Foodstuffs - Determination of deoxynivalenol in cereals, cereal products and cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup and UV detection

This European Standard specifies a method for the determination of deoxynivalenol (DON) in cereals (grain and flour), cereal based foods and cereal based foods for infants and young children by high performance liquid chromatography (HPLC) with immunoaffinity cleanup and UV detection. This method has been validated in three interlaboratory studies. The first study was for the analysis of samples of wheat, rice flour, oat flour, maize, polenta, and wheat based breakfast cereal ranging from 85,4 μg/kg to 1 768 μg/kg, the second study was for wheat and maize ranging from 165 μg/kg to 4 700 μg/kg and the third study was for cereal based foods for infants and young children ranging from 58 μg/kg to 452 μg/kg. For further information on the validation, see Clause 9 and Annex B.

Lebensmittel - Bestimmung von Deoxynivalenol in Getreide, Getreideerzeugnissen und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und UV-Detektion

