1ICS 67.180.10Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published
4、under the authority of theStandards Policy and Strategy Committee on 30 November 2010.Amendments issued since publicationDate Text affectedBS EN 15911:2010EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15911 October 2010 ICS 67.180.10 English Version Foodstuffs - Simultaneous determination of
5、nine sweeteners by high performance liquid chromatography and evaporative light scattering detection Denres alimentaires - Dtermination simultane de neuf dulcorants par chromatographie liquide haute performance et dtection diffusion de lumire Lebensmittel - Gleichzeitige Bestimmung von neun Sungsmit
6、teln mit Hochleistungs-Flssigchromatographie und Verdampfungs-Lichtstreu-Detektion This European Standard was approved by CEN on 18 September 2010. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of
7、 a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German)
8、. A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, C
9、yprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION
10、 COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15911:2010: EBS EN 15911:2010EN 15911:2010 (E) 2 Contents Page
11、 Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .55 Apparatus and equipment 76 Procedure .87 Calculation of results . 118 Precision 129 Test report . 14Annex A (informative) Table A.1 Suitable method conditions 16Annex B (informative) Examples of chromatograms 17Annex C (informat
12、ive) Precision data . 19Annex D (informative) Present EU limits for the nine sweeteners 29Bibliography . 30BS EN 15911:2010EN 15911:2010 (E) 3 Foreword This document (EN 15911:2010) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is
13、held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by April 2011, and conflicting national standards shall be withdrawn at the latest by April 2011. Attention is drawn to the possibility th
14、at some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound t
15、o implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Swed
16、en, Switzerland and the United Kingdom. BS EN 15911:2010EN 15911:2010 (E) 4 1 Scope This European Standard specifies a method for the simultaneous determination of nine sweeteners in beverages and canned or bottled fruits by high performance liquid chromatography (HPLC) with evaporative light scatte
17、ring detection (HPLC-ELSD). This method has been validated in an interlaboratory study via the analysis of spiked samples on the following matrices: acesulfame-K (ACS-K) (from 38,3 mg/l to 383,5 mg/l) in beverages and (from 38,4 mg/kg to 391,3 mg/kg) in canned fruits; alitame (ALI) (from 31,1 mg/l t
18、o 114,5 mg/l) in beverages and (from 36 mg/kg to 175,2 mg/kg) in canned fruits; aspartame (ASP) (from 38,1 mg/l to 702 mg/l) in beverages and (from 37,2 mg/kg to 1 120,2 mg/kg) in canned fruits; cyclamic acid (CYC) (from 28,3 mg/l to 307,2 mg/l) in beverages and (from 27,5 mg/kg to 1 100,6 mg/kg) in
19、 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 canned fruits; 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) in canned fruits; neohesperidine dihydrochalcone (NHDC) (from 31,4 mg/l to 59
20、,3 mg/l) in beverages and (from 35,3 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) in canned fruits; sucralose (SCL) (from 36,8 mg/l to 346,8 mg/l) in beverages and (from 35,3 mg/kg to 462,4 mg/kg) in canned fru
21、its. 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 IUPAC Harmonised Protocol 2, on analyte-matrix combinations of all nine sweeteners in beverages and canned or bottled fruits. 2 Normative
22、references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical
23、 laboratory use Specification and test methods (ISO 3696:1987) 3 Principle 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. BS EN 15911:2010EN 15911:2010 (E) 5 4 Reagents During th
24、e 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 3696:1995. When preparing solutions, the purity of the substances shall be taken into account. 4.1 Acesulfame-K, with a mass fraction w of at lea
25、st 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 Trieth
26、ylamine, (C2H5)3N, 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 (4.10) in 5 l of water. Adjust to pH 4,5 with approximately 12,5 ml triethylamine (4.11). 4.15 HPLC mobile phase A, methanol/buffer solution/acetone 69:24:7 (v/
27、v/v). Mix 690 ml of methanol (4.12) with 240 ml of buffer solution (4.14) and with 70 ml of acetone (4.13). Degas by sonication for 10 min. 4.16 HPLC Mobile phase B, methanol/buffer solution/acetone 11:82:7 (v/v/v). Mix 110 ml of methanol (4.12) with 820 ml of buffer solution (4.14) and with 70 ml o
28、f acetone (4.13). Degas by sonication for 10 min. 4.17 Mixed stock solution, containing ACS-K, ALI, ASP, CYC-Na, DUL, NEO, NHDC, SAC-Na and SCL; mass concentration (sweetener i) = 30 g/ml to 250 g/ml. Prepare a mixed stock solution of all nine sweeteners by weighing the given masses of the individua
29、l sweetener standards (Table 1) first into a 100 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 (4.14). Mix thoroughly by son
30、ication until complete dissolution. BS EN 15911:2010EN 15911:2010 (E) 6 Table 1 Masses of individual standards for preparation of mixed stock solution Standard Mass weighed into 500 ml volumetric flask cmg Final mass concentration of sweetener i in mixed stock standard g/ml Acesulfame-K (ACS-K) 45 9
