1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 12856:1999 The Euro
2、pean Standard EN 12856:1999 has the status of a British Standard ICS 67.040 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Foodstuffs Determination of acesulfame-K, aspartame and saccharin High performance liquid chromatographic methodThis British Standard, having been prepar
3、ed under the 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 BSI 09-1999 ISBN 0 580 32593 8 BS EN 12856:1999 Amendments issued since publication Amd. No. Date Comments National f
4、oreword This British Standard is the English language version of EN 12856:1999. The UK participation in its preparation was entrusted to Technical Panel AW/-/3, Food analysis Horizontal methods, which has the responsibility to: aid enquirers to understand the text; present to the responsible Europea
5、n committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can be obtained on request to its secretary. Cros
6、s-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 using the “Find” facility of the BSI Standards Electroni
7、c 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 from legal obligations. Summary of pages This docume
8、nt 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 indicates when the document was last issued.EUROPEAN STANDARD EN 12856 NORME EUROPENNE EUROPISCHE NORM April 1999 ICS 67.050 English version Foods
9、tuffs Determination of acesulfame-K, aspartame and saccharin High performance liquid chromatographic method Produits alimentaires Dosage dacsulfame-K, daspartame et du saccharine Mthode par chromatographie liquide haute performance Lebensmittel Bestimmung von Acesulfam-K, Aspartam und Saccharin Hoch
10、leistungs- flssigchromatographisches Verfahren This European Standard was approved by CEN on 16 April 1999. 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 alterat
11、ion. 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 European Standard exists in three official versions (English, French, German). A version in any other language made by translat
12、ion 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 national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Ita
13、ly, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN European Committee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart, 36 B-1050 Brussels 1999 CEN All rights of exploitation in any f
14、orm and by any means reserved worldwide for CEN national Members. Ref. No. EN 12856:1999 EPage 2 EN 12856:1999 BSI 09-1999 Contents Page Foreword 2 1 Scope 3 2 Normative references 3 3 Principle 3 4 Reagents 3 5 Apparatus and equipment 5 6 Procedure 5 7 Calculation 8 8 Precision 8 9 Test report 10 A
15、nnex A (normative) Examples for chromatographic conditions which have been proven to lead to satisfactory results 11 Annex B (informative) Figures 12 Annex C (informative) Precision data 14 Annex D (informative) Bibliography 17 Foreword This European Standard has been prepared by Technical Committee
16、 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 endorsement, at the latest by October 1999, and conflicting national standards shall be wi
17、thdrawn at the latest by October 1999. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Ital
18、y, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom.Page 3 EN 12856:1999 BSI 09-1999 1 Scope This European Standard specifies a high performance liquid chromatographic (HPLC) method for the determination of acesulfame-K, aspartame and saccharin, see 1, 2 a
19、nd 3. It also allows the determination of caffeine, sorbic acid and benzoic acid in foodstuffs. Interlaboratory tests have been carried 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
20、, orange flavoured beverage, cola, jam, and preparation for flan, and with sodium saccharin in marzipan, yogurt, fruit yogurt, orange juice, orange juice beverage, cola, cream and jam. 2 Normative references This European Standard incorporates by dated or undated reference, provisions from other pub
21、lications. These normative references are cited at the appropriate places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this European Standard only when incorporated in it by amendment or revis
22、ion. For undated references the latest edition of the publication referred to 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 intense sweet
23、eners is purified on a solid phase extraction column or with Carrez reagents. The intense sweeteners in the sample test solution are separated on an HPLC-reversed phase chromatography column and determined spectrometrically at a wavelength of 220 nm. 4 Reagents During the analysis, unless otherwise
24、stated, use only reagents of recognized analytical grade for HPLC analysis and water of at least grade 1 as defined in EN ISO 3696. When preparing solutions, the purity of the substances shall be taken into account. 4.1 Acetonitrile, for HPLC. 4.2 Methanol, for HPLC. 4.3 Potassium dihydrogen orthoph
25、osphate (KH 2 PO 4 ). 4.4 Dipotassium hydrogen orthophosphate (K 2 HPO 4 ). 4.5 Phosphoric acid, r 20 (H 3 PO 4 ) = 1,71 g/ml, w(H 3 PO 4 ) = 85 % 1) . 4.6 Phosphoric acid, w(H 3 PO 4 ) = 5 %. Carefully pipette 6 ml of phosphoric acid (4.5) into a 100 ml volumetric flask, which already contains 80 m
26、l of water. Dilute to the mark with water. 4.7 Carrez solution No. 1 Dissolve 15 g potassium hexacyanoferrate (II) (K 4 Fe(CN) 6 3H 2 O) in water and dilute to 100 ml. 4.8 Carrez solution No. 2 Dissolve 30 g zinc sulfate (ZnSO 4 7H 2 O) in water and dilute to 100 ml.1)w is the mass fraction.Page 4 E
27、N 12856:1999 BSI 09-1999 4.9 Phosphate buffer solution I, c(KH 2 PO 4 ) = 0,02 mol/l, pH 4,3 2) Dissolve 2,72 g of potassium dihydrogen orthophosphate (4.3) and 800 ml of water in a 1 000 ml beaker. Adjust to pH 4,3 with phosphoric acid (4.6). Transfer the solution to a 1 000 ml volumetric flask and
