EN 15835:2010
(Main)Foodstuffs - Determination of ochratoxin A in cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup and fluorescence detection
Foodstuffs - Determination of ochratoxin A in cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup and fluorescence detection
This European Standard specifies a method for the determination of ochratoxin A in cereal based foods for infants and young children by high performance liquid chromatography (HPLC) with immunoaffinity column cleanup and fluorescence detection. This method has been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samples ranging from 0,050 µg/kg to 0,217 µg/kg. For further information on the validation see Clause 8 and Annex B. Additional studies have shown that this method is applicable to cereal based baby foods containing 8 different types of cereals, honey and cocoa, at levels up to 3,540 µg/kg, see Annex C and [6].
Lebensmittel - Bestimmung von Ochratoxin A in Säuglings- und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion
Dieses Dokument legt ein Verfahren zur Bestimmung von Ochratoxin A in Säuglings- und Kleinkindernahrung auf Getreidebasis durch Hochleistungsflüssigchromatographie (HPLC) mit Reinigung an einer Immuno¬affinitätssäule und Fluoreszenzdetektion fest. Dieses Verfahren wurde in einem Ringversuch an sowohl natürlich kontaminierten als auch aufgestockten Proben mit Gehalten von 0,050 µg/kg bis 0,217 µg/kg validiert. Weitere Informationen zur Validierung, siehe Abschnitt 8 und Anhang B. In weiteren Untersuchungen wurde nachgewiesen, dass das Verfahren auf Beikost, die 8 verschiedene Getreidearten, Honig und Kakao enthält, bis zu Gehalten von 3,540 µg/kg anwendbar ist, siehe Anhang C und [6].
WARNUNG - Bei der Anwendung dieser Norm ist es möglich, dass gefährliche Substanzen, Arbeits¬gänge und Geräte angewendet werden. Diese Norm erhebt nicht den Anspruch, dass alle mit ihrer Anwendung verbundenen Sicherheitsprobleme angesprochen werden. Es liegt in der Verantwortung des Anwenders dieser Norm, geeignete Vorkehrungen für den Arbeits- und Gesundheitsschutz zu treffen und vor der Anwendung die Anwendbarkeit einschränkender Vorschriften zu bestimmen.
Produits alimentaires - Dosage de l'ochratoxine A dans les aliments à base de céréales pour nourrissons et jeunes enfants - Méthode CLHP avec purification sur colonne d'immuno-affinité et détection par fluorescence
Le présent document spécifie une méthode pour déterminer la teneur en ochratoxine A dans les aliments à
base de céréales pour nourrissons et jeunes enfants, par chromatographie liquide à haute performance
(CLHP) avec purification sur colonne d’immuno-affinité et détection fluorimétrique. La présente méthode a été
validée dans une étude interlaboratoires via l’analyse d’échantillons naturellement contaminés et
d’échantillons dopés à des concentrations allant de 0,050 μg/kg à 0,217 μg/kg. Pour de plus amples
informations sur la validation voir l’Article 8 et l’Annexe B. Des études supplémentaires ont démontré que
cette méthode est applicable aux aliments à base de céréales pour nourrissons contenant 8 types différents
de céréales, miel et noix de coco, à des niveaux jusqu’à 3,540 μg/kg, voir l’Annexe C et [6].
AVERTISSEMENT — L’utilisation de la présente norme peut impliquer l’utilisation de produits et la
mise en oeuvre de modes opératoires et d’appareillages à caractère dangereux. La présente norme n’a
pas pour but d’aborder tous les problèmes de sécurité liés à son utilisation. Il incombe à l’utilisateur
de la présente norme d’établir, avant de l’utiliser, des pratiques d’hygiène et de sécurité appropriées
et de déterminer l’applicabilité des restrictions réglementaires.
Živila - Določevanje ohratoksina A v žitnih kašicah za dojenčke in majhne otroke - Metoda HPLC s čiščenjem z imunoafinitetno kolono in fluorescenčno detekcijo
Ta evropski standard opredeljuje metodo za določevanje ohratoksina A v hrani na osnovi žita za dojenčke in majhne otroke z metodo s tekočinsko kromatografijo visoke ločljivosti (HPLC) s čiščenjem z imunoafinitetno kolono in fluorescenčno detekcijo. Ta metoda je bila potrjena v medlaboratorijski študiji preko analize tako naravno kontaminiranih kot vzorcev z internimi dodatki v razponu od 0,050 μg/kg do 0,217 μg/kg. Za nadaljnje informacije o potrjevanju glej Klavzulo 8 in Dodatek B. Dodatne študije so pokazale, da je ta metoda uporabna za otroško hrano na osnovi žita, ki vsebuje 8 različnih vrst žitaric, med in kakav, v stopnjah do 3,540 μg/kg (glej Dodatek C).
