EN 15829:2010
(Main)Foodstuffs - Determination of ochratoxin A in currants, raisins, sultanas, mixed dried fruit and dried figs - HPLC method with immunoaffinity column cleanup and fluorescence detection
Foodstuffs - Determination of ochratoxin A in currants, raisins, sultanas, mixed dried fruit and dried figs - HPLC method with immunoaffinity column cleanup and fluorescence detection
This European Standard specifies a method for the determination of ochratoxin A in currants, raisins, sultanas, mixed dried fruit and dried figs by high performance liquid chromatography (HPLC) with immunoaffinity 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 1,1 µg/kg to 11 µg/kg.
For further information on the validation, see Clause 9 and Annex B.
WARNING - 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.
Lebensmittel - Bestimmung von Ochratoxin A in Korinthen, Rosinen, Sultaninen, gemischtem Trockenobst und getrockneten Feigen - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion
Diese Europäische Norm legt ein Verfahren zur Bestimmung von Ochratoxin A in Johannisbeeren, Rosinen, Sultaninen, gemischtem Trockenobst und getrockneten Feigen durch Hochleistungsflüssigchromatographie (HPLC) mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion fest. Dieses Verfahren wurde in einem Ringversuch durch die Untersuchung sowohl von natürlich kontaminierten als auch von aufgestockten Proben mit Gehalten von 1,1 µg/kg bis 11 µg/kg validiert.
Weitere Informationen zur Validierung siehe Abschnitt 9 und Anhang B.
WARNUNG — Bei der Anwendung dieser Norm ist es möglich, dass gefährliche Substanzen und Geräte angewendet bzw. gefährliche Arbeitsgänge durchgeführt 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 Vor¬schriften zu bestimmen.
Produits alimentaires - Dosage de l'ochratoxine A dans les raisins de Corinthe, les raisins secs, les raisins secs de Smyrne, les mélanges de fruits secs et les figues sèches - Méthode CLHP avec purification sur colonne d'immuno-affinité et détection par fluorescence
La présente Norme Européenne spécifie une méthode pour déterminer la teneur en ochratoxine A dans les raisins de Corinthe, raisins secs, raisins secs de Smyrne, mélanges de fruits secs et figues sèches par chromatographie liquide à haute performance (CLHP) avec purification sur colonne d’immuno-affinité et détection par fluorescence. Cette méthode a été validée lors d’une étude interlaboratoires par l’analyse d’échantillons naturellement contaminés et d’échantillons dopés, de 1,1 µg/kg à 11 µg/kg.
Pour plus d’informations sur la validation, voir l’Article 9 et l’Annexe B.
AVERTISSEMENT - L’utilisation de la présente norme peut impliquer l’utilisation de produits et la mise en œuvre 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 korintah, rozinah, sultaninah, mešanem sušenem sadju in sušenih figah - Metoda HPLC s čiščenjem z imunoafinitetno kolono in fluorescenčno detekcijo
Ta evropski standard določametodo za določevanje ohratoksina A v korintah, rozinah, sultaninah, mešanem sušenem sadju in sušenih figah z metodo 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 1,1 μg/kg do 11 μg/kg. Za nadaljnje informacije o potrjevanju glej Klavzulo 9 in dodatek B.
