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.

Lebensmittel - Bestimmung von Ochratoxin A in Johannisbeeren, Rosinen, Sultaninen, gemischten Trockenfrüchten und getrockneten Feigen - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion

Dieser europäische Normentwurf legt ein Verfahren zur Bestimmung von Ochratoxin A in Johannisbeeren, Rosinen, Sultaninen, gemischten Trockenfrüchten und getrockneten Feigen durch Hochleistungsflüssigkeits¬chromatographie (HPLC) mit Reinigung an einer Immunoaffinitätssäule fest. Dieses Verfahren wurde in einem Ringversuch durch die Untersuchung sowohl von natürlich kontaminierten als auch von aufgestockten Proben mit Gehalten von 1,0 µg/kg bis 11 µg/kg validiert.

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

Le présent projet de 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. Cette méthode a été validée lors d’une étude interlaboratoires par l’analyse d’échantillons naturellement contaminés et d’échantillons dopés, de 1,0 g/kg à 11 g/kg.

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

Status
Published
Publication Date
18-Feb-2010
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
19-Feb-2010
Due Date
26-Apr-2010
Completion Date
19-Feb-2010

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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



SIST EN 15829:2010



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



EN 15829:2010 (E) 2 Contents Page Foreword .31Scope .42Normative references .43Principle .44Reagents .45Apparatus .76Procedure .87HPLC analysis .98Calculation . 109Precision . 1110Test report . 12Annex A (informative)
Typical chromatogram . 13Annex B (informative)
Precision data . 14Bibliography . 15
SIST EN 15829:2010



EN 15829:2010 (E) 3 Foreword This document (EN 15829:2010) 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 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



EN 15829:2010 (E) 4 1 Scope 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. 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 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



EN 15829:2010 (E) 5 4.7 Sodium hydroxide 4.8 Ammonium hydroxide solution, substance concentration c(NH4OH) = 1,1 mol/l, for post-column pH shift Prepare fresh when required (optional, see 7.2). 4.9 Hydrochloric acid solution, mass fraction w(HCl) = 37 % in water 4.10 Phosphoric acid solution, c(H3PO4) = 0,1 mol/l 4.11 Hydrochloric acid solution, c(HCl) = 0,1 mol/l Dilute 8,28 ml of hydrochloric acid solution (4.9) to 1 l with water. 4.12 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l Dissolve 4 g of sodium hydroxide (4.7) in 1 l of water. 4.13 Phosphate buffered saline (PBS) solution Dissolve 8,0 g of sodium chloride (4.6), 1,2 g of anhydrous disodium hydrogen phosphate or 2,9 g of Na2HPO4·12 H2O (4.3), 0,2 g of potassium dihydrogen phosphate (4.5) and 0,2 g of potassium chloride (4.4) in 900 ml of water. After dissolution, adjust the pH to 7,4 with hydrochloric acid solution (4.11) or sodium hydroxide solution (4.12) as appropriate, then dilute to 1 l with water. Alternatively, a PBS solution with equivalent properties can be prepared from commercially available PBS material. 4.14 Acetonitrile WARNING — Acetonitrile is hazardous and samples shall be blended using an explosion proof blender which is housed within a fume cupboard. After blending, samples shall be filtered inside a fume cupboard. 4.15 Glacial acetic acid, w(CH3COOH) ≥ 98 % 4.16 Methanol 4.17 Toluene 4.18 Injection solvent Mix 80 parts per volume of water with 20 parts per volume of acetonitrile (4.14) and two parts per volume of acetic acid (4.15). 4.19 HPLC mobile phase Mix 99 parts per volume of water with 99 parts per volume of acetonitrile (4.14) and two parts per volume of glacial acetic acid (4.15). Degas the mobile phase solvent with for example helium (4.2). SIST EN 15829:2010



EN 15829:2010 (E) 6 4.20 Mixture of toluene and glacial acetic acid Mix 99 parts per volume of toluene (4.17) with one part per volume of glacial acetic acid (4.15). 4.21 Immunoaffinity column The immunoaffinity column contains 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 70 % when 5 ng of ochratoxin A is applied in a solution of five parts per volume of acetonitrile (4.14) and 95 parts per volume phosphate buffered saline (4.13). 4.22 Surface silanising fluid (optional) Mix one part per volume of the surface silanising fluid with 19 parts per volume of toluene (4.17). 4.23 Ochratoxin A, in crystal form or as a film in ampoules 4.24 Ochratoxin A stock solution 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. 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.
SIST EN 15829:2010



EN 15829:2010 (E) 7 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. 4.26 Ochratoxin A standard solution Transfer 500 µl of the ochratoxin A spiking solution (4.25) to a 5 ml volumetric flask, make up to volume with methanol (4.16). This solution contains 0,25 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 bin
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