31、0 Alitame (ALI) 25 50 Aspartame (ASP) 125 250 Sodium cyclamate (CYC-Na) 140 a Cyclamic acid (CYC) (free acid) 249,42 Dulcin (DUL) 25 50 Neotame (NEO) 25 50 Neohesperidine dihydrochalcone (NHDC) 15 30 Saccharin, sodium salt dihydrate (SAC-Na2H2O) 35 b Saccharin (SAC) (free imide) 53,17 Sucralose (SCL
32、) 50 100 aEquivalent to 124,71 mg free cyclamic acid; conversion factor to calculate mass of free cyclamic acid = 0,890 8; mCYC= 0,890 8 mCYC-Na. bEquivalent to 26,58 mg free saccharin; conversion factor to calculate mass of free saccharin = 0,759 5; mSAC= 0,759 5 mSAC-Na2H2O. cFirst weigh into 100
33、ml beaker, dissolve in 50 ml of a methanol:water (1:1) mixture and then transfer quantitatively into 500 ml volumetric flask. NOTE In case of cyclamic acid and saccharin, their sodium salts are used, since they are either not available in free form or poorly soluble. The final concentrations of the
34、individual sweeteners in micrograms per millilitre in the mixed stock solution have to be calculated by using the actually weighed masses. 4.18 Standard solutions. From the mixed stock solution (4.17) prepare a series of standard solutions containing the sweeteners at levels fitting appropriate limi
35、ts, 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 curr
36、ent EU legislation (ALI, DUL and NEO) fictitious maximum usable dosages (MUD) are assumed at approximately 200 mg/l or 200 mg/kg. The user of the standard has to check whether the limits in Table D.1 are still valid. If not, the mass concentration of the standard substance in the calibration solutio
37、n shall be adjusted to meet the current MUDs. NOTE The present procedure is simplified by preparing one calibration series for both food matrices. The described calibration series is fitted to canned fruits as the MUDs for canned fruits are in some cases higher than the MUDs for beverages. In case o
38、nly the latter matrix is analysed the calibration series can be fitted to the MUDs of beverages. Pipette the following volumes (see Table 2) from the mixed stock solution (4.17) into appropriate volumetric flasks (10 ml to 50 ml) and make up to the mark with buffer solution (4.14) and shake thorough
39、ly. BS EN 15911:2010EN 15911:2010 (E) 7 Table 2 Preparation of series of standard solutions Calibration solution Volume of volumetric flask ml Volume taken from mixed stock solution (4.17) ml Volume taken from buffer solution (4.14) ml 1a10 10 02 10 8 2 3 10 6 4 4 10 4 6 5 10 2 8 6 25 3 22 7 50 478
40、50 1,5 48,5aUndiluted mixed stock solution (4.17). Table 3 details the concentration of sweetener i in each calibration standard following preparation described in Table 2. If not all of the sweeteners covered by this standard are the subject of analysis in routine use of the method, when applied to
41、 a particular set of samples consideration may be given to reduce the levels of the calibration solutions used for those samples. Table 3 Concentration of sweetener i in the individual standard solutions Sweetener Calibration solutionmg/ml 1 2 3 4 5 6 7 8 ACS-K 90,0 72,0 54,0 36,0 18,0 10,8 5,4 2,7
42、aALI 50,0 40,0 30,0 20,0 10,0 6,0 3,0 a1,5 aASP 250,0 200,0 150,0 100,0 50,0 30,0 15,0 7,5 CYC 249,4 199,5 149,7 99,8 49,9 29,9 15,0 7,5 DUL 50,0 40,0 30,0 20,0 10,0 6,0 a3,0 a1,5 aNEO 50,0 40,0 30,0 20,0 10,0 6,0 3,0 a1,5 aNHDC 30,0 24,0 18,0 12,0 6,0 3,6 a1,8 a0,9 aSAC 53,2 42,5 31,9 21,3 10,6 6,4
43、 3,2 a1,6 aSCL 100,0 80,0 60,0 40,0 20,0 12,0 6,0 3,0 aaThe concentration level might be below the limit of quantification (LOQ). If yes, the result obtained by HPLC analysis is not included in the construction of the calibration graph, e.g. in case of ACS-K a seven point calibration is performed, i
44、gnoring the result obtained for calibration solution 8. 5 Apparatus and equipment Usual laboratory apparatus and, in particular, the following: 5.1 Common laboratory glassware, such as graduated cylinders, volumetric pipettes, glass beakers. 5.2 Analytical balance, capable of weighing to 0,01 mg. 5.
45、3 Laboratory balance, capable of weighing to 0,01 g. 5.4 Positive displacement pipette, or equivalent, capable of delivering 1 ml to 10 ml (variable volume). BS EN 15911:2010EN 15911:2010 (E) 8 5.5 Volumetric flasks, of suitable capacity, e.g. 10 ml, 25 ml, 50 ml, 100 ml and 500 ml. 5.6 Centrifuge t
46、ubes, made of polypropylene, of suitable capacity, e.g. 50 ml. 5.7 Graduated test tubes, of suitable capacity, e.g. 5 ml. 5.8 Food blender, suitable for homogenisation of food samples. 5.9 Ultrasonic bath. 5.10 Centrifuge, capable of maintaining 4 000 min-1. 5.11 SPE Vacuum system, or equivalent. 5.
47、12 Equipment for solvent evaporation. 5.13 pH meter. 5.14 C18SPE cartridges. 5.15 Analytical reverse phase column, fully end-capped, allowing sufficient separation of all nine sweeteners. E.g. with: an RP C 18 stationary phase of 5 m; a length of 250 mm; internal diameter of 3 mm. 5.16 HPLC system,
48、equipped with a binary pump capable of maintaining a flow rate of 0,5 ml/min, preferably an automatic injection system, and an evaporative light scattering detector. Other detection systems such as MS as substitute for ELSD or UV and DAD when substances do absorb in the UV region can also be used pr
49、ovided that the equivalent performance characteristics can be obtained. 5.17 Data acquisition and analysis software. 6 Procedure 6.1 General Comminute the entire test sample to give a homogenous suspension (5.8). Liquid samples can be subjected directly to the extraction procedure. 6.2 Preparation of test sample 6.2.1 Step 1 Weigh approximately 5 g (M1, recorded to two decimal places) of the homogenised test sample (6.1) into a volumetric flask o
