Foodstuffs - Determination of zearalenone in maize based baby food, barley flour, maize flour, polenta, wheat flour and 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 zearalenone in maize based baby food, barley flour, maize flour, polenta, wheat flour and cereal based foods for infants and young children by high performance liquid chromatography (HPLC) with immunoaffinity cleanup and fluorescence detection. This method has been validated in two interlaboratory studies. The first study was for the analysis of samples of maize based baby food, barley flour, maize flour, polenta and wheat flour ranging from 10 µg/kg to 335 µg/kg, and the second study was for samples of cereal based foods for infants and young children ranging from 9 µg/kg to 44 µg/kg. Further information on validation, see Clause 9 and Annex B.

Lebensmittel - Bestimmung von Zearalenon in Säuglingsnahrung auf Maisbasis, Gerstenmehl, Maismehl, Maisgrieß, Weizenmehl und Lebensmittel auf Getreidebasis für Säuglinge und Kleinkinder - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion

Dieser europäische Normentwurf legt ein Verfahren zur Bestimmung von Zearalenon durch Hochleistungs¬flüssigkeitschromatographie (HPLC) mit Reinigung an einer Immunoaffinitätssäule fest. Dieses Verfahren wurde in einem Ringversuch durch Analyse von Proben von Gerste, Mais, Weizen, Maisgrieß und Säuglings¬nahrung auf Maisbasis mit Gehalten von 10 µg/kg bis 335 µg/kg und von Proben von Säuglings- und Klein¬kindernahrung auf Getreidebasis mit Gehalten von 9 µg/kg bis 44 µg/kg validiert.

Produits alimentaires - Dosage de la zéaralénone dans les aliments à base de maïs pour bébés, dans la farine d'orge, dans la farine de maïs, dans la polenta, dans la farine de blé et dans les aliments à base de céréales pour nourrissons et enfants en bas âge - Méthode par chromatographie liquide haute performance avec purification sur colonne d'immuno-affinité et détection par fluorescence

Le présent projet de Norme européenne spécifie une méthode pour le dosage de la zéaralenone par chromatographie liquide haute performance (CLHP) avec purification par immunoaffinité. Cette méthode a été validée suite à une étude interlaboratoire via l’analyse d’échantillons d’orge, de maïs, de blé, de polenta et d’aliments pour nourrissons à base de maïs variant de 10 g/kg à 335 g/kg, et d’échantillons d’aliments pour nourrissons et jeunes enfants à base de céréales variant de 9 g/kg à 44 g/kg.

Živila - Določevanje zearalenona v hrani za dojenčke in majhne otroke na osnovi koruze, ječmenove moke, koruzne moke, polente, pšenične moke ter v žitnih kašicah - Metoda HPLC z imunoafinitetnim kolonskim čiščenjem in fluorescenčno detekcijo

Ta evropski standard določa metodo za določevanje zearalenona v hrani za dojenčke na osnovi koruze, ječmenove moke, koruzne moke, polente, pšenične moke in žitnih kašicah za dojenčke in majhne otroke z tekočinsko kromatografijo visoke ločljivosti (HPLC) z imunoafinitetnim čiščenjem in fluorescenčno detekcijo. Ta metoda je bila potrjena v dveh medlaboratorijskih raziskavah. Prva raziskava je bila izvedena za analizo vzorcev hrane za dojenčke na osnovi koruze, ječmenove moke, koruzne moke, polente in pšenične moke v razponu od 10 µg/kg do 335 µg/kg, druga raziskava pa je bila za vzorce hrane za dojenčke in majhne otroke na osnovi žit v razponu od 9 µg/kg do 44 µg/kg. Za dodatne informacije, glej Klavzulo 9 in Dodatek B.

