1、BSI Standards PublicationBS EN 16187:2015Foodstuffs Determination offumonisin B1 and fumonisin B2in processed maize containingfoods for infants and youngchildren HPLC methodwith immunoaffinity columncleanup and fluorescencedetection after pre-columnderivatisationBS EN 16187:2015 BRITISH STANDARDNati
2、onal forewordThis British Standard is the UK implementation of EN 16187:2015. It supersedes DD CEN/TS 16187:2011 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this comm
3、ittee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 80963 7 ICS 67.23
4、0 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 June 2015.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dEUROPEAN STANDARD NORME
5、EUROPENNE EUROPISCHE NORM EN 16187 June 2015 ICS 67.230 Supersedes CEN/TS 16187:2011English Version Foodstuffs - Determination of fumonisin B1 and fumonisin B2 in processed maize containing foods for infants and young children - HPLC method with immunoaffinity column cleanup and fluorescence detecti
6、on after pre-column derivatisation Denres alimentaires - Dosage de la fumonisine B1 et de la fumonisine B2 dans les aliments pour nourrissons et jeunes enfants contenant du mas transform - Mthode par CLHP avec purification sur colonne dimmunoaffinit et dtection de fluorescence aprs drivation prcolon
7、ne Lebensmittel - Bestimmung von Fumonisin B1 und Fumonisin B2 in Suglings- und Kleinkindernahrung auf Maisbasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinittssule und Fluoreszenzdetektion nach Vorsulenderivatisierung This European Standard was approved by CEN on 7 May 2015. CEN members are
8、 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 standards may be obtained on application to t
9、he 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 notified to the CEN-CENELEC Management Cen
10、tre 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuan
11、ia, 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 CEN-CENELEC Management Centre: Avenue Marnix 17, B-10
12、00 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 EBS EN 16187:2015EN 16187:2015 (E) 2 Contents Page Foreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents .4 5 Apparatus .7 6 Procedure .8
13、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 in this document and EN 14352:2004 and EN 13585:2001 on fumonisins in maize 18 Bibliography . 19 BS EN 161
14、87:2015EN 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 by DIN. This European Standard shall be given the status of a national standard, either by publication of an id
15、entical 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 that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be
16、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 into this final draft European Standard. WARNING The use of this document can involve hazardous materials, op
17、erations 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 appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Accord
18、ing to the CEN/CENELEC Internal Regulations, the national standards organisations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany,
19、Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16187:2015EN 16187:2015 (E) 4 1 Scope This European Standard specifies a method for the
20、 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 immunoaffinity cleanup and fluorescence detection (FLD). This method has been validated in an interlaboratory study via t
21、he 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 information on the validation, see Clause 8 and Annex B. 2 Normative references The following documents, in whole or in
22、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 edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laborato
23、ry 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. The filtered extract is diluted with water and applied to an immunoaffinity column containing antibodies specific to
24、 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 Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696:1995, unless otherwi
25、se specified. Solvents shall be of quality for HPLC analysis, unless otherwise specified. Commercially available solutions with equivalent properties to those listed may be used. WARNING Dispose of waste solvents according to applicable environmental rules and regulations. Decontamination procedures
26、 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 shaken using an orbital shaker which is housed within a fume cupboard. After shaking, samples shall be filtered inside a
27、 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 1 l. 4.5 Disodium hydrogen phosphate solution, c(Na2HPO4) = 0,2 mol/l Dissolve 28,4 g of Na2HPO4in water and dilute
28、 to 1 l. BS EN 16187:2015EN 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 disodium hydrogen phosphate solution (4.5). 4.8 Extraction solvent Mix 2 parts per volume of citrate-phosphate buffer
29、 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) = 137 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 commerc
30、ially 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 to prepare spiking solutions. 4.12 Mixture of acetonitrile and water B Mix 3 parts per volume of acetonitrile (4.1)
31、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(Na2B4O710H2O) = 0,1 mol/l Dissolve 3,8 g of Na2B4O710H2O in 100 ml of water. 4.14 OPA reagent solution Dissolve 40 mg
32、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 stable 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 mobi
33、le 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 per volume of acetonitrile (4.1) with 39 parts per volume of water and 1 part per volume of glacial acetic acid (4.9)
34、. 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 give a recovery of not less than 80 % for the sum of FB1and FB2when applied as a standard solution in PBS containin
35、g 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 BS EN 16187:2015EN 16187:2015 (E) 6 5 000 g/kg of total fumonisins. The use of columns with a capacity less than 5 g of fumonisins will reduce the working ra
36、nge 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 of water (e.g. Biopure RK 002003 1)or equivalent) 4.18 Certified standard solution of fumonisin B2(FB2), (FB2) = 50 g/m
37、l 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 carcinogenic to rats and mice and classified as possible human carcinogen by IARC. These compounds should be treated with ex
38、treme 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 FB2stock solution Prepare a mixed FB1and FB2stock solution by pipetting 2 000 l of the FB1certified standard solution (4.17) a
39、nd 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 Diluted mixed FB1and FB2stock solution Pipette 500 l of the mixed stock solution (4.19) into a 10 ml calibrated volumetric f
40、lask. 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. Store the solution at less than 4 C. This solution is stable for at least 6 months at these conditions. 4.21 Mixed FBs cali
41、bration 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) according to Table 1. Make up each calibration solution to volume (5 ml) with the mixture of acetonitrile and water B (4.12) a
42、nd 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 HPLC calibration solution Diluted mixed FBs stock solution (4.20) Final concentration of mixed FBs calibration solution
43、s (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 0,10 200,0 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 wa
44、ter 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 Standard and does not constitute an endorsement by CEN of these products. Equivalent products may be used if they can be
45、shown to lead to the same results. BS EN 16187:2015EN 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.
46、5.4 Conical flasks, of 250 ml capacity with screw caps. 5.5 Orbital shaker. 5.6 Centrifuge, capable of a centrifugal force up to 3 000 g. 5.7 Centrifuge bottles, of 250 ml capacity with screw caps. 5.8 Calibrated microliter pipettes or microliter syringes, of 100 l, 200 l or 1 000 l capacity. 5.9 Di
47、splacement pipettes, of 5 ml, 10 ml or 25 ml capacity. 5.10 Vacuum manifold, to accommodate immunoaffinity columns (4.16). 5.11 Reservoirs (of 25 ml capacity) and attachments to fit to columns. 5.12 Vacuum pump. 5.13 Filter paper, e.g. qualitative, strong, fast flow, 24 cm diameter, 30 m pore size,
48、prefolded or equivalent. 5.14 Glass microfibre filter paper, e.g. 1,6 m pore size or equivalent. 5.15 Heating block with nitrogen or air gas supply. 5.16 Vials, of 4 ml to 12 ml capacity with screw caps. 5.17 HPLC autosampler vials, of 1,8 ml capacity with caps. 5.18 Glass flat bottom vial insert, o
49、f 250 l volume capacity. 5.19 Vortex mixer, or equivalent. 5.20 HPLC apparatus, comprising the following: 5.20.1 Injection system. 5.20.2 Mobile phase pump, (binary, ternary or quaternary pump), capable of generating a binary gradient at 1 ml/min. 5.20.3 Autosampler, capable of performing automated pre-column derivatisation with OPA reagent according to 6.4.1. 5.20.4 Analytical reverse-phase HPLC separating column, e.g. C18 reverse-phase column, 150 mm x 4,6 mm, 5 m preceded by a suitable pre-column or guard fil
