1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 16155:2012Foodstuffs Determination ofsucralose High performanceliquid chromatographic methodBS EN 16155:2012 BRITISH STANDARDNational forewordThis British Standard is the U
2、K implementation of EN 16155:2012. The UK participation in its preparation was entrusted to TechnicalCommittee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all
3、 the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2012. Published by BSI Standards Limited 2012ISBN 978 0 580 72441 1 ICS 67.050 Compliance with a British Standard cannot confer immunity from legal obligations.This British S
4、tandard was published under the authority of the Standards Policy and Strategy Committee on 30 April 2012.Amendments issued since publicationDate T e x t a f f e c t e dBS EN 16155:2012EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16155 April 2012 ICS 67.050 English Version Foodstuffs - Deter
5、mination of sucralose - High performance liquid chromatographic method Produits alimentaires - Dosage du sucralose - Mthode par chromatographie liquide haute performance Lebensmittel - Bestimmung von Sucralose - Hochleistungsflssigchromatographisches Verfahren This European Standard was approved by
6、CEN on 25 February 2012. 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 alteration. Up-to-date lists and bibliographical references concerning such national stand
7、ards may be obtained on application to the CEN-CENELEC Management Centre 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 no
8、tified to the CEN-CENELEC Management Centre has the same status as the 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, Lithua
9、nia, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, 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
10、 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16155:2012: EBS EN 16155:2012EN 16155:2012 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus and equipment 56 Procedure .66.1
11、Sample preparation .66.2 Preparation of the sample test solutions 66.2.1 Soluble samples (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 chromatograph
12、y (HPLC) 66.4 Identification .66.5 Quantitative determination .77 Calculation 78 Precision .78.1 General 78.2 Repeatability .78.3 Reproducibility .89 Test report 8Annex A (informative) Statistical data of the inter-laboratory trial 9Bibliography . 11BS EN 16155:2012EN 16155:2012 (E) 3 Foreword This
13、document (EN 16155:2012) 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 publication of an identical text or by endorsement, at the la
14、test by October 2012, and conflicting national standards shall be withdrawn at the 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
15、 such patent rights. According to the CEN/CENELEC Internal Regulations, the national 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, Hung
16、ary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16155:2012EN 16155:2012 (E) 4 1 Scope This European Standard specifies a method for the determinati
17、on of sucralose in foodstuffs by high performance liquid chromatography (HPLC) 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
18、/kg) in spiked samples of ketchup, mayonnaise, biscuits, yoghurt, instant beverage 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
19、 for its application. For dated references, only the edition cited applies. 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 Depen
20、ding on their consistency, the samples are either dissolved in water or diluted 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
21、content is determined by high-performance liquid chromatography (HPLC) by means 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 b
22、y applying the external standard method. The content of sucralose in foodstuffs 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 re
23、cognized analytical grade and water complying with grade 1 of EN ISO 3696:1995, 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 Su
24、cralose, (C12H19Cl3O8, MW: 397,63). 4.2 Potassium hexacyanoferrate(II), K4Fe(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
25、of water and dilute to the calibration mark with water. Prepare the solution fresh every day. The water content and the purity of the standard substance shall be taken into consideration. BS EN 16155:2012EN 16155:2012 (E) 5 4.6 Standard solutions (Suc= 20 mg/l to 100 mg/l). The concentrations of the
26、 standard solution given in the following are examples only and may be 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 solut
27、ions by diluting in such a way that sucralose concentrations of e.g., 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
28、hexacyanoferrate(II) (4.2) in water and make up to 500 ml. 4.7.2 Solution 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 Usu
29、al laboratory apparatus, in particular: 5.1 Membrane filter, for sample 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,
30、 an ELSD; a column oven; an evaluation system. 5.5 Analytical C-18 reversed-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
31、 100 RP-18 is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. BS EN 16155:2012EN 16155:2012 (E) 6 6 Procedure 6.1 Sample preparation Homogenize the
32、 test material in a suitable manner. Liquid samples, for example, 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
33、a modified Carrez solution (4.7), if necessary. Some samples may 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
34、ml volumetric flask and make up with water to the stated volume. 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 2
35、5 ml of water in a 50 ml volumetric flask and stir for 30 min at 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 so
36、lution to room temperature and make up to the mark with water. Filter 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 samp
37、le solution given in 6.2.1 or 6.2.2 respectively onto the conditioned 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). C
38、ollect in a 10 ml volumetric flask until the columns are dry, and 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 parameter
39、s has proven useful: injection volume: up to 100 l eluent: (4.8) 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. Iden
40、tify sucralose in the sample by comparing the retention time in the 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. BS EN 16155:2012EN 16155:2012 (E) 7 6.5 Quantitative determination
41、 Quantitative determination is performed by integrating the 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
42、the linear function and a is the value where the calibration 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
43、 (suc) by integration of the peak area (R) obtained from the 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
44、of the calibration line (6.5); b is the slope of the calibration 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
45、 sucralose were obtained in 2007/2008 in two inter-laboratory 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 val
46、ues derived from the inter-laboratory test may not be applicable 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
47、 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 of the analyte in the sample. BS EN 16155:2012EN 16155:2012 (E) 8 Ketchup x = 128,83 mg/kg r = 8,38 mg/kg Mayonnaise 1 x = 463,03 mg/k
48、g r = 38,58 mg/kg Mayonnaise 2 x = 175,84 mg/kg r = 27,87 mg/kg Biscuits x = 737,30 mg/kg r = 31,12 mg/kg Yoghurt x = 87,53 mg/kg r = 7,88 mg/kg Instant beverage powder 1 x = 249,52 mg/kg r = 24,61 mg/kg Instant beverage powder 2 x = 225,21 mg/kg r = 16,88 mg/kg Sweets 1 x = 471,14 mg/kg r = 25,30 m
49、g/kg Sweets 2 x = 139,35 mg/kg r = 10,73 mg/kg 8.3 Reproducibility The absolute difference between two single test results on identical test material reported by two laboratories will exceed the reproducibility limit R in not more than 5 % of the cases. Ketchup x = 128,83 mg/kg R = 18,16 mg/kg Mayonnaise 1 x = 463,03 mg/kg R = 69,92 mg/kg Mayonnaise 2 x = 175,84 mg/kg R = 53,55 mg/kg Biscuits x = 737,30 mg/kg R = 130,89 mg/kg Yoghurt x
