1、STD-BSI BS EN L285b-ENGL 1999 m Lb24bb9 079bLbL 203 m BRITISH STANDARD Foodstuffs - Determination of acesulfame-K, aspartame and saccharin - High performance liquid chromatographic method The European Standard EN 128561999 has the status of a British Standasd ICs 67.040 BS EN 12856:1999 U- NO COPYIN
2、G WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW STD-BSI BS EN 1285b-ENGL 1777 = lb24bb7 077bLb2 14T direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 September 1999 Amd. No. O BSI 0
3、9-1999 BS EN 12856:1999 Date Comments National foreword This British Standard is the English hguage version of EN 12856:1999. The UK participation in its preparation was entrusted to Technical Panel AW/-B, Food analysis - Horizontal methods, which has the responsibility to: - aid enquirers to unders
4、tand the text; - present to the responsible European committee any enquiries on the interpretation, or proposals for change, and keep the UK interesls informed; - monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this commit
5、tee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by usi
6、ng the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity
7、 from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 17 and a back cover. The BSI copyright notice displayed in this document inicates when the document was last issued. ISBN O 580 32593 8 STD-BSI BS EN 2285b-ENGL L777
8、E lb24bb9 077bLb3 OBb D EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 12856 April 1999 English version Foodstuffs - Determination of acesulfame-K, aspartame and saccharin - High performance liquid chromatographic method Produits alimentaires - Dosage dacesulfame-K, daspartame et du saccharine
9、 - Mthode par chromatographie liquide haute performance Lebensmittel - Bestimmung von Acesulfam-K, Aspartam und Saccharin - Hochleistungs- flssigchromatographisches Verfahren This European Standard was approved by CEN on 16 April 1999. CEN members are bound to comply with the CENICENELEC Internal Re
10、gulations 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 may be obtained on application to the Central Secretariat or to any CEN member. This
11、 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 to the Central Secretariat has the same status as the official versions. CEN members are the
12、 national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden. Switzerland and United Kingdom. CEN European Committee for Standardization Comit Europen de Normalisation Euro
13、pisches Komitee fr Normung Central Secretariat: rue de Stassart, 36 B-1050 Brussels Q 1999 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12856:1999 E Page 2 EN 12856: 1999 Con tents Foreword 1 Scope 2 Normative references 3 Princ
14、iple 4 Reagents 5 Apparatus and equipment 6 Procedure 7 Calculation 8 Precision 9 Test report Annex A (normative) Examples for chromatographic conditions which have been proven to lead to satisfactory results Annex B (informative) Figures Annex C (informative) Precision data Annex D (informative) Bi
15、bliography Page 2 3 3 3 3 5 5 8 8 10 11 12 14 17 Foreword This European Standard 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 public
16、ation of an identical text or by endorsement, at the latest by October 1999, and conflicting national standards shall be withdrawn at the latest by October 1999. According to the CENKENELEC Internal Regulations. the national standards organizations of the following countries are bound to implement t
17、his European Standard: Austria, Belgium. Czech Republic, Denmark. Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden. Switzerland and the United Kingdom. STD-BSI BS EN L285b-ENGL 1777 W Lb2qbb7 077bLb5 757 = Page 3 EN 12856:1999 1 Scop
18、e This European Standard specifies a high performance liquid chromatographic (HPLC) method for the determination of acesulfame-K, aspartame and saccharin, see i, 2 and 3. It also allows the determination of caffeine, sorbic acid and benzoic acid in foodstuffs. Interlaboratory tests have been carried
19、 out with acesulfame-K in marzipan, yogurt, fruit yogurt, orange juice beverage, cola, cream and jam, with aspartame in marzipan, fruit yogurt, orange juice beverage, orange flavoured beverage, cola, jam, and preparation for flan, and with sodium saccharin in marzipan, yogurt, fruit yogurt, orange j
20、uice, orange juice beverage, cola, cream and jam. 2 Normative references This European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text and the publications are listed hereafter. For da
