DIN EN 16187-2015 Foodstuffs - Determination of fumonisin B1 and fumonisin B2 in processed maize containing foods for infants and young children - HPLC method with immunoaffinity cr.pdf

1、September 2015 Translation by DIN-Sprachendienst.English price group 12No 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).

2、ICS 67.230!%F0/“2351312www.din.deDDIN EN 16187Foodstuffs Determination of fumonisin B1 and fumonisin B2 inprocessed maize containing foods for infants and young children HPLC method with immunoaffinity column cleanup and fluorescencedetection after precolumn derivatization;English version EN 16187:2

3、015,English translation of DIN EN 16187:201509Lebensmittel Bestimmung von Fumonisin B1 und Fumonisin B2 in Suglings- undKleinkindernahrung auf Maisbasis HPLC-Verfahren mit Reinigung an einer Immunoaffinittssule und Fluoreszenzdetektionnach Vorsulenderivatisierung;Englische Fassung EN 16187:2015,Engl

4、ische bersetzung von DIN EN 16187:2015-09Denres alimentaires Dosage de la fumonisine B1 et de la fumonisine B2 dans les alimentspour nourrissons et jeunes enfants contenant du mas transform Mthode par CLHP avec purification sur colonne dimmunoaffinit et dtection defluorescence aprs drivation prcolon

5、ne;Version anglaise EN 16187:2015,Traduction anglaise de DIN EN 16187:2015-09SupersedesDIN CEN/TS 16187(DIN SPEC 91259):2011-06www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 21 pages 08.15 DIN EN 16187:2015-09 2 A comma is used as the

6、decimal marker. National foreword This document (EN 16187:2015) has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was the DIN-Normenausschuss Lebensmittel und landwirtschaftliche

7、 Produkte (DIN Standards Committee Food and Agricultural Products), Working Committee NA 057-01-03 AA Biotoxine. Amendments This standard differs from DIN CEN/TS 16187 (DIN SPEC 91259):2011-06 as follows: a) the status of the Technical Specification has been changed to that of a European Standard; b

8、) the standard has been editorially revised. Previous editions DIN CEN/TS 16187 (DIN SPEC 91259): 2011-06 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16187 June 2015 ICS 67.230 Supersedes CEN/TS 16187:2011English Version Foodstuffs - Determination of fumonisin B1and fumonisin B2in processed

9、 maize containing foods for infants and young children -HPLC method with immunoaffinity column cleanup and fluorescence detection after pre-column derivatisation Denres alimentaires - Dosage de la fumonisine B1et de la fumonisine B2dans les aliments pour nourrissons et jeunes enfants contenant du ma

10、s transform - Mthode par CLHP avec purification sur colonne dimmunoaffinit et dtection de fluorescence aprs drivation prcolonne Lebensmittel - Bestimmung von Fumonisin B1und Fumonisin B2in Suglings- und Kleinkindernahrung auf Maisbasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinittssule und

11、Fluoreszenzdetektion nach Vorsulenderivatisierung This European Standard was approved by CEN on 7 May 2015. 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

12、ion. Up-to-date lists and bibliographical references concerning such national standards 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 b

13、y 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 the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

14、 Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARD

15、IZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16187:2015 EEN 16187:2015 (E) 2 Contents Pa

16、ge Foreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents .4 5 Apparatus .7 6 Procedure .8 7 Calculation . 10 8 Precision 11 9 Test report . 13 Annex A (informative) Typical chromatograms 14 Annex B (informative) Precision data . 15 Annex C (informative) Comparison between the method

17、 in this document and EN 14352:2004 and EN 13585:2001 on fumonisins in maize 18 Bibliography . 19 DINEN 16187:2015-09EN 16187:2015 (E) 3 Foreword This document (EN 16187:2015) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held b

18、y 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 December 2015, and conflicting national standards shall be withdrawn at the latest by December 2015. Attention is drawn to the possibility th

19、at 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. This document supersedes CEN/TS 16187:2011. No technical change has been introduced during the conversion of CEN/TS 16187:2011

20、 into this final draft European Standard. WARNING The use of this document can involve hazardous materials, operations and equipment. This document does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this document to establish appropri