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.ILQLWHWQRLebensmittel - Bestimmung von Ochratoxin A in Johannisbeeren, Rosinen, Sultaninen, gemischten Trockenfrüchten und getrockneten Feigen - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und FluoreszenzdetektionProduits alimentaires - Dosage de l'ochratoxine A dans les raisins de Corinthe, les raisins secs, les raisins secs de Smyrne, les mélanges de fruits secs et les figues sèches - Méthode CLHP avec purification sur colonne d'immuno-affinité et détection par fluorescenceFoodstuffs - Determination of ochratoxin A in currants, raisins, sultanas, mixed dried fruit and dried figs - HPLC method with immunoaffinity column cleanup and fluorescence detection67.080.10Sadje in sadni proizvodiFruits and derived products67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 15829:2010SIST EN 15829:2010en,fr,de01-april-2010SIST EN 15829:2010SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15829
January 2010 ICS 67.050; 67.080.10 English Version
Foodstuffs - Determination of ochratoxin A in currants, raisins, sultanas, mixed dried fruit and dried figs - HPLC method with immunoaffinity column cleanup and fluorescence detection
Produits alimentaires - Dosage de l'ochratoxine A dans les raisins de Corinthe, les raisins secs, les raisins secs de Smyrne, les mélanges de fruits secs et les figues sèches - Méthode CLHP avec purification sur colonne d'immuno-affinité et détection par fluorescence
Lebensmittel -Bestimmung von Ochratoxin A in Korinthen, Rosinen, Sultaninen, gemischtem Trockenobst und getrockneten Feigen - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion This European Standard was approved by CEN on 18 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 15829:2010: ESIST EN 15829:2010
Typical chromatogram . 13Annex B (informative)
Precision data . 14Bibliography . 15
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 give to CEN by the European Commission and the European Free Trade Association. 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. SIST EN 15829:2010
Specification and test methods (ISO 3696:1987) 3 Principle A test portion is extracted with a mixture of methanol and phosphoric acid. The extract is filtered, diluted with phosphate buffered saline, and applied to an immunoaffinity column containing antibodies specific for ochratoxin A. The ochratoxin A is isolated, purified and concentrated on the column then released with elution solvent. Ochratoxin A is quantified by reverse-phase high performance liquid chromatography (HPLC) with fluorescence detection. 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 [1]. 4.2 Helium purified compressed gas 4.3 Disodium hydrogen phosphate, anhydrous or Na2HPO4·12 H2O 4.4 Potassium chloride 4.5 Potassium dihydrogen phosphate 4.6 Sodium chloride SIST EN 15829:2010
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. Dissolve 1 mg of the ochratoxin A or the contents of 1 ampoule (if ochratoxin A has been obtained as a film) in solvent mixture (4.20) to give a solution containing approximately 20 µg/ml to 30 µg/ml of ochratoxin A. To determine the exact concentration, record the absorption curve between a wavelength of 300 nm and 370 nm in a 1 cm quartz cell with solvent mixture (4.20) as reference using the spectrometer (5.12). Identify the wavelength for maximum absorption. Calculate the mass concentration of ochratoxin A, ota, in micrograms per millilitre using Equation (1):
bMA×××=ερ100maxota (1) where Amax is the absorption determined at the maximum of the absorption curve (here: at 333 nm); M is the molar mass, in grams per mole, of ochratoxin A (M = 403,8 g/mol); 0 is the molar absorption coefficient, in square metres per mole, of ochratoxin A in the solvent mixture (4.20) (here: 544 m2/mol, see [2]); b
is the optical path length, in centimetres, of the quartz cell.
Store this solution in a freezer at approximately - 18 °C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the concentration of the solution if it is older than six months. 4.25 Ochratoxin A spiking solution Transfer an aliquot of the stock solution (4.24) containing 12,5 µg of ochratoxin A to a 5 ml volumetric flask. Evaporate to dryness under nitrogen at no more than 50 °C. Redissolve immediately in methanol (4.16) and make up to volume. This solution contains 2,5 µg/ml ochratoxin A.
Store this solution in a freezer at approximately - 18 °C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the concentration of the solution if it is older than six months. 5
Apparatus 5.1 General Usual laboratory glassware and equipment and, in particular the following. 5.2 Silanised glass vials (optional) Prepare the vials by filling them with the silanising reagent (4.22) and leave this reagent in the vial for 1 min. Rinse the vial first with a solvent of low polarity, for example toluene (4.17) then with methanol (4.16) and dry before use. WARNING — The use of silanised glassware may prevent ochratoxin A binding to glass during evaporation. 5.3 High speed blender or homogenizer 5.4 Analytical balance, capable of
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