21、ted references, subsequent amendments to or revisions of any of these publications apply to this European Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies. EN IS0 3696, Water,for analvtical laboratop use
22、- Specijication and test methods. (IS0 3696: 1987). 3 Principle The sample is extracted or diluted with water. If necessary, the sample solution with the intense sweeteners is purified on a solid phase extraction column or with Carrez reagents. The intense sweeteners in the sample test solution are
23、separated on an HPLC-reversed phase chromatography column and determined spectrometrically at a wavelength of 220 nm. 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognized analytical grade for HPLC analysis and water of at least grade 1 as defined in EN IS0 3696. W
24、hen preparing solutions, the purity of the substances shall be taken into account. 4.1 Acetonitrile. for HPLC. 4.2 Merhanoi, for HPLC. 4.3 Potassiirm dihvdrogen orthophosphate ( KHzP04). 4.4 Dipotassiuni hydrogen orthophosphate ( K2HP04). 4.6 Phosphoric acid, w(H3P01) = 5 %. Carefully pipette 6 mi o
25、f phosphoric acid (4.5) into a 100 mi volumetric flask, which already contains 80 ml of water. Dilute to the mark with water. 4.7 Carrez sohrtion No. I Dissolve 15 g potassium hexacyanoferrate (II) ( - length of 100 mm to 300 mm; - inner diameter of 3 mm to 4 mm; - a guard column, RP C i 8 (optional
26、, but usually recommended especially for all solid sample materiais). Performance criterion for suitable separation columns is the baseline resolution of the respective analyte. Examples for suitable columns or appropriate chromatographic conditions are given in annex A. Whenever interferences are i
27、dentified with a diode array detector or by measurement at a second wavelength, an alternative chromatographic condition shall be chosen. 6 Procedure 6.1 Preparation of the sample test solution 6.1.1 Clear liquid products (ag. lemonades, cola, beverages) Dilute 20 ml of the sample in a 100 mi volume
28、tric flask with water. Filter the solution through a membrane filter of pore size 0,45 pm before injection. C BSI 09-1999 Page 6 EN 12856:1999 6.1.2 Cloudy liquid products (e.g. juices, flavoured milk drinks) Dilute 20 mi of the homogenized sample in a 100 mi volumetric flask with 50 ml water, add 2
29、 ml of Carrez solution No. 1 (4.7), mix and add 2 ml of Carrez solution No. 2 (4.8). Shake vigorously and allow the solution to stand at room temperature for 10 min. Dilute to the mark with water. Filter through a fluted filter paper, discarding the first 10 ml of the filtrate. Pass the filtrate thr
30、ough a membrane filter of pore size 0.45 pm before injection. To make allowance for the volume of any precipitate, if the fat-free insoluble matter in the sample volume (here 20 mi) exceeds approximately 3 g. it is advisable to centrifuge the clarified sample mixture for 10 min at at least 1 400g be
31、fore filtering it quantitatively into the 100 ml volumetric flask. Wash the settled matter twice with water and centrifuge again, collect each of the supernatants in the 100 ml volumetric flask and then dilute the solution to the mark with water. This procedure may also be followed when the amount o
32、f insoluble matter is less than 3 g. 6.1 -3 Jams, preserves, marmalades and related products (except fruit curds) Weigh, to the nearest 1 mg, about 20 g of homogenized sample into a 100 ml volumetric flask. Add about 60 ml of water and place the flask in an ultrasonic bath at 40 “C for 20 min. The t
33、emperature shall not exceed 40 “C since aspartame can be degraded. Cool the solution to room temperature. Add 2 ml of Carrez solution No. 1 (4.7), mix and then add 2 ml of Carrez solution No. 2 (4.8). Shake vigorously and allow the solution to stand at room temperature for IO min. Dilute to the mark
34、 with water. Filter the solution through a fluted filter paper. discarding the first 10 ml of the filtrate. Pass the filtrate through a membrane filter of pore size 0,45 pm before injection. To make allowance for the volume of any precipitate, if the fat-free insoluble matter in the initial sample m
35、ass exceeds approximately 3 g, it is advisable to centrifuge the clarified sample mixture for 10 min at least 1 400g before filtering it quantitatively into the 100 ml volumetric flask. Wash the settled matter twice with water and centrifuge again, collect each of the supernatants in the 100 ml volu