28、 dilute to the mark with water. 4.10 Phosphate buffer solution II, c(KH 2 PO 4 ) = 0,0125 mol/l, pH 3,5 Dissolve 1,70 g of potassium dihydrogen orthophosphate (4.3) and 800 ml of water in a 1 000 ml beaker. Adjust to pH 3,5 with phosphoric acid (4.6). Transfer the solutions to a 1 000 ml volumetric
29、flask and dilute to the mark with water. 4.11 Mobile phase, phosphate buffer and acetonitrile Add, carefully measured, the required amounts of the selected phosphate buffer to acetonitrile as given in A.5 and mix. Filter through suitable membrane filters, e.g. of pore size 0,45 mm, and degas, e.g. f
30、or 5 min in an ultrasonic bath. Prepare the mobile phase on the day of use. 4.12 Control solution (optional) The control solution contains acesulfame-K, sodium saccharin, aspartame, 5-benzyl-3,6-dioxo-2-piperazine acetic acid (diketopiperazine), aspartylphenylalanine, phenylalanine, caffeine, sorbic
31、 acid, benzoic acid, theobromine, hydroxymethylfurfural and vanillin. In a 100 ml volumetric flask, weigh to the nearest 0,1 mg, 30 mg of acesulfame-K, 20 mg of sodium saccharin, 220 mg of aspartame, 60 mg of caffeine, 100 mg of sorbic acid, 100 mg of benzoic acid, 100 mg of vanillin, 10 mg of diket
32、opiperazine, 10 mg of phenylalanine, 10 mg of aspartylphenylalanine, 20 mg of hydroxymethylfurfural and 70 mg of theobromine. Dissolve and dilute to the mark with water. Pipette 20 ml of this solution into a 100 ml volumetric flask and dilute to the mark with water. 4.13 Stock solution Weigh, to the
33、 nearest 0,1 mg, 100 mg of acesulfame-K, 100 mg of sodium saccharin and 100 mg of aspartame, in the same 100 ml volumetric flask. Dissolve and dilute to the mark with water. This solution contains 1g/l of each sweetener. 4.14 Standard solutions (optional) 4.14.1 Standard solution I Pipette 10 ml of
34、the stock solution (4.13) into a 100 ml volumetric flask and dilute to the mark with water. This solution contains 100 mg/l of each sweetener. 4.14.2 Standard solution II Pipette 5 ml of the stock solution (4.13) into a 100 ml volumetric flask and dilute to the mark with water. This solution contain
35、s 50 mg/l of each sweetener. 4.14.3 Standard solution III Pipette 1 ml of the stock solution (4.13) into a 100 ml volumetric flask and dilute to the mark with water. This solution contains 10 mg/l of each sweetener.2)c is the substance concentration.Page 5 EN 12856:1999 BSI 09-1999 5 Apparatus and e
36、quipment Usual laboratory apparatus and, in particular, the following: 5.1 High speed blender, or homogenizer. 5.2 Volumetric flasks, of suitable capacities, e.g. 1 000 ml, 500 ml and 100 ml. 5.3 Beaker, 1 000 ml. 5.4 Pipettes, of suitable capacities, e.g. 100 ml, 25 ml, 20 ml, 10 ml, 5 ml and 1 ml.
37、 5.5 Micropipette, 1 000 ml. 5.6 Graduated cylinder, 1 000 ml. 5.7 Fluted filter papers, medium fast, qualitative. 5.8 Ultrasonic bath. 5.9 Centrifuge with centrifuge tubes of suitable capacity, capable of producing a centrifugal acceleration of at least 1 400g at the base of the centrifuge tubes. 5
38、.10 Degassing system, for solvents (optional, instead of ultrasonic degassing). 5.11 Membrane filters, of pore size 0,45 mm or smaller. 5.12 Filter holder for membrane filters, with suitable syringe. 5.13 Solid phase extraction column, with RP C 18 cartridge (optional), e.g. with 500 mg filling. 5.1
39、4 High performance liquid chromatograph, equipped with an ultraviolet (UV) detector (capable of operating at a wavelength of 220 nm, preferably a diode array detector) and equipped with a recorder and/or integrator which allows the measurement of peak heights and peak areas. 5.15 Chromatographic col
40、umn for HPLC, type reversed phase (RP), e.g. with a RP C 18 stationary phase of 3 mm to 10 mm; length of 100 mm to 300 mm; inner diameter of 3 mm to 4 mm; a guard column, RP C 18 (optional, but usually recommended especially for all solid sample materials). Performance criterion for suitable separat
41、ion 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 identified with a diode array detector or by measurement at a second wavelength, an alternative chromatographic c
42、ondition shall be chosen. 6 Procedure 6.1 Preparation of the sample test solution 6.1.1 Clear liquid products (e.g. lemonades, cola, 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 mm before injection.Page
43、 6 EN 12856:1999 BSI 09-1999 6.1.2 Cloudy liquid products (e.g. juices, flavoured milk drinks) Dilute 20 ml of the homogenized sample in a 100 ml volumetric flask with 50 ml water, add 2 ml of Carrez solution No. 1 (4.7), mix and add 2 ml of Carrez solution No. 2 (4.8). Shake vigorously and allow th
44、e 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 through a membrane filter of pore size 0,45 mm before injection. To make allowance for the volume of any precipitate,
45、 if the fat-free insoluble matter in the sample volume (here 20 ml) 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 100 ml volumetric flask. Wash the settled matter twice with water and ce
46、ntrifuge 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 of insoluble matter is less than 3 g. 6.1.3 Jams, preserves, marmalades and related products (except fruit curds) W
47、eigh, 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 temperature shall not exceed 40 C since aspartame can be degraded. Cool the solution to room temperature. Add 2 ml of
48、 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 10 min. Dilute to the mark with water. Filter the solution through a fluted filter paper, discarding the first 10 ml of the filtrate. Pass the
49、filtrate through a membrane filter of pore size 0,45 mm before injection. To make allowance for the volume of any precipitate, if the fat-free insoluble matter in the initial sample mass 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 volumetric flask and then dilute the solut
