1、June 2012 Translation by DIN-Sprachendienst.English price group 9No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67
2、.050!$|vo“1898376www.din.deDDIN EN 16155Foodstuffs Determination of sucralose High performance liquid chromatographic methodEnglish translation of DIN EN 16155:2012-06Lebensmittel Bestimmung von Sucralose Hochleistungsflssigchromatographisches VerfahrenEnglische bersetzung von DIN EN 16155:2012-06Pr
3、oduits alimentaires Dosage du sucralose Mthode par chromatographie liquide haute performanceTraduction anglaise de DIN EN 16155:2012-06www.beuth.deDocument comprises 13 pagesIn case of doubt, the German-language original shall be considered authoritative.06.12 DIN EN 16155:2012-06 2 A comma is used
4、as the decimal marker. National foreword This standard has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). Preliminary work was done by Working Group WG 2 “Sweeteners”. The responsible German body involved in its preparation was the Nor
5、menausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-01-11 AA Sungsmittel. The DIN Standard corresponding to the International Standard referred to in this document is as follows: EN ISO 3696 DIN ISO 3696 National A
6、nnex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16155 April 2012 ICS 67.050 English Version Foodstuffs - Determination of sucralose - High performance liquid chromatographic metho
7、d Produits alimentaires - Dosage du sucralose - Mthode par chromatographie liquide haute performance Lebensmittel - Bestimmung von Sucralose - Hochleistungsflssigchromatographisches Verfahren This European Standard was approved by CEN on 25 February 2012. CEN members are bound to comply with the CEN
8、/CENELEC Internal Regulations 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 CEN-CENELEC Management Cen
9、tre or to any CEN member. This 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 CEN-CENELEC Management Centre has the same status as th
10、e official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romani
11、a, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any means res
12、erved worldwide for CEN national Members. Ref. No. EN 16155:2012: EEN 16155:2012 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus and equipment 56 Procedure .66.1 Sample preparation .66.2 Preparation of the sample test solutions 66.2.1 Soluble samp
13、les (e. g. hard candies and similar products) 66.2.2 Incompletely soluble samples (e. g. pastries, chewing gum, yoghurt, ketchup, mayonnaise) .66.2.3 Solid phase extraction 66.3 High-performance liquid chromatography (HPLC) 66.4 Identification .66.5 Quantitative determination .77 Calculation 78 Prec
14、ision .78.1 General 78.2 Repeatability .78.3 Reproducibility .89 Test report 8Annex A (informative) Statistical data of the inter-laboratory trial 9Bibliography . 11DIN EN 16155:2012-06 EN 16155:2012 (E) 3 Foreword This document (EN 16155:2012) has been prepared by Technical Committee CEN/TC 275 “Fo
15、od 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 2012, and conflicting national standards shall be withdrawn at t
16、he latest by October 2012. 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 nati
17、onal standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands,
18、 Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN EN 16155:2012-06 EN 16155:2012 (E) 4 1 Scope This European Standard specifies a method for the determination of sucralose in foodstuffs by high performance liquid chromatography (HP
19、LC) by means of elution from a reversed-phased (RP) column using aqueous methanol, followed by RI detection 1. This method has been validated in an inter-laboratory study via the analysis of sucralose (from 83 mg/kg to 737 mg/kg) in spiked samples of ketchup, mayonnaise, biscuits, yoghurt, instant b
20、everage powder and sweets. For further information on the validation results, see Annex A. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies.
21、 For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) 3 Principle Depending on their consistency, the samples are either dissolved in water or dil
22、uted with water and, if appropriate, they are filtered or clarified with modified Carrez solutions. Afterwards, they are extracted on a solid phase extraction column and eluted with a mixture of methanol/water. The sucralose content is determined by high-performance liquid chromatography (HPLC) by m
23、eans of elution from a reversed-phased (RP) column using aqueous methanol, followed by RI (refractive index) detection. Alternatively, an ELSD (Evaporative Light Scattering Detector) may be used. Quantification is performed by applying the external standard method. The content of sucralose in foodst
24、uffs means identifying the content of 1,6-dichloro-1,6-dideoxy-D-fructofuranosyl-4-chloro-4-deoxy-D-galactopyranoside, as determined in accordance with the method described in this document. 4 Reagents Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696:1
25、995, unless otherwise specified. Solvents shall be of the same quality used for HPLC analysis, unless otherwise specified. Commercially available solutions with equivalent properties to the reagents listed may be used. 4.1 Sucralose, (C12H19Cl3O8, MW: 397,63). 4.2 Potassium hexacyanoferrate(II), K4F
26、e(CN)6 3H2O. 4.3 Zinc nitrate, Zn(NO3)2 6H2O. 4.4 Methanol for HPLC. 4.5 Stock solution (Suc 1 000 mg/l). Weigh approximately 100 mg of sucralose to the nearest 0,1 mg in a 100 ml volumetric flask; dissolve in a small amount of water and dilute to the calibration mark with water. Prepare the solutio
27、n fresh every day. The water content and the purity of the standard substance shall be taken into consideration. DIN EN 16155:2012-06 EN 16155:2012 (E) 5 4.6 Standard solutions (Suc= 20 mg/l to 100 mg/l). The concentrations of the standard solution given in the following are examples only and may be
28、 changed depending on the devices sensitivity and the concentration range to be covered. Care shall be taken not to exceed the linear range of the detector system. Prepare from the stock solution (4.5) at least five standard solutions by diluting in such a way that sucralose concentrations of e.g.,
29、20 mg/l, 40 mg/l, 60 mg/l, 80 mg/l and 100 mg/l are obtained. Prepare these solutions fresh every day of the analysis. 4.7 Modified Carrez solutions. 4.7.1 Solution A (potassium hexacyanoferrate(II). Dissolve 53,45 g of potassium hexacyanoferrate(II) (4.2) in water and make up to 500 ml. 4.7.2 Solut
30、ion B (Zinc nitrate). Dissolve 148,75 g of zinc nitrate (4.3) in water and make up to 500 ml. 4.8 Elution solution for HPLC. Mix one volume fraction of methanol (4.4) with 3 volume fractions of water. 5 Apparatus and equipment Usual laboratory apparatus, in particular: 5.1 Membrane filter, for sampl
31、e filtration, pore size: 0,45 m maximum. 5.2 Pleated filter. 5.3 C18 SPE cartridges, 500 mg. 5.4 High-performance liquid chromatograph, comprising: a pump; a sample injector; a temperature-controlled RI detector or, alternatively, an ELSD; a column oven; an evaluation system. 5.5 Analytical C-18 rev
32、ersed-phase separating column, 250 mm 4 mm, e.g., Lichrospher1)100 RP-18, 5 m, or a similar column. A guard column with a similar packing material should be used in order to protect the analytical separating column. 1) Lichrospher 100 RP-18 is an example of a suitable product available commercially.