36、metric flask and then dilute the solution to the mark with water. This procedure may also be followed when the amount of insoluble matter is less than 3 g. 6.1.4 Semi solid and solid products (e.g. curd cheese desserts, Yoghurt products, delicatessen salads except custard po wder) Weigh, to the near
37、est 1 mg, about 10 g to 20 g of the thoroughly homogenized sample into a 100 ml volumetric flask. Add about 50 ml of water and place the volumetric flask in the ultrasonic bath at 40 “C for 20 min. The temperature shall not exceed 40 OC since aspartame can be degraded. Cool the solution to room temp
38、erature. Add 2 ml of Carrez solution No. 1 (4.7), mix, add 2 ml of Carrez solution No. 2 (4.8). Shake vigorously and allow the solution to stand at room temperature for 10 min. Dilute to the mark with water. Filter through a fluted filter paper, discarding the first i0 ml of the filtrate. In the cas
39、e of very complex matrices, additional purification using the solid phase extraction column (5.13) may be necessary to protect the separating column, since colourings, flavourings and fat cannot be separated by Carrez clarification. In this case, add 2 ml of the clarified filtrate to the cartridge,
40、previously activated with 3 ml of methanol (4.2) and 20 mi of water, and elute with about 20 mi of mobile phase (4.1 1). Pass the filtrate through a membrane filter of pore size 0,45 pm before injection. To make allowance for the volume of any precipitate, if the fat-free insoluble matter in the ini
41、tial sample mass exceeds approximately 3 g. it is advisable to centrifuge the clarified sample mixture for 10 min at at least i 400g before filtering it quantitatively into the i00 ml volumetric flask. Wash the settled matter twice with water and centrifuge again, collect each of the supernatants in
42、 the 100 ml volumetric flask and then dilute the solution to the mark with water. This procedure may also be followed when the amount of insoluble matter is less than 3 g. O BSI 09-1999 STD.BS1 BS EN 1285b-ENGL 1979 m 1624bb 079blb9 5T4 m Page 7 EN 12856:1999 6.1.5 Custardpowder Weigh, to the neares
43、t 1 mg, about 10 g of the sample into a 500 ml volumetric flask. Add about 400 ml of water and proceed as described above, .e. add 6 ml of Carrez solution No. 1 (4.7), mix, add 6 ml of Carrez solution No. 2 (4.8) for clarification. To make allowance for the volume of any precipitate, if the fat-free
44、 insoluble matter in the initial sample mass exceeds approximately 3 g, it is advisable to centrifuge the clarified sample mixture for 10 min at at least 1 400g before filtering it quantitatively into the 500 ml volumetric flask. Wash the settled matter twice with water and centrifuge again, collect
45、 each of the supernatants in the 500 ml volumetric flask and then dilute the solution to the mark with water. This procedure may also be followed when the amount of insoluble matter is less than 3 g. 6.2 Identification Identify the intense sweeteners in the sample solution by comparing the retention
46、 times of the analyte concerned in the sample solution with that of the standard substance, or by simultaneous injection of the standard solution and the sample test solution, or by adding the standard solution to the sample test solution and recording an absorption curve in the relevant wavelength
47、range. Inject equal volumes of the sample test and standard solutions. Intervals between successive injections of the standard solutions should not be less than 15 min. To minimize the risk that substances eluted from earlier injections will be confused with components from subsequent samples, succe
48、ssive injections of the sample test solutions should be made at sufficiently long intervals. In case of possible interferences, washing of the columns is recommended. A suitable mobile phase for washing would have the following composition: 50 parts per volume of mobile phase (4.1 1) + 50 parts per
49、volume of acetonitrile. Suitable chromatographic conditions for identification are described in annex A. Specimen chromatograms are given for information in annex B. 6.3 Determination For the determination by the external standard method, integrate the peak areas or determine the peak heights and compare the results with the corresponding values for the standard substance with the nearest peak area/height, or use a calibration graph. To prepare a calibration graph, inject a suitable amount of standard solutions of appropriate mass concentrations. Plot the peak heights or pea
