EN 14352:2004

Foodstuffs - Determination of fumonisin B1 and B2 in maize based foods - HPLC method with immunoaffinity column clean up

This document specifies a method for the detemination of fumonisin B1 (FB1) and fumonisin B2 (FB2) in maize based foods using high performance liquid chromatography (HPLC) and immunoaffinity clean-up, see [1], [2], [3].
The method has been successfully validated in a collaborative study according to AOAC Guidelines for collaborative study procedures [4] to validate characteristics of a method of analysis for the determination of fumonisins in maize flour and corn flakes containing 323 µg/kg to 1414 µg/kg FB1 and 90 µg/kg to 558 µg/kg FB2.

Lebensmittel - Bestimmung von Fumonisin B1 und B2 in Maiserzeugnissen - HPLC-Vefahren mit Immunoaffinitätssäulen-Reinigung

Dieses Europäische Dokument legt ein Verfahren zur Bestimmung des Gehaltes von Fumonisin B1 (FB1) und Fumonisin B2 (FB2) in Maiserzeugnissen mit Hochleistungs-Flüssigchromatographie (HPLC) und Immunoaffinitätssäulen-Reinigung fest, siehe [1], [2] und [3].
Das Verfahren wurde erfolgreich in einem Ringversuch nach den "AOAC-Guidelines for Collaborative Studies to validate characteristics of a method of analysis" [4] für die Untersuchung von Fumonisinen in Maismehl und Cornflakes, die 323 µg/kg bis 1 414 µg/kg FB1 und 90 µg/kg bis 558 µg/kg FB2 enthalten, validiert.

Produits alimentaires - Dosage des fumonisines B1 et B2 dans des aliments à base de maïs - Méthode CLHP avec purification par colonne d'immunoaffinité

Le présent document spécifie une méthode de dosage de la fumonisine B1 (FB1) et de la fumonisine B2 (FB2) dans les aliments à base de maïs, par chromatographie en phase liquide haute performance (CLHP) et par purification par immunoaffinité (voir [1], [2], [3].
Cette méthode, validée par un essai interlaboratoires selon les Lignes Directrices AOAC pour Essais Interlaboratoires [4], consiste à doser les fumonisines dans la farine de maïs et les flocons de maïs contenant 323 µg/kg à 1414 µg/kg de FB1 et 90 µg/kg à 558 µg/kg de FB2.

Živila - Določevanje fumonizina B1 in B2 v živilih na osnovi koruze - Metoda HPLC s čiščenjem z imunoafinitetno kolono

General Information

• Status: Published
• Publication Date: 27-Jul-2004
• Withdrawal Date: 30-Jan-2005
• ICS: 67.060 - Cereals, pulses and derived products
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 5 - Biotoxins
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Completion Date: 28-Oct-2020
• Ref Project: SIST EN 14352:2005 - Foodstuffs - Determination of fumonisin B1 and B2 in maize based foods - HPLC method with immunoaffinity column clean up

Standards Content (Sample)

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Fumonisin B1 und B2 in Maiserzeugnissen - HPLC-Vefahren mit Immunoaffinitätssäulen-ReinigungProduits alimentaires - Dosage des fumonisines B1 et B2 dans des aliments a base de mais - Méthode CLHP avec purification par colonne d'immunoaffinitéFoodstuffs - Determination of fumonisin B1 and B2 in maize based foods - HPLC method with immunoaffinity column clean up67.060QMLKCereals, pulses and derived productsICS:Ta slovenski standard je istoveten z:EN 14352:2004SIST EN 14352:2005en01-januar-2005SIST EN 14352:2005SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14352July 2004ICS 67.060English versionFoodstuffs - Determination of fumonisin B1 and B2 in maizebased foods - HPLC method with immunoaffinity column cleanupProduits alimentaires - Dosage des fumonisines B1 et B2dans des aliments à base de maïs - Méthode CLHP avecpurification par colonne d'immunoaffinitéLebensmittel - Bestimmung von Fumonisin B1 und B2 inMaiserzeugnissen - HPLC-Vefahren mitImmunoaffinitätssäulen-ReinigungThis European Standard was approved by CEN on 30 April 2004.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2004 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14352:2004: ESIST EN 14352:2005