33、 This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. DIN EN 16155:2012-06 EN 16155:2012 (E) 6 6 Procedure 6.1 Sample preparation Homogenize the test material in a suitable manner. Liquid samples, for example,
34、 may be stirred, solid products such as hard candies may be crushed, and products such as chewing gum deep-frozen and then crushed. Semi-solid products such as yoghurt or ketchup may be homogenized by stirring, and then clarified with a modified Carrez solution (4.7), if necessary. Some samples may
35、require pre-treatment using SPE cartridges. 6.2 Preparation of the sample test solutions 6.2.1 Soluble samples (e. g. hard candies and similar products) Dissolve about 5 g to the nearest 1 mg of the homogenized sample in water in a 50 ml volumetric flask and make up with water to the stated volume.
36、Filter through a membrane filter if the solution is turbid. 6.2.2 Incompletely soluble samples (e. g. pastries, chewing gum, yoghurt, ketchup, mayonnaise) Mix about 5 g to the nearest 1 mg of the homogenized sample with approximately 25 ml of water in a 50 ml volumetric flask and stir for 30 min at
37、about 40 C to 60 C (magnetic stirrer) or treat in the ultrasonic bath. Mix protein-containing samples with 1 ml each of the modified Carrez solutions (4.7.1 and 4.7.2) and shake after each addition. Afterwards, bring the sample test solution to room temperature and make up to the mark with water. Fi
38、lter through a pleated filter and then through the membrane filter if the solution is turbid. 6.2.3 Solid phase extraction Condition the solid phase extraction columns (5.3) with 5 ml each of methanol and water. Inject 5 ml of the sample solution given in 6.2.1 or 6.2.2 respectively onto the conditi
39、oned columns. Afterwards, wash the cartridges three times with 5 ml water, taking care not to let the columns run dry during these steps. Subsequently, elute the sucralose from the column with 5 ml of the HPLC elution solution (4.8). Collect in a 10 ml volumetric flask until the columns are dry, and
40、 make up to volume. Filter the eluate through a membrane filter (5.1) and fill into HPLC glass vials. 6.3 High-performance liquid chromatography (HPLC) When using a column in accordance with 5.5, compliance with the following parameters has proven useful: injection volume: up to 100 l eluent: (4.8)
41、column oven temperature: 35 C RI-detector temperature: 30 C flow: approximately 1,2 ml/min. 6.4 Identification Inject an aliquot of the sample test solution into the HPLC-System, preferably under the condition as mentioned in 6.3. Identify sucralose in the sample by comparing the retention time in t
42、he sample with that of the standard sucralose solution. An additional identification consists of adding sucralose to the sample and in determining if the peaks overlap. DIN EN 16155:2012-06 EN 16155:2012 (E) 7 6.5 Quantitative determination Quantitative determination is performed by integrating the
43、peak area of sucralose and relating it to concentration via a calibration function. The obtained peak areas are plotted against the concentrations. A straight line (y = a + bx) is fitted to the results, where b is the value of the slope of the linear function and a is the value where the calibration
44、 function intercepts the y-axis. The appropriate character of the calibration function shall be checked. 7 Calculation Quantify the mass concentration sucin milligram per litre or the mass fraction wsucin milligram per kilogram of sucralose (suc) by integration of the peak area (R) obtained from the
45、 analysis of the injected sample solution. The concentration of sucralose in the sample is then calculated using the calibration function according to Formula (1): mbVaRw=)(or sucsuc(1) where R is the peak area response; a is the intercept of the calibration line (6.5); b is the slope of the calibra
46、tion line (6.5); V is the total volume of the sample solution (e.g. 50 ml); m is the mass of the sample (e.g. 5 g). The result is expressed rounded with no decimal point. 8 Precision 8.1 General The statistical data for the determination of sucralose were obtained in 2007/2008 in two inter-laborator
47、y trials on the following products: ketchup, mayonnaise with high and low sucralose concentrations, biscuits, yoghurt, 2 types of instant beverage powders and 2 compressed tablets (sweets) with different flavours and concentrations. The values derived from the inter-laboratory test may not be applic
48、able to analyte concentration ranges and matrices other than those detailed in Annex A. 8.2 Repeatability The absolute difference between two single test results determined on identical test material by one operator using the same apparatus within the shortest feasible time interval will exceed the repeatability limit r in not more than 5 % of cases. The repeatability depends on the concentration level o
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