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Ochratoxin A in Säuglings- und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und FluoreszenzdetektionProduits alimentaires - Dosage de l'ochratoxine A dans les aliments à base de céréales pour nourrissons et jeunes enfants - Méthode CLHP avec purification sur colonne d'immuno-affinité et détection par fluorescenceFoodstuffs - Determination of ochratoxin A in cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup and fluorescence detection67.230Predpakirana in pripravljena hranaPrepackaged and prepared foods67.060QMLKCereals, pulses and derived productsICS:Ta slovenski standard je istoveten z:EN 15835:2010SIST EN 15835:2010en,fr,de01-maj-2010SIST EN 15835:2010SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15835
February 2010 ICS 67.050; 67.230 English Version
Foodstuffs - Determination of ochratoxin A in cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup and fluorescence detection
Produits alimentaires - Dosage de l'ochratoxine A dans les aliments à base de céréales pour nourrissons et jeunes enfants - Méthode CLHP avec purification sur colonne d'immuno-affinité et détection par fluorescence
Lebensmittel - Bestimmung von Ochratoxin A in Säuglings-und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion This European Standard was approved by CEN on 25 December 2009.
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 standards may be obtained on application to the CEN 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 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, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15835:2010: ESIST EN 15835:2010
Typical chromatograms . 14Annex B (informative)
Precision data . 15Annex C (informative)
Data on the in-house study . 16Bibliography . 18 SIST EN 15835:2010
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 August 2010, and conflicting national standards shall be withdrawn at the latest by August 2010. 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. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. Annexes A, B and C are informative. 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, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom.
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 3696:1995, Water for analytical laboratory use
Specification and test methods (ISO 3696:1987) 3 Principle A test portion is extracted with tert-butyl methyl ether after addition of 0,5 mol/l phosphoric acid / 2 mol/l sodium chloride solution. The extract is evaporated and redissolved in methanol and phosphate buffered saline (PBS) solution. After removal of lypophilic compounds with hexane, the extract is applied to an immunoaffinity column containing antibodies specific to ochratoxin A. The toxin is eluted from the column with methanol. Ochratoxin A is determined by HPLC with enhanced fluorescence detection involving post column reaction with ammonia. NOTE Some investigations indicate that HPLC can be also performed without the use of ammonia although this results in at least a two-fold decrease of the response for ochratoxin A. In this case, the fluorescence detection conditions need to be changed (excitation wavelength = 333 nm, emission wavelength = 460 nm). 4 Reagents 4.1 General Use 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. 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 for laboratory wastes have been reported by the International Agency for Research on Cancer (IARC), see [4]. 4.2 Helium purified compressed gas 4.3 Nitrogen SIST EN 15835:2010
4.21 Mixture of methanol and acetic acid solution Mix 72 parts per volume of methanol (4.19) with 28 parts per volume of acetic acid solution (4.17). 4.22 Tert-butyl methyl ether WARNING — Tert-butyl methyl ether is hazardous and samples shall be blended using an explosion proof blender which is housed within a fume cupboard. Centrifugation of extracts shall be performed at cool temperature (4 °C to 8 °C). 4.23 Mixture of toluene and glacial acetic acid Mix 99 parts per volume of toluene (4.20) with one part per volume of glacial acetic acid (4.16). 4.24 HPLC mobile phase A Acetic acid solution (4.17). 4.25 HPLC mobile phase B Methanol (4.19). Degas the mobile phases A and B with for example helium (4.2). Helium can be pumped into the reservoirs of both mobile phases A and B. The pumping rate should be 50 ml/min to 100 ml/min. The use of a degasser is also an acceptable option. 4.26 Immunoaffinity columns The immunoaffinity column contain antibodies raised against ochratoxin A. The column shall have a capacity of not less than 100 ng of ochratoxin A and shall give a recovery of not less than 85 % when applied as a standard solution of ochratoxin A in a mixture of 15 parts per volume of methanol (4.19) and 85 parts per volume of PBS solution (4.14) containing 3 ng of ochratoxin A. 4.27 Ochratoxin A, in crystal form or as a film in ampoules WARNING — Ochratoxin A is a potent nephrotoxin with immunotoxic, teratogenic and potential genotoxic properties. The International Agency for Research on Cancer (IARC) has classified ochratoxin A as a possible human carcinogen (group 2B). Protective clothing, gloves and safety glasses should be worn at all times, and all standard and sample preparation stages should be carried out in a fume cupboard. 4.28 Ochratoxin A stock solution Prepare a stock solution of ochratoxin A in the mixture of toluene and glacial acetic acid (4.23) with a nominal concentration of 10 µg/ml. To determine the exact concentration, record the absorption curve between a wavelength of 300 nm and 370 nm in 5 nm steps in 1 cm quartz cells (5.22) in a spectrometer with the solvent mixture (4.23) as reference. Identify the wavelength for maximum absorption and calculate the mass concentration of ochratoxin A, ota, in micrograms per millilitre, using Equation (1): bMA×××=ερ100maxota (1) SIST EN 15835:2010
Store this solution in a freezer at approximately - 18 °C. Allow to reac
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