1、DD CEN/TS15606:2009ICS 67.050NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWDRAFT FOR DEVELOPMENTFoodstuffs Determinationof acesulfame-K, aspartame,neohesperidine-dihydrochalcone andsaccharin Highperformance liquidchromatographicmethodThis Draft for Developmentwas published un
2、derthe authority of theStandards Policy andStrategy Committee on 30November 2009. BSI 2009ISBN 978 0 580 57999 8Amendments/corrigenda issued since publicationDate CommentsDD CEN/TS 15606:2009National forewordThis Draft for Development is the UK implementation of CEN/TS15606:2009.This publication is
3、not to be regarded as a British Standard.It is being issued in the Draft for Development series of publications andis of a provisional nature. It should be applied on this provisional basis,so that information and experience of its practical application can beobtained.Comments arising from the use o
4、f this Draft for Development arerequested so that UK experience can be reported to the internationalorganization responsible for its conversion to an international standard.A review of this publication will be initiated not later than 3 years afterits publication by the international organization so
5、 that a decision can betaken on its status. Notification of the start of the review period will bemade in an announcement in the appropriate issue of Update Standards.According to the replies received by the end of the review period,the responsible BSI Committee will decide whether to support thecon
6、version into an international Standard, to extend the life of theTechnical Specification or to withdraw it. Comments should be sent tothe Secretary of the responsible BSI Technical Committee at BritishStandards House, 389 Chiswick High Road, London W4 4AL.The UK participation in its preparation was
7、entrusted to TechnicalCommittee AW/-/3, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct ap
8、plication.Compliance with a British Standard cannot confer immunityfrom legal obligations.DD CEN/TS 15606:2009TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 15606 September 2009 ICS 67.050 English Version Foodstuffs - Determination of acesulfame-K, aspartame, neohespe
9、ridine-dihydrochalcone and saccharin - High performance liquid chromatographic method Produits alimentaires - Dosage de lacsulfame-K, de laspartame, de la saccharine et de la nohespridine dihydrochalcone - Mthode par chromatographie liquide haute performance Lebensmittel - Bestimmung von Acesulfam-K
10、, Aspartam, Neohesperidin-Dihydrochalcon und Saccharin - Hochleistungsflssigchromatographisches Verfahren This Technical Specification (CEN/TS) was approved by CEN on 20 June 2009 for provisional application. The period of validity of this CEN/TS is limited initially to three years. After two years
11、the members of CEN will be requested to submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard. CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available promptly at nation
12、al level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cypr
13、us, 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 CO
14、MIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TS 15606:2009: EDD CEN/TS 15606:2009CEN/TS 15606:2009 (E) 2 Cont
15、ents Page Foreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents .4 5 Apparatus and equipment 7 6 Procedure .8 7 Calculations . 11 8 Precision 12 9 Test report . 14 Annex A (normative) Example for chromatographic conditions which have been proven to lead to satisfactory results . 15
16、Annex B (informative) Figures 16 Annex C (informative) Precision data . 18 Bibliography . 21 DD CEN/TS 15606:2009CEN/TS 15606:2009 (E) 3 Foreword This document (CEN/TS 15606:2009) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is he
17、ld by DIN. 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 identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards o
18、rganizations of the following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Por
19、tugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DD CEN/TS 15606:2009CEN/TS 15606:2009 (E) 4 1 Scope This Technical Specification (CEN/TS 15606:2009) specifies a high performance liquid chromatographic (HPLC) method with UV-detection for the determination of ace
20、sulfame-K, aspartame, neohesperidine-dihydrochalcone and saccharin in foodstuffs. The method has been fully validated 1 for the dialysis procedure through collaborative trial (see 8.2, 8.3 and Annex C), according to the IUPAC Harmonised Protocol 2, on the following analyte matrix combinations: acesu
