DIN EN 15911-2011 Foodstuffs - Simultaneous determination of nine sweeteners by high performance liquid chromatography and evaporative light scattering detection German version EN .pdf

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1、January 2011 Translation by DIN-Sprachendienst.English price group 15No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).IC

2、S 67.180.10!$m3X“1741653www.din.deDDIN EN 15911Foodstuffs Simultaneous determination of nine sweeteners by high performanceliquid chromatography and evaporative light scattering detectionEnglish translation of DIN EN 15911:2011-01Lebensmittel Gleichzeitige Bestimmung von neun Sungsmitteln mitHochlei

3、stungs-Flssigchromatographie und Verdampfungs-Lichtstreu-DetektionEnglische bersetzung von DIN EN 15911:2011-01Denres alimentaires Dtermination simultane de neuf dulcorants par chromatographie liquide hauteperformance et dtection diffusion de lumireTraduction anglaise de DIN EN 15911:2011-01www.beut

4、h.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritative.320 .11 2DIN EN 15911:2011-01 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Se

5、cretariat: DIN, Germany). Preliminary work was done by Working Group WG 2 “Sweeteners”. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-01-11 A

6、A Sungsmittel. Because the excessive consumption of sweeteners can have a laxative effect, foodstuffs must be appropriately labelled if they are to be placed on the European market. The use of sweeteners such as acesulfame-K, aspartame, cyclamic acid, neohesperidine dihydrochalcone, saccharin and su

7、cralose is governed by European Directives. This standard is intended to help those involved in the food trade, the official control of foodstuffs, or in private laboratories determine whether or not a foodstuff meets the requirements of European legislation. Furthermore, this method can be used for

8、 the determination of the sweeteners alitame, dulcin, and neotame. The DIN Standard corresponding to the International Standard referred to in this document is as follows: EN ISO 3696 DIN ISO 3696 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specific

9、ation and test methods 2 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15911 October 2010 ICS 67.180.10 English Version Foodstuffs - Simultaneous determination of nine sweeteners by high performance liquid chromatography and evaporative light scattering detection Denres alimentaires - Dtermin

10、ation simultane de neuf dulcorants par chromatographie liquide haute performance et dtection diffusion de lumire Lebensmittel - Gleichzeitige Bestimmung von neun Sungsmitteln mit Hochleistungs-Flssigchromatographie und Verdampfungs-Lichtstreu-Detektion This European Standard was approved by CEN on 1

11、8 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 a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards ma

12、y 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). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified

13、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, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Lu

14、xembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2010 CEN All r

15、ights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15911:2010: EEN 15911:2010 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .55 Apparatus and equipment 76 Procedure .87 Calculation of results . 118 Prec

16、ision 129 Test report . 14Annex A (informative) Table A.1 Suitable method conditions 16Annex B (informative) Examples of chromatograms 17Annex C (informative) Precision data . 19Annex D (informative) Present EU limits for the nine sweeteners 29Bibliography . 30DIN EN 15911:2011-01 EN 15911:2010 (E)

17、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 held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endors

18、ement, 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 that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifyin

19、g any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany,

20、Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15911:2011-01 EN 15911:2010 (E) 4 1 Scope This European Standard specifies a method for the si

21、multaneous determination of nine sweeteners in beverages and canned or bottled fruits by high performance liquid chromatography (HPLC) with evaporative light scattering detection (HPLC-ELSD). This method has been validated in an interlaboratory study via the analysis of spiked samples on the followi

22、ng 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 to 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 beverage

23、s 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 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

24、(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 59,3 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 a

25、nd (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 fruits. For further information on the validation see Clause 8 and Annex C. NOTE The method has been fully validated 1 through collaborativ

26、e 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 references The following referenced documents are indispensable for the application of this document. For dated references, only the edi

27、tion cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) 3 Principle The procedure involves extraction of the nine sweeteners with

28、 a buffer solution, sample clean-up using solid-phase extraction cartridges followed by HPLC-ELSD analysis. DIN EN 15911:2011-01 EN 15911:2010 (E) 5 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognised analytical grade for HPLC analysis and water of at least grade

29、 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 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 dihydroc

30、halcone, 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, (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 for

31、mic 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/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

32、 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 of acetone (4.13). Degas by sonication for 10 min. 4.17 Mixed stock solution, containing ACS-K, ALI, ASP, CYC-Na, DUL, NEO, NHDC, SA

33、C-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 individual sweetener standards (Table 1) first into a 100 ml beaker and dissolving them in 50 ml of methanol/water (1:1) until complete diss

34、olution. 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 sonication until complete dissolution. DIN EN 15911:2011-01 EN 15911:2010 (E) 6 Table 1 Masses of individual standards for preparation

35、 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 90 Alitame (ALI) 25 50 Aspartame (ASP) 125 250 Sodium cyclamate (CYC-Na) 140 a Cyclamic acid (CYC) (free acid) 249,42 Dulcin (D

36、UL) 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) 50 100 aEquivalent to 124,71 mg free cyclamic acid; conversion factor to calculate mass of free cyclamic acid = 0,890 8; mCY

37、C= 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 ml beaker, dissolve in 50 ml of a methanol:water (1:1) mixture and then transfer quantitatively into 500 ml volumetric flask.

38、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 individual sweeteners in micrograms per millilitre in the mixed stock solution have to be calculated by using the actually wei

39、ghed 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 limits, e.g. the highest concentration of the calibration shall be at least equivalent to 125 % of the given limits, such as those

40、 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/

41、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 solution shall be adjusted to meet the current MUDs. NOTE The present procedure is simplified by preparing one calibration series for

42、 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 only the latter matrix is analysed the calibration series can be fitted to the MUDs of beverages. Pipette the following volumes

43、 (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 thoroughly. DIN EN 15911:2011-01 EN 15911:2010 (E) 7 Table 2 Preparation of series of standard solutions Calibration solution Volume o

44、f 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 50 1,5 48,5aUndiluted mixed stock solution (4.17). Table 3 details the concentration of sweetener i in each calibration s

45、tandard 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 a particular set of samples consideration may be given to reduce the levels of the calibration solutions used for those

46、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 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,

47、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 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 quantificatio

48、n (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, ignoring the result obtained for calibration solution 8. 5 Apparatus and equipment Usual laboratory apparatus and, in part

49、icular, 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. 5.3 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). DIN EN 15911:2011-01 EN 15911:2010 (E) 8 5.5 Volumetric flasks, of suitable capacity, e.g. 10 ml, 25