21、ate safety and health practices and determine the applicability of regulatory limitations prior to use. According to the CEN/CENELEC Internal Regulations, the national standards organisations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croati

22、a, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

23、 United Kingdom. DIN EN 16187:2015-09 EN 16187:2015 (E) 4 1 Scope This European Standard specifies a method for the determination of fumonisin B1(FB1) and fumonisin B2(FB2) in processed maize-containing foods for infants and young children by high performance liquid chromatography (HPLC) with immuno

24、affinity cleanup and fluorescence detection (FLD). This method has been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samples ranging from 112 g/kg to 458 g/kg for FB1+FB2, 89 g/kg to 384 g/kg for FB1and 22 g/kg to 74 g/kg for FB2. For further infor

25、mation on the validation, see Clause 8 and Annex B. 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. For undated references, the latest edi

26、tion 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 Fumonisins are extracted from the sample with a mixture of citrate-phosphate buffer with methanol and acetonitrile. T

27、he filtered extract is diluted with water and applied to an immunoaffinity column containing antibodies specific to fumonisins. Fumonisins are eluted from the column with methanol and water and quantified by HPLC-FLD with pre-column derivatisation with o-phthaldialdehyde (OPA) reagent. 4 Reagents Us

28、e only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696:1995, unless otherwise specified. Solvents shall be of quality for HPLC analysis, unless otherwise specified. Commercially available solutions with equivalent properties to those listed may be used. WARNIN

29、G Dispose of waste solvents according to applicable environmental rules and regulations. Decontamination procedures for laboratory wastes have been reported by the International Agency for Research on Cancer (IARC), see 1. 4.1 Acetonitrile WARNING Acetonitrile is hazardous and samples shall be shake

30、n using an orbital shaker which is housed within a fume cupboard. After shaking, samples shall be filtered inside a fume cupboard. 4.2 Methanol 4.3 O-phthaldialdehyde (OPA) 4.4 Citric acid solution, substance concentration c(C6H8O7H2O) = 0,1 mol/l Dissolve 21,0 g of C6H8O7H2O in water and dilute to

31、1 l. 4.5 Disodium hydrogen phosphate solution, c(Na2HPO4) = 0,2 mol/l Dissolve 28,4 g of Na2HPO4in water and dilute to 1 l. DINEN 16187:2015-09EN 16187:2015(E)5 4.6 2-mercaptoethanol 4.7 Citrate-phosphate buffer solution Mix 1 part per volume of citric acid solution (4.4) with 1 part per volume of d

32、isodium hydrogen phosphate solution (4.5). 4.8 Extraction solvent Mix 2 parts per volume of citrate-phosphate buffer solution (4.7) with 1 part per volume of methanol (4.2) and 1 part per volume of acetonitrile (4.1). 4.9 Glacial acetic acid 4.10 Phosphate buffered saline (PBS) solution, c(NaCl) = 1

33、37 mmol/l, c(KCl) = 2,7 mmol/l, c(phosphate buffer) = 10 mmol/l, pH = 7,4 at T = 25 C Dissolve one tablet of commercially available PBS material in 200 ml of water. 4.11 Mixture of acetonitrile and water A Mix 1 part per volume of acetonitrile (4.1) with 1 part per volume of water. Use this solvent

34、to prepare spiking solutions. 4.12 Mixture of acetonitrile and water B Mix 3 parts per volume of acetonitrile (4.1) with 7 parts per volume of water. Use this solvent to prepare calibration solutions and to redissolve dried extracts from immunoaffinity cleanup. 4.13 Sodium tetraborate solution, c(Na

35、2B4O710H2O) = 0,1 mol/l Dissolve 3,8 g of Na2B4O710H2O in 100 ml of water. 4.14 OPA reagent solution Dissolve 40 mg of OPA (4.3) in 1 ml of methanol (4.2) and dilute with 5 ml of sodium tetraborate solution (4.13). Add 50 l of 2-mercaptoethanol (4.6) and mix for 1 min. This reagent solution is stabl

36、e for up to one week at room temperature in the dark in a capped amber vial. 4.15 HPLC mobile phase 4.15.1 HPLC mobile phase A Mix 30 parts per volume of acetonitrile (4.1) with 69 parts per volume of water and 1 part per volume of glacial acetic acid (4.9). 4.15.2 HPLC mobile phase B Mix 60 parts p