21、lfame-K (from 86 mg/l to 331 mg/l) and aspartame (from 97 mg/kg to 610 mg/l)in water-based drink, fruit-based drink, cheesecake with biscuit base, canned soup and instant chocolate drink saccharin (from 70 mg/l to 97 mg/kg) in water-based drink, fruit-based drink, cheesecake with biscuit base and ca
22、nned soup; neohesperidine-dihydrochalcone (from 27 mg/l to 57 mg/kg)in water-based drink, fruit-based drink and canned soup. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For und
23、ated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) 3 Principle The sample is extracted or diluted with water. If necessary, the sample solution with the int
24、ense sweeteners is purified with Carrez reagents. Alternatively, solid samples are slurried in NaCl/HCl solution and the sweeteners extracted by dialysis using cellulose acetate (molecular weight cut-off of approximately 12 000). The purified extract is analysed by ion-pair reversed-phase HPLC with
25、UV detection. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only reagents of recognised analytical grade for HPLC analysis and water suitable for HPLC or of at least grade 1 as defined in EN ISO 3696. When preparing solutions, the purity of the substances shall be taken in
26、to account. 4.2 Methanol, HPLC grade. 4.3 Hydrochloric acid, w(HCl) 36 % 1. 4.4 Phosphoric acid, c(H3PO4) = 5 mol/l 2. 1w is the mass fraction. 2c is the substance concentration. DD CEN/TS 15606:2009CEN/TS 15606:2009 (E) 5 4.5 Sodium hydroxide, c(NaOH) = 5 mol/l. 4.6 Saccharin, w(C7H5NO3S): 98,0 % t
27、o 101,0 %. or w(C7H4NNaO3S x 2H2O): 99,0 % to 101,0 %. 4.7 Neohesperidine-dihydrochalcone, w(C28H34O15) 99,0 %. 4.8 Acesulfame-K, w(C4H4KNO4S) 99,0 %. 4.9 Aspartame, w(C14H18N2O5) 98 %. 4.10 Sodium chloride 4.11 Tetrapropylammonium hydrogensulphate (TPrA-HSO4) 4.12 HPLC mobile phases 4.12.1 Mobile p
28、hase 1 (5 % methanol in water) Pipette (5.4) 50 ml of methanol (4.2) into a 1 l volumetric flask (5.2). Make up to the mark with water. Add 0,24 g 0,01 g of TPrA-HSO4(4.11) and shake. Adjust to pH 3,5 with drop-wise addition of either 5 mol/l sodium hydroxide (4.5) or 5 mol/l phosphoric acid (4.4).
29、4.12.2 Mobile phase 2 (75 % methanol in water) Transfer 750 ml methanol (4.2) from a measuring cylinder into a 1 l volumetric flask (5.2) and make up to the mark with water. Add 0,24 g 0,01 g TPrA-HSO4(4.11) and shake. Adjust to pH 3,5 with drop-wise addition of either a 5 mol/l sodium hydroxide (4.
30、5) or 5 mol/l phosphoric acid (4.4). 4.12.3 Isocratic elution mobile phase (15 % methanol in water) Transfer 150 ml of methanol (4.2) from a measuring cylinder into a 1 l volumetric flask (5.2) and make up to the mark with water. Add 0,24 g 0,01 g of TPrA-HSO4(4.11) and shake. Adjust to pH 3,5 with
31、drop-wise addition of either 5 mol/l of sodium hydroxide (4.5) or 5 mol/l of phosphoric acid (4.4). Filter (5.5) the solutions and sonicate (5.9) for 30 min. 4.13 Dialysing solution, 1,71 mol/l NaCl and 0,01 mol/l HCl, optional. Dissolve 100 g of NaCl (4.10) and 1 ml of concentrated HCl (4.3) to 1 l
32、 in a volumetric flask (5.2) with water. 4.14 Dialysate solution, 0,01 mol/l HCl, optional. Dissolve 1 ml of concentrated HCl (4.3) to 1 l in a volumetric flask (5.2) with water. 4.15 Stock solutions of acesulfame-K, aspartame, saccharin and neohesperidine-dihydrochalcone Prepare 1 000 mg/l solution
33、s of acesulfame-K (4.8) and aspartame (4.9) in water. Prepare a solution of saccharin (4.6) equivalent to 1 000 mg/l free imide (e.g. 1 126 mg/l of anhydrous sodium salt) in water and a 1 000 mg/l solution of neohesperidine-dihydrochalcone (4.7) in a mixture of 50 % methanol (4.2) and 50 % water. DD
34、 CEN/TS 15606:2009CEN/TS 15606:2009 (E) 6 4.16 Mixed calibration solutions containing acesulfame-K, aspartame, saccharin and neohesperidine-dihydrochalcone From the stock solutions (4.15) prepare a series of mixed standards containing the sweeteners at concentrations equivalent to a maximum level of
35、 150 % of the appropriate limit (for example, those given in Commission Directives 94/35/EC and 96/83/EC, as amended) whilst taking the dilution steps within the procedure into account. EXAMPLE Calibration standards for measurement of sweeteners in a dairy-based dessert. The present EU limits for th
