1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 1378 : 1997 The Eur
2、opean Standard EN 1378 : 1996 has the status of a British Standard ICS 67.180.10 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Foodstuffs Determination of aspartame in table top sweetener preparations Method by high performance liquid chromatographyBS EN 1378 : 1997 This Bri
3、tish Standard, having been prepared under the direction of the Consumer Products and Services Sector Board, was published under the authority of the Standards Board and comes into effect on 15 May 1997 BSI 1997 The following BSI references relate to the work on this standard: Committee reference AW/
4、-/3 Draft for comment 94/501926 DC ISBN 0 580 27122 6 Amendments issued since publication Amd. No. Date Text affected Committees responsible for this British Standard The preparation of this British Standard was entrusted to Technical Panel AW/-/3, Food analysis Horizontal methods, upon which the fo
5、llowing bodies were represented: Association of Public Analysts Food and Drink Federation Institute of Food Science and Technology Laboratory of the Government Chemist Ministry of Agriculture, Fisheries and Food Royal Society of ChemistryBS EN 1378 : 1997 BSI 1997 i Contents Page Committees responsi
6、ble Inside front cover National foreword ii Foreword 2 Text of EN 1378 3ii BSI 1997 BS EN 1378 : 1997 National foreword This British Standard has been prepared by Technical Panel AW/-/3 and is the English language version of EN 1378 : 1996 Foodstuffs Determination of aspartame in table top sweetener
7、 preparations Method by high performance liquid chromatography, published by the European Committee for Standardization (CEN). EN 1378 was produced as a result of international discussions in which the United Kingdom took an active part. Cross-references Publication referred to Corresponding British
8、 Standard EN ISO 3696 : 1995 BS EN ISO 3696 : 1995 Water for analytical laboratory use. Specification and test methods ISO 5725 : 1986, to which informative reference is made in the text, has been superseded by ISO 5725-1 : 1994, ISO 5725-2 : 1994, ISO 5725-3 : 1994, ISO 5725-4 : 1994 and ISO 5725-6
9、 : 1994 which are identical with BS ISO 5725 Accuracy (trueness and precision) of measurement methods and results, BS ISO 5725-1 : 1994 General principles and definitions, BS ISO 5725-2 : 1994 Basic method for the determination of repeatability and reproducibility of a standard measurement method, B
10、S ISO 5725-3 : 1994 Intermediate measures of the precision of a standard measurement method, BS ISO 5725-4 : 1994 Basic method for the determination of the trueness of a standard measurement method, and BS ISO 5725-6 : 1994 Use in practice of accuracy values. Compliance with a British Standard does
11、not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, the EN title page, pages 2 to 8, an inside back cover and a back cover.CEN European Committee for Standardization Comite Europe en de Normalisation Eur
12、opa isches Komitee fu r Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1996 Copyright reserved to CEN members Ref. No. EN 1378 : 1996 E EUROPEAN STANDARD EN 1378 NORME EUROPE ENNE EUROPA ISCHE NORM September 1996 ICS 67.180.10 Descriptors: Food products, intense sweeteners, chemica
13、l analysis, determination of content, high performance liquid chromatography English version Foodstuffs Determination of aspartame in table top sweetener preparations Method by high performance liquid chromatography Produits alimentaires Dosage de laspartame dans les e dulcorants de table Me thode p
14、ar chromatographie liquide a haute performance Lebensmittel Bestimmung von Aspartam in Tafelsu en Hochleistungs- flu ssigkeitschromatographisches Verfahren This European Standard was approved by CEN on 1996-06-09. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipul
15、ate 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 European Standard exi
16、sts 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 national standards bo
17、dies of Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Page 2 EN 1378 : 1996 BSI 1997 Foreword This European Standard has been prepared by the Technical Committee CEN/TC 27
18、5, 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 March 1997, and conflicting national standards shall be withdrawn at t
19、he latest by March 1997. According to the CEN/CENELEC Internal Regulations, the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerla
20、nd and the United Kingdom. Contents Page Foreword 2 1 Scope 3 2 Normative references 3 3 Principle 3 4 Reagents 3 5 Apparatus and equipment 3 6 Procedure 4 7 Expression of results 4 8 Precision 5 9 Test report 5 Annexes A (informative) Figures 6 B (informative) Precision data 8 C (informative) Bibli
21、ography 8Page 3 EN 1378 : 1996 BSI 1997 1) c is the substance concentration. 1 Scope This European Standard specifies a high performance liquid chromatography (HPLC) method for the determination of aspartame in table top sweetener preparations. An inter-laboratory test has been carried out on sweete
22、ner tablets 1. 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 dated references, subsequent amendmen
23、ts 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 ISO 3696 Water for analytical laboratory use Specification and test methods 3 Pr
24、inciple Determination of aspartame in an appropriate solution of table top sweetener preparation in water by HPLC and subsequent photometric detection in the ultraviolet (UV) range. Identification of the aspartame on the basis of the retention time and determination by the external standard method u
25、sing peak areas or peak heights. 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and water of at least grade 1 as defined in EN ISO 3696. 4.1 Aspartame standard substance, with a known content of at least 98 % and not more than 102 % in dry m
26、atter (see 6.2). The loss in mass on drying shall not exceed 4,5 %. The material shall be chromatographically pure and be kept cool and dry. NOTE. For further information on identification and purity, see 2. 4.2 Potassium dihydrogen orthophosphate solution, c (KH 2 PO 4 ) = 0,0125 mol/l. 1) 4.3 Meth
27、anol, suitable for HPLC analysis. 4.4 Orthophosphoric acid. 4.5 Mobile phase for HPLC. Mix 70 parts by volume of the potassium dihydrogen orthophosphate solution (4.2) and 30 parts by volume of methanol (4.3) and adjust the pH to 4,5 with phosphoric acid (4.4). Remove particles by membrane filtratio
28、n (5.2). In order to avoid corrosion problems due to prolonged contact with phosphate containing eluents and as a precaution against blockages occurring due to precipitation of phosphate, water should be pumped through the HPLC equipment after carrying out this method. 4.6 Aspartame standard solutio
29、n. Finely grind at least about 1,5 g of the aspartame standard substance (4.1), then, without delay, dissolve about 200 mg (m 1 ) of the undried, ground aspartame standard substance, weighed to the nearest 0,1 mg, in the mobile phase (4.5) in a 1000 ml (V 2 ) volumetric flask and dilute to the mark
30、with the mobile phase. Reserve remaining finely ground aspartame standard substance for the determination of loss in mass on drying (L D ) (see 6.2). The determination of the loss in mass on drying is carried out immediately. Prepare the standard solution on the day of the examination. Additional so
31、lutions with concentrations within the linear range may be prepared for the calibration graph. Calculate the mass concentration, r, of anhydrous aspartame in milligrams per litre of the standard solution, using the following equation: (1) r = m 1 3 (1002 L D ) 100 where: m 1 is the mass of undried a
32、spartame standard substance used for the stock solution, in milligrams; L D is the loss in mass on drying, in percent. 5 Apparatus and equipment Usual laboratory apparatus and, in particular, the following. 5.1 Filtration unit, e.g. glass vacuum filtration unit consisting of a glass sintered disk (d
33、iameter 50 mm), a 250 ml top section and a 1 l conical flask, all with ground glass joints. 5.2 Membrane filter, suitable for the mobile phase (4.5), pore size# 5mm.Page 4 EN 1378 : 1996 BSI 1997 5.3 High performance liquid chromatograph consisting of a pump, a sample applicator, a UV detector with
34、variable wavelength setting and an evaluation system e.g. a chart recorder or integrator. 5.4 Analytical reversed phase separating column, e.g. C 18 reversed phase, particle size 10mm, diameter 4 mm, length 250 mm. Other particle sizes than specified in this standard may be used. Separation paramete
35、rs have to be adapted to such materials to guarantee equivalent results. NOTE. Minimal theoretical plates at the retention volume of the resolved analyte should preferably be not less than 1900 under the conditions of chromatography employed. 6 Procedure 6.1 Determination of the average tablet mass