General Information

Status
Published
Publication Date
15-Aug-2010
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
08-Jun-2010
Due Date
13-Aug-2010
Completion Date
16-Aug-2010

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.OXRUHVFHQþQRGHWHNFLMRLebensmittel - Bestimmung von Zearalenon in Säuglingsnahrung auf Maisbasis, Gerstenmehl, Maismehl, Maisgrieß, Weizenmehl und Lebensmittel auf Getreidebasis für Säuglinge und Kleinkinder - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und FluoreszenzdetektionProduits alimentaires - Dosage de la zéaralénone dans les aliments à base de maïs pour bébés, dans la farine d'orge, dans la farine de maïs, dans la polenta, dans la farine de blé et dans les aliments à base de céréales pour nourrissons et enfants en bas âge - Méthode par chromatographie liquide haute performance avec purification sur colonne d'immuno-affinité et détection par fluorescenceFoodstuffs - Determination of zearalenone in maize based baby food, barley flour, maize flour, polenta, wheat flour and 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 15850:2010SIST EN 15850:2010en,fr,de01-september-2010SIST EN 15850:2010SLOVENSKI

STANDARD
SIST EN 15850:2010
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15850
April 2010 ICS 67.060 English Version

Foodstuffs - Determination of zearalenone in maize based baby food, barley flour, maize flour, polenta, wheat flour and cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup and fluorescence detection

Produits alimentaires - Dosage de la zéaralénone dans la farine d'orge, de maïs et de blé, la polenta et les produits pour nourrissons et jeunes enfants à base de céréales - Méthode par chromatographie liquide haute performance avec purification sur colonne d'immunoaffinité et détection par fluorescence

Lebensmittel - Bestimmung von Zearalenon in Säuglingsnahrung auf Maisbasis, Gerstenmehl, Maismehl, Maisgrieß, Weizenmehl und Lebensmittel auf Getreidebasis für Säuglinge und Kleinkinder - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion This European Standard was approved by CEN on 27 February 2010.

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 15850:2010: ESIST EN 15850:2010

EN 15850:2010 (E) 2 Contents Page Foreword ..............................................................................................................................................................31Scope ......................................................................................................................................................42Normative references ............................................................................................................................43Principle ..................................................................................................................................................44Reagents .................................................................................................................................................45Apparatus ...............................................................................................................................................76Procedure ...............................................................................................................................................97HPLC analysis ..................................................................................................................................... 108Calculation ........................................................................................................................................... 129Precision .............................................................................................................................................. 1210Test report ........................................................................................................................................... 13Annex A (informative)

Typical chromatograms ............................................................................................ 15Annex B (informative)

Precision data ............................................................................................................. 16Bibliography ..................................................................................................................................................... 18 SIST EN 15850:2010

EN 15850:2010 (E) 3 Foreword This document (EN 15850:2010) 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 October 2010, and conflicting national standards shall be withdrawn at the latest by October 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. 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. 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 15850:2010

EN 15850:2010 (E) 4 1 Scope This European Standard specifies a method for the determination of zearalenone in maize based baby food, barley flour, maize flour, polenta, wheat flour and cereal based foods for infants and young children by high performance liquid chromatography (HPLC) with immunoaffinity cleanup and fluorescence detection. This method has been validated in two interlaboratory studies. The first study was for the analysis of samples of maize based baby food, barley flour, maize flour, polenta and wheat flour ranging from 10 µg/kg to 335 µg/kg, and the second study was for samples of cereal based foods for infants and young children ranging from 9 µg/kg to 44 µg/kg. Further information on validation, see Clause 9 and Annex B. 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 aqueous acetonitrile or methanol according to the products analyzed. The extract is then diluted with phosphate buffered saline (PBS) to give an aqueous extract that is applied to an immunoaffinity column containing antibodies specific for zearalenone. Zearalenone is purified and concentrated on the column and removed from the antibodies using acetonitrile or methanol as eluent. Zearalenone is quantified by reverse-phase high performance liquid chromatography (RP-HPLC) with fluorescence detection. 4 Reagents 4.1 General Use only reagents of recognised analytical grade and water complying with grade 1 of EN ISO 3696:1995, unless otherwise specified. Solvents shall be of quality for HPLC analysis, unless otherwise specified. Commercially available solutions with equivalent properties to those listed may be used. 4.2 Disodium hydrogen phosphate, Na2HPO4 anhydrous or Na2HPO4·12 H2O. 4.3 Potassium chloride (KCl). 4.4 Potassium dihydrogen phosphate, KH2PO4. 4.5 Sodium chloride (NaCl). 4.6 Sodium hydroxide (NaOH). 4.7 Hydrochloric acid solution, mass fraction w(HCl) = 37 % in water. 4.8 Hydrochloric acid solution, substance concentration c(HCl) = 0,1 mol/l. Dilute 8,28 ml of hydrochloric acid solution (4.7) to 1 l with water. SIST EN 15850:2010

