1、 STD-BSI BS EN 1378-ENGL 1797 Lh2qbbS 0b332 BRITISH STANDARD Foodstfls - Determination of aspartame in table top sweetener preparations - Method by high performance liquid chromatography The hopean Standard EN 1378 : 1996 has the status of a British Standard ICs 67.180.10 NO COPYING WITHOUT BSI PERM
2、ISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW 383 BS EN 1378 : 1997 BS EN 1378 : 1997 Amd. No. This British 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 19
3、97 O BSI 1997 Date Text affected Committees responsible for this British Standard The preparation of this British Standard was entrusted to Technical Panel AW/-B, Food analysis - Horizontal methods, upon which the following bodies were represented: Association of Public Analyss Food and Drink Federa
4、tion Mtuk of Food Science and Technology Laboratory of the Government Chemist Muustry of Agriculture, Fisheries and Food Royal Society of Chemistry The following BSI references relate to the work on this standard Committee reference AW/-/3 Draft for comment 94601926 DC ISBN O 680 27122 6 STD=BSI BS
5、EN L378-ENGL 1997 Lb2LibbS Ob33298 15b W BS EN 1378 : 1997 Contents Committees responsible Page Inside front cover National foreword ii Foreword 2 Text of EN 1378 3 * m * O BSI 1997 i STD.BSI BS EN 1378-ENGL 1777 Lb2libb b33277 072 D BS EN 1378 : 1997 National foreword This British Standard has been
6、 prepared by Technical Panel AW/B and is the English ianguage version of EN 1378 : 1996 Foodstufls - DetemL.ination of aspartume in table top sweetenerprepamtions - Method by high pm$onnance liquid chnnnutogmphy, published by the European Committee for Standardization (CEN). EN 1378 was produced as
7、a resuit of international discussions in which the United Kingdom took an active part. Cross-references Publication referred to EN IS0 3696 : 1995 Corresponing British Standard BS EN IS0 3696 : 1995 Water for analyticai labomtow use. Spehficatwn and test methods IS0 5725 : 1986, to which informative
8、 reference is made in the text, has been superseded by IS0 57251 : 1994, IS0 57252 : 1994, IS0 5725-3 : 1994, IS0 57254 : 1994 and IS0 5725-6 : 1994 which are identicai with BS IS0 5725 Acm (trueness und precision) of rn.4msu-t methods and results, BS IS0 57251 : 1994 General principles and definiti
9、ons, Bs IS0 57252 : 1994 Bmic method for ULe detemnination of -peataoil.ity and repmdmibility of a stundud meclsurement method, BS IS0 57253 : 1994 Inte?naediate measures of the precision of a standavd rrtmsurement method, BS IS0 57254 : 1994 Basic method for LD is the loss in mass on drying, in per
10、cent 6 Apparatus and equipment Usual laboratory apparatus and, in particular, the following. 6.1 Filtration unit, e.g. glass vacum lWon unit consisting of a giasS sintered disk (diameter 50 mm), a 250 mi top section and a 1 1 conical flask, ail with ground glass joints. 6.2 Membrane fzlter, suitable
11、 for the mobile phase (4.Q pore size 5 5 pn. c is the substance concentration. O BSI 1997 STD.BSI BS EN 1378-ENGL 1797 LbZIbb9 Ob33303 31i3 Page 4 EN 1378 : 1996 6.3 High pqlnmum liguid chwmatograph consisting of a pump, a sample appiicahr, a UV detedor with variable wavelength setting and an evalua
12、tion system e.g. a chart recorder or integrator. 6.4 Analytical reversed phclse separating column, e.g. C 18 reversed phase, particle size 10 pm, diameter 4 mm, length 250 mm. other particle sizes than specified in this standard may be used. Separation parameters have to be adapted to such materiais
13、 to guarantee equivalent results. NOTE. Minimal theoretical piates 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 Determine the mass of at least 20 sweet
14、ener tablets to the nearest 0,l 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,l mg, about 1,0 g of the remaining nely ground aspartame stana
15、rd substance (4.1). Dry this portion to constant mass at (105 f 2) “C and determine the loss in mass on drying (LD) in per cent by weighing. 6.3 Preparation of the sample test solution Dissolve an amount of nely ground (mo) table top sweetener prepamlion ment to about 400 mg aspartame (corresponduig
16、 to 20 times the average mass of a tablet), and after tsansferring it to a 200 ml volumetric flask, dissolve in water and dilute to the mark mute 20,O mi of this solution with the mobile phase (4.6) to 200,O mi (VI) and lter through a membrane lter. Prepare the sample test solution on the day of the
17、 analysis. 6.4 Identification by HPLC Identify the sweetener to be determined either by comparing the retention thne in the sample with that of the standard substance, or by comparing the absorption properties of the sample with those of the standard substance after either recording the absorption c
18、urve or talnng measurements at different wavelengths in the relevant wavelength range for both sample and standard NOTE 1. if the separating column (6.4) and the mobile phase (4.6) are used, it has been found satisfactory to adopt the following experimental conditions (see figure A. 1). Flow 1,5 mlm