36、Determine the mass of at least 20 sweetener tablets to the nearest 0,1 mg and calculate the average mass of one tablet. NOTE. For improved accuracy the use of 100 tablets is recommended. 6.2 Determination of loss in mass on drying of standard substance Weigh, to the nearest 0,1 mg, about 1,0 g of th
37、e remaining finely ground aspartame standard substance (4.1). Dry this portion to constant mass at (105 2) C and determine the loss in mass on drying (L D ) in per cent by weighing. 6.3 Preparation of the sample test solution Dissolve an amount of finely ground (m 0 ) table top sweetener preparation
38、 equivalent to about 400 mg aspartame (corresponding to 20 times the average mass of a tablet), and after transferring it to a 200 ml volumetric flask, dissolve in water and dilute to the mark. Dilute 20,0 ml of this solution with the mobile phase (4.5) to 200,0 ml (V 1 ) and filter through a membra
39、ne filter. Prepare the sample test solution on the day of the analysis. 6.4 Identification by HPLC Identify the sweetener to be determined either by comparing the retention time in the sample with that of the standard substance, or by comparing the absorption properties of the sample with those of t
40、he standard substance after either recording the absorption curve or taking measurements at different wavelengths in the relevant wavelength range for both sample and standard. NOTE 1. If the separating column (5.4) and the mobile phase (4.5) are used, it has been found satisfactory to adopt the fol
41、lowing experimental conditions (see figure A.1). Flow 1,5 ml/min UV detection 217 nm Volume injected 20ml NOTE 2. When detecting at 210 nm, a higher sensitivity can be achieved. Possible interferences with decomposition products from aspartame such as diketopiperazine or phenylalanine can occur. 6.5
42、 Determination by HPLC To carry out the determination by the external standard method, integrate the peak areas or determine the peak heights and compare the results with the corresponding value for the standard substance or use a calibration graph. Inject equal volumes of the sample and standard te
43、st solutions. NOTE. The chromatogram shown in figure A.1 in was prepared using a C 18 reversed phase, particle size 10mm, diameter 4,0 mm, length 250 mm column. 7 Expression of results 7.1 Graphical evaluation (optional) Plot the absorption values of the aspartame standard solutions (4.6) on millime
44、tre graph paper against the aspartame concentrations in milligrams per litre. The calibration graph should be linear. Read off the aspartame concentration, x, in milligrams per litre, corresponding to the absorption of the sample test solution from the calibration graph. 7.2 Calculation for graphica
45、l evaluation 7.2.1 If the calculation is based on the calibration graph, calculate the mass fraction, w 1 , of anhydrous aspartame, in milligrams per kilogram table top sweetener preparation, using the following equation: w 1 = (2) x3 10 6 3 2 m 0 where: x is the concentration of anhydrous aspartame
46、 in the sample test solution, read off from the calibration graph, in milligrams per litre; 10 6 is the conversion factor from milligrams to kilograms; m 0 is the initial sample mass, in milligrams. 7.2.2 Calculate the mass fraction, w 2 , of anhydrous aspartame, in milligrams per tablet, using the
47、following equation: (3) w 2 = x3 m 2 3 2 m 0 where: m 2 is the average tablet mass (6.1), in milligrams; x, m 0 see equation (2). An alternative calculative evaluation using the regression graph may be used.Page 5 EN 1378 : 1996 BSI 1997 7.3 Calculation for routine analysis 7.3.1 The calculation may
48、 either be carried out based on a calibration graph or, having demonstrated the linear range of response for standard solutions, for routine and repeat analyses. Calculate the mass fraction, w 1 , of anhydrous aspartame, in milligrams per kilogram of the sample, using the following equation (externa
49、l standard method): w 1 = (4) A 1 3 V 1 3 m3 10003 F A 2 3 V 2 3 m 0 where: A 1 is the peak area or peak height for the aspartame in the sample test solution, in units of area or length; A 2 is the peak area or peak height for the aspartame in the standard test solution, in units of area or length; V 1 is the total volume of the sample test solution (see 6.2), in millilitres (here: 200 ml); V 2 is the total volume of the standard test solution (see 4.6), in millilitres (here: 1000 ml); m is the mass of aspartame contained i