Precision data.14 Annex B (informative)
Typical chromatograms.16 Bibliography.18
The use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. SIST EN 14352:2005

1 Scope This document specifies a method for the detemination of fumonisin B1 (FB1) and fumonisin B2 (FB2) in maize based foods using high performance liquid chromatography (HPLC) and immunoaffinity clean-up, see [1], [2], [3]. The method has been successfully validated in a collaborative study according to AOAC Guidelines for collaborative study procedures [4] to validate characteristics of a method of analysis for the determination of fumonisins in maize flour and corn flakes containing 323 µg/kg to 1414 µg/kg FB1 and 90 µg/kg to 558 µg/kg FB2. 2 Normative references The following referenced documents are indispensable for the application of this document. 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, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987). 3 Principle Fumonisins are extracted from the sample with a mixture of water, methanol and acetonitrile. The filtered extract is purified by immunoaffinity column and fumonisins are eluted with methanol. The extract is evaporated and the residue is redissolved in a mixture of acetonitrile and water and o-phthaldialdehyde-2-mercaptoethanol (OPA-MCE) is added to form fluorescent fumonisin derivatives. The derivatives are analysed by reverse-phase high performance liquid chromatography (RP-HPLC) with fluorescence detection. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only distilled water or water of grade 1 according to EN ISO 3696. Solvents shall be of quality for HPLC analysis. 4.2 Methanol 4.3 Acetonitrile 4.4 o-phosphoric acid, volume fraction ϕ(H3PO4) = 85 % 4.5 o-phthaldialdehyde (OPA) 4.6 2-mercaptoethanol (MCE) 4.7 Sodium dihydrogen phosphate solution, substance concentration c(NaH2PO4·2H2O) = 0,1 mol/l
Dissolve 15,6 g of NaH2PO4·2H2O in 1 l of distilled water. SIST EN 14352:2005

and, in particular, the following: 5.2 Orbital shaker 5.3 Centrifuge bottle of 250 ml capacity with screw cap 5.4 Centrifuge capable of a centrifugal force up to 2 500 g 5.5 Filter papers, with a pore size of 20 µm to 25 µm 5.6 Glass microfiber filter papers, with a pore size of 11 µm 5.7 Reservoir, 25 ml with luer tip connector for immunoaffinity column (IAC) 5.8 Microlitre syringe(s) or calibrated micropipette(s), 25 µl to 1 000 µl 5.9 Laboratory balance, capable of weighing to the nearest 0,01 g 5.10 Analytical balance capable of weighing to the nearest 0,1 mg 5.11 Vacuum manifold to accommodate immunoaffinity columns 5.12 Vortex mixer 5.13 Solvent evaporator, with heating module, or similar. 5.14 Membrane filter for aqueous solutions, with a pore size of 0,45 µm. 5.15 HPLC apparatus, comprising the following 5.15.1 HPLC pump, isocratic, suitable for e.g. 1 ml/min constant flow rate 5.15.2 Injection system capable to deliver e.g. 20 µl 5.15.3 Analytical reverse-phase separating column, for example octyldecylsilane (ODS), which ensures a baseline resolution of the fumonisin peaks from all other peaks, with the following characteristics:  stainless steel;  a length of 150 mm;  an inner diameter of 4,6 mm;  a stationary phase with particle size of 5 µm;  a suitable corresponding reverse-phase guard column. SIST EN 14352:2005

Mix the two extract filtrates and pipette 10 ml of filtrate into 100 ml flask. Add 40 ml of PBS (4.16) to the 10 ml filtrate and mix well. Filter the diluted extract through microfiber filter (5.6) and collect 10 ml of filtrate (equivalent to 0,4 g test sample) that will be cleaned-up through immunoaffinity column (4.17). 7.3 Immunoaffinity clean-up Remove top cap fr
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