37、er volume of acetonitrile (4.1) with 39 parts per volume of water and 1 part per volume of glacial acetic acid (4.9). 4.16 Immunoaffinity column The immunoaffinity column shall contain antibodies raised against FB1and FB2. The column shall have a capacity of not less than 5 g of fumonisins and shall

38、 give a recovery of not less than 80 % for the sum of FB1and FB2when applied as a standard solution in PBS containing 5 g of fumonisins. The columns shall be warmed up to room temperature before use. With these columns the working range of the method reaches DINEN 16187:2015-09EN 16187:2015 (E) 6 5

39、000 g/kg of total fumonisins. The use of columns with a capacity less than 5 g of fumonisins will reduce the working range of the method. 4.17 Certified standard solution of fumonisin B1(FB1), mass concentration (FB1) = 50 g/ml in a mixture of 1 part per volume of acetonitrile and 1 part per volume

40、of water (e.g. Biopure RK 002003 1)or equivalent) 4.18 Certified standard solution of fumonisin B2(FB2), (FB2) = 50 g/ml in a mixture of 1 part per volume of acetonitrile and 1 part per volume of water (e.g. Biopure RK 002004 1)or equivalent) WARNING Fumonisins are nephrotoxic, hepatotoxic and carci

41、nogenic to rats and mice and classified as possible human carcinogen by IARC. These compounds should be treated with extreme caution. Gloves and safety glasses shall be worn at all times and all standard and sample preparation stages shall be carried out in a fume cupboard. 4.19 Mixed FB1and FB2stoc

42、k solution Prepare a mixed FB1and FB2stock solution by pipetting 2 000 l of the FB1certified standard solution (4.17) and 500 l of the FB2certified standard solution (4.18) into a vial. Cap the vial and shake well to obtain a stock solution containing 40,0 g/ml of FB1and 10,0 g/ml of FB2. 4.20 Dilut

43、ed mixed FB1and FB2stock solution Pipette 500 l of the mixed stock solution (4.19) into a 10 ml calibrated volumetric flask. Fill up to the mark with the mixture of acetonitrile water B (4.12) and shake well to obtain a diluted mixed stock solution containing 2,0 g/ml of FB1and 0,5 g/ml of FB2. Stor

44、e the solution at less than 4 C. This solution is stable for at least 6 months at these conditions. 4.21 Mixed FBs calibration solutions for HPLC Prepare five HPLC mixed calibration solutions in 5 ml calibrated volumetric flasks by further diluting the diluted mixed FBs stock solution (4.20) accordi

45、ng to Table 1. Make up each calibration solution to volume (5 ml) with the mixture of acetonitrile and water B (4.12) and mix well. Store the solution at less than 4 C. This solution is stable for at least 6 months at these conditions. Table 1 Preparation of mixed FBs calibration solutions for HPLC

46、HPLC calibration solution Diluted mixed FBs stock solution (4.20) Final concentration of mixed FBs calibration solutions (4.21) Sample equivalent levels of FB1and FB2al FB1g/ml FB2g/ml FB1g/kg FB2g/kg 1 100 0,04 0,01 20,0 5,0 2 200 0,08 0,02 40,0 10,0 3 400 0,16 0,04 80,0 20,0 4 1 000 0,40 0,10 200,

47、0 50,0 5 2 400 0,96 0,24 480,0 120,0 aFor a sample extract reconstituted in 500 l of the mixture of acetonitrile and water B (4.12). 1) Biopure RK 002003 and RK 002004 are examples of suitable products available commercially. This information is given for the convenience of users of this European St

48、andard and does not constitute an endorsement by CEN of these products. Equivalent products may be used if they can be shown to lead to the same results. DIN EN 16187:2015-09 EN 16187:2015 (E) 7 5 Apparatus Usual laboratory glassware and equipment and, in particular, the following: 5.1 Analytical balance, capable of weighing to 0,000 1 g. 5.2 Laboratory balance, capable of weighing to 0,1 g. 5.3 Thermostated water bath. 5.4 Conical flasks, of 250 ml capacity with screw caps. 5.