36、e four sweeteners in dairy-based desserts are: acesulfame-K 350 mg/kg; saccharin 100 mg/kg (free imide); aspartame 1 000 mg/kg; neohesperidine dihydrochalcone 50 mg/kg. 150 % of the limits are: acesulfame K 525 mg/kg; saccharin 150 mg/kg (free imide); aspartame 1 500 mg/kg; neohesperidine-dihydrocha
37、lcone 75 mg/kg. Sweeteners from 20 g of sample are extracted in 200 ml of dialysate (10:1 dilution). Thus, calibration should be carried out for extracts containing up to the following concentrations of sweetener: acesulfame-K 50 mg/l; saccharin 15 mg/l (free imide); aspartame 150 mg/l; neohesperidi
38、ne-dihydrochalcone 7 mg/l. For a seven point calibration, take the volumes as given in Table 1 from the 1 000 mg/l stock solutions (4.15) and make up to 50 ml with water in volumetric flasks (5.2). Table 1 Preparation of the calibration graph Acesulfame-K ml Saccharin l Aspartame ml Neohesperidine-
39、dihydrochalcone l 1 0,25 75 0,75 50 2 0,50 150 1,50 100 3 0,75 225 2,25 150 4 1,00 300 3,00 200 5 1,50 450 4,50 250 6 2,00 600 6,00 300 7 2,50 750 7,50 350 Table 2 details the concentration of sweeteners in each calibration standard following dilution: DD CEN/TS 15606:2009CEN/TS 15606:2009 (E) 7 Tab
40、le 2 Concentration of sweeteners in each calibration solution Acesulfame-K mg/l Saccharin (free imide) mg/l Aspartame mg/l Neohesperidine-dihydrochalcone mg/l 1 5 1,5 15 1 2 10 3 30 2 3 15 4,5 45 3 4 20 6 60 4 5 30 9 90 5 6 40 12 120 6 7 50 15 150 7 4.17 Blank and fortified samples for determination
41、 of recovery If the sample preparation is carried out by dialysis, a homogenised sample of the same type as the test sample should be prepared. Divide the sample into two portions. Add a known quantity of sweeteners to one portion (gravimetrically as solid or volumetrically in solution as appropriat
42、e). 4.18 Carrez solution No 1 Dissolve 15 g potassium hexacyanoferrate (II) (K4Fe(CN)6 3 H2O) in water and dilute to 100 ml. 4.19 Carrez solution No 2 Dissolve 30 g zinc sulfate (ZnSO4 7 H2O) in water and dilute to 100 ml. 5 Apparatus and equipment 5.1 General Usual laboratory equipment and glasswar
43、e. The following items are also required. 5.2 Volumetric flasks, e.g. 50 ml and smaller. 5.3 HPLC vials, clear, 2 ml. 5.4 Pipette, 50 ml and smaller. 5.5 Mobile phase glass filter equipment 5.6 Screw cap bottles, 250 ml. 5.7 Glass funnels 5.8 Homogeniser, Ultra Turrax or equivalent. 5.9 Ultrasonic b
44、ath DD CEN/TS 15606:2009CEN/TS 15606:2009 (E) 8 5.10 Magnetic stirrer, multi-place preferred (or roller mixer, or some other means of mixing the dialysate). 5.11 Mini-magnets 5.12 Membrane syringe filters, 13 mm, 0,45 m (PVDF or equivalent). 5.13 Mobile phase filters, diameter 4,8 cm, pore size 0,45
45、 m. 5.14 Dialysis tubing, cellulose acetate, planar width 4,8 cm; molecular weight cut-off approximately 12 000, optional. 5.15 pH meter 5.16 High performance liquid chromatograph, equipped with an analytical column (5.17), a pump system (gradient capability), an autosampler, an ultraviolet (UV) det
46、ector (capable of operating at a wavelength of 220 nm and 280 nm, preferably a diode array detector) and equipped with a recorder and/or integrator which allows the measurement of peak heights and peak areas. 5.17 HPLC-reversed phase column, e.g. ODS3 10 m; 250 mm x 3,2 mm or equivalent (10 m partic
47、le size 250 mm x 3,2 mm). NOTE Column dimensions other than those specified in this Technical Specification can be used (e.g. of diameter 3,0 mm to 4,6 mm, length 100 mm to 250 mm). Separation parameters (flow, injection volume) should be adapted to guarantee equivalent results. 5.18 HPLC System sui
48、tability Sweeteners acesulfame K, saccharin, aspartame and neohesperidine-dihydrochalcone should be resolved (Rs = 1,3) from each other and from other components of the matrix. Two commonly encountered critical pairs are (a) acesulfame-K and theobromine; and (b) aspartame and caffeine. 6 Procedure 6
49、.1 Sample preparation without dialysis 6.1.1 Clear liquid products such as lemonades, cola or beverages Dilute 20 ml of the sample in a 100 ml volumetric flask with water. Filter the solution through a membrane filter of pore size 0,45 m before injection. 6.1.2 Cloudy liquid products such as juices or flavoured milk drinks Dilute 20 ml of the homogenised sample in a 100 ml volumetric flask with 50 ml water, add 2 ml of Carrez solution No 1 (4.18), mix and add 2 ml of Carrez solution No 2 (4.19). Shake vi