19、in UV detection 217 nm Volume iIyected 20 pl 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 OcCui: 6.6 Determination by HPLC lb carry out the determination by the
20、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 dibraon graph. Inject equal volumes of the sample and standard test solutions. NOTE. The chromatogram shown in figure Al in was pr
21、epared using a C 18 reversed phase, particle size 10 pm, diameter 4,O mm, length 250 nun column. 7 Expression of results 7.1 Graphical evaluation (optional) Plot the absorption values of the aspartsune standard solutions (4.6) on millimetre graph paper against the aspartame concentrations in mihgram
22、 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 graphical evaluation 7.2.1 If the calculation is based on the calibration
23、graph, caiculak the mass fraction, w1, of anhydrous aspartame, in milligrams per kilogram table top sweetener preparation, using the following equation: xx106x2 w1= mo where: x is the concentration of anhydrous aspartame in the sample test solution, read off from the caJibration graph, in milligrams
24、 per litre; io6 is the conversion factor from milligrams to mo is the initial sample mass, in milligrams. kilograms; 7.2.2 Calculate the mass fraction, w2, of anhydrous aspartame, in milligrams per tablet, using the following equation: XXrrQX2 w2 = mo (3) where: m2 x, m see equation (2). is the aver
25、age tablet mass (6.1), in milligrams; An altemative caiculative evaluation using the regression graph may be used. O BSI 1997 STD=BSI BS EN 1378-ENGL 1777 1b24bb7 Ob33304 28T = 7.3 Calculation for routine analysis 7.3.1 The calculation may either be carried out based on a calibration graph or, havin
26、g demonstsated the hear range of response for standard solutions, for routine and repeat analyses. Calculate the mass Mon, w1, of anhydrous aspartame, in milligrams per kilogram of the sample, using the following equation (exteniai standard method): (4) Al X VI X m X lo00 X F w1= A2 x v2 x mo where:
27、 Al A2 Vl V2 m mo lo00 F Report is the peak area or peak height for the aspartame in the sample test solution, in units of area or length; is the peak area or peak height for the aspartame in the stanard test solution, in units of area or length; is the totai volume of the sample test solution (see
28、6.2), in miililitres (here: 200 mi); is the totai volume of the standard test solution (see 4.6), in millilitres (here: lo00 ml); is the mass of aspartame contained in V2, correcd for the loss in mass on drying, in is the jniia sample mass of 20 tablets, in grams; is the conversion factor from grams
29、 to kilograms; is the dilution factor (here: 10). the result after rouning to one decimal place. Page 6 EN 1378 : 1996 8 Precision Details of the inter-laboratory test of the precision of the method accordmg to IS0 5725 : 1986 3 are summarized in annex B. The values derived from the inter-laboratory
30、 te - a reference to this European Standard or to the method used; - the results and the units in which the results have been - if the repeatability of the method has been veA xmo where: w1, mo see equation 4. - any particular points observed in the course of the - any operations not specified in th
31、e method or regarded as optional which might have affected the results. (5) test; O ES1 1997 Page 6 EN 1378 : 1996 0,080 AU Annex A (informative) Figures 1 8,30 I o, O70 0,060 t 0,050 c O *L 0,040 a k 0,030 VI 2 0,020 0,010 I I I I I I I O 2 4 6 8 10 12 14 min time - sepamhg column Mobile phase Migr
32、ation rate 1,5 dmin UV detection 217 nm diameter 4 mm, length 250 mm; NucleosilB lo) C 18, particle size 10 pm poassium dihydrogenphosphate solution (c = 0,0125 mol/l)/methanol for HPLC (7030 V/V) and phosphoric acid H = 4,5) Volume injected 20 p1 Aspartame retention time 8,30 min Figure A.1. Exampl
33、e for an HPLC chromatogram of 200 mg/i aspartame standard solution Nucleosii 100 is a trade-name of a product supplied by Marcherey-Nagel, Dttren. This infomation is given for the convenience of users of this draft European Standard and does not constitute an endorsement by CEN of this product named
34、. Equivalent products may be used if they can be shown to lead to the same results. O BSI 1997 STD-BSI BS EN 1378-ENGL 1797 m 1b2LibbS Ob3330b 052 m Page 7 EN 1378 : 1996 Sample k4p-e Soivent Mobile phase (4.0) Concentration 200 mp/i Function Absorption Wavelengh range 200nmt0300nm 2,1?l 1 ,?3?2 1 1
35、,2973 c a a i 0,85?36 2? .- Methods of sampling and analysis of foods, tobacco pmducts, cosmetics and commodity goods, Federal Health Once) Loseblattausgabe, Stand Mai 1994. Bd. 1 (Loose-leaf edition of 1999-05 Vol. I.) Berlin, Kln: Beuth Verlag GmbH 2 FA0 Food and Nutrition Paper 19 of the Joint FA
36、O/WHO Expert Committee on Food Additives, Apri 1981. 3 Is0 5725 : 1986precision of test methods -Determination of repeutubiCy and vvpmd Fax: 0181 996 7400. BSI offers members an inividual updating service called PLUS which ensures that subscribers automatically receive the latest editions of standar
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