EN 15850:2010 (E) 5 4.9 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l. Dissolve 4 g of sodium hydroxide (4.6) in 1 l of water. 4.10 Phosphate buffered saline (PBS) solution, c(NaCl) = 120 mmol/l, c(KCl) = 2,7 mmol/l, c(phosphate buffer) = 10 mmol/l, pH = 7,4. Dissolve 8,0 g of sodium chloride (4.5), 1,2 g of anhydrous disodium hydrogen phosphate or 2,9 g of Na2HPO4·12 H2O (4.2), 0,2 g of potassium dihydrogen phosphate (4.4) and 0,2 g of potassium chloride (4.3) in 900 ml of water. After dissolution, adjust the pH to 7,4 with hydrochloric acid solution (4.8) or sodium hydroxide solution (4.9) as appropriate, then dilute to 1 l with water. Alternatively, a PBS solution with equivalent properties can be prepared from commercially available PBS material. 4.11 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. 4.12 Methanol, HPLC grade. 4.13 Methanol,

technical grade. 4.14 Extraction solvent A. Mix 75 parts per volume of acetonitrile (4.11) with 25 parts per volume of water. 4.15 Injection solvent A for HPLC analysis. Mix four parts per volume of acetonitrile (4.11) with six parts per volume of water. 4.16 HPLC mobile phase A. Mix 53 parts per volume of acetonitrile (4.11) with 47 parts per volume of water. Filter and degas the HPLC mobile phase before use. 4.17 Extraction solvent B. Mix 75 parts per volume of methanol (4.13) with 25 parts per volume of water. 4.18 Washing solvent. Mix 15 parts per volume of methanol (4.12) with 85 parts per volume of PBS (4.10). 4.19 Injection solvent B for HPLC analysis. Mix five parts per volume of methanol (4.12) with five parts per volume of water. 4.20 HPLC mobile phase B. Mix 75 parts per volume of methanol (4.12) with 25 parts per volume of water. Filter and degas the HPLC mobile phase before use. SIST EN 15850:2010

EN 15850:2010 (E) 6 4.21 Immunoaffinity column. The immunoaffinity column shall contain antibodies raised against zearalenone. The column shall have a capacity of not less than 1 500 ng of zearalenone and shall give a recovery of not less than 80 % when 75 ng of zearalenone is applied in 10 ml of a mixture of 15 parts per volume of methanol and 85 parts per volume of PBS. 4.22 Zearalenone, in crystal form or as a film in ampoules, purity not less than 98 % mass fraction or in form of commercially available Zearalenone solution. WARNING — Zearalenone is an oestrogenic compound and should be treated with extreme 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. 4.23 Zearalenone stock solution, c ≈ 200 µg/ml. Add 4,0 ml of acetonitrile (4.11) to 5 mg of zearalenone (4.22) to form a solution with a mass concentration of approximately 1,25 mg/ml. Dilute 800 µl of this solution to 5 ml with acetonitrile (4.11) to form a stock solution with a concentration of approximately 200 µg/ml. Store this solution in a freezer at - 18 °C to - 20 °C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the mass concentration of the solution if it is older than six months. 4.24 Zearalenone spiking solution, c ≈ 10 µg/ml. Dilute 250 µl of stock solution (4.23) with 4,75 ml of acetonitrile (4.11) to form a solution with a mass concentration of approximately 10 µg/ml. To determine the exact concentration, record the absorption curve of this solution between 200 nm to 300 nm in a 1 cm quartz cell in the spectrometer (5.25) with acetonitrile (4.11) as reference. Identify the wavelength for maximum absorption ( is approximately 274 nm). Calculate the mass concentration of zearalenone, zon, in micrograms per millilitre using Equation (1):

bMA×××=ερ100maxzon (1) where Amax is the absorption determined at the maximum of the absorption curve (274 nm); M is the molar mass, in grams per mole, of zearalenone (M = 318,4 g/mol); 0 is the molar absorption coefficient, in square metres per mole of zearalenone in acetonitrile (4.11) ( 1 262 m2/mol, see [1]); b is the optical path length, in centimetres, of the quartz cell. Store this solution in a freezer at - 18 °C to - 20 °C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the mass concentration of the solution if it is older than six months. 4.25 Zearalenone standard solution A,

= 2 µg/ml, for maize based baby food, barley flour, maize flour, polenta and wheat flour. Transfer an aliquot of the spiking solution (4.24) equivalent to 10 µg of zearalenone into either a vial (5.9) or a calibrated volumetric flask (5.10). Add acetonitrile (4.11) to make the total volume up to 5 ml.

SIST EN 15850:2010

EN 15850:2010 (E) 7 Store this solution in a freezer at - 18 °C to - 20 °C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the mass concentration of the solution if it is older than six months. 4.26 Zearalenone standard solution B,

= 0,4 µg/ml, for cereal based foods for infants and young children. Transfer an aliquot of the spiking solution (4.24) equivalent to 2 µg of zearalenone into either a vial (5.9) or a calibrated volumetric flask (5.10). Add acetonitrile (4.11) to make the total volume up to 5 ml.

Store this solution in a freezer at - 18 °C to - 20 °C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the mass 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 High speed blender or homogenizer. 5.3 Analytical balance, capable of weighing to 0,000 1 g. 5.4 Laboratory balance, capable of weighing to 0,1 g. 5.5 Adjustable vertical or horizontal shaker. 5.6 Vortex mixer, or equivalent. 5.7 Mills, various screens. 5.8 Tumble mixer. 5.9 Glass vials, of various sizes 5.10 Volumetric flasks, of 3 ml or 5 ml and 10 ml capacity. 5.11 Beaker, of 250 ml capacity. 5.12 Conical flask, with screw cap or glass stopper of 100 ml, 250 ml and 500 ml capacity. 5.13 Filter paper, e.g., qualitative, strong, fast flow, 24 cm diameter, 30 µm pore size, prefolded or equivalent. 5.14 Glass microfibre filter, e.g. 1,6 µm retention size or equivalent. 5.15 Pipettes, of e.g. 25 µl to 250 µl, 1 ml, 5 ml and 10 ml capacity. 5.16 Displacement micropipettes or syringes, gas tight of e.g. 100 µl, 500 µl, 1 000 µl capacity. SIST EN 15850:2010

EN 15850:2010 (E) 8 5.17 Vacuum manifold or automated system, to accommodate immunoaffinity columns. 5.18 Reservoirs, of 50 ml to 75 ml capacity, and attachments for immunoaffinity columns. 5.19 Plastic syringes, 5 ml. 5.20 Vacuum pump, capable of for example pulling a vacuum of 1 kPa and or pumping 18 l/min. 5.21 Solvent vacuum filtration system, fitted with 47 mm glass microfibre filter. 5.22 Disposable syringe filter unit, nylon with a pore size of 0,45 µm. Prior to usage, verify that no zearalenone losses occur during filtration (recovery testing). NOTE There is a possibility that various filter materials retain zearalenone. 5.23 Ultrasonic bath. 5.24 HPLC apparatus, comprising the following: 5.24.1 Injection system, capable of injecting e.g. 100 µl to 300 µl. 5.24.2 Mobile phase pump, pulse free, capable of maintaining a volume flow rate of 0,5 ml/min to 1,5 ml/min. 5.24.3 Analytical reverse-phase HPLC separating column, that allows a sufficient baseline separation of zearalenone from other interfering components. The maximum overlap shall be less than 10 % peak height.

Phenomenex Prodigy® ODS 3 1) (150 mm × 4,6 mm internal diameter, 5 µm particle size, 25 nm pore size) or Spherisorb® 1) ODS-2 Excel (250 mm × 4,6 mm internal diameter, 5 µm particle size, 25 nm pore size) have been found to be suitable when used with mobile phase A (4.16). Supelcosil® 1) (C18), fully end capped (250 mm × 4,6 mm internal diameter, 5 µm particle size, 18 nm pore size), carbon loading of 12 %, or similar, has been found to be suitable when used with mobile phase B (4.20). 5.24.4 Pre-column, with preferably the same stationary phase material as the analytical column, an internal diameter of 4 mm, 5 µm particle size. 5.24.5 Fluorescence detector, fitted with a flow cell and suitable for measurements with excitation wavelength of 274 nm or 275 nm and emission at 446 nm or 450 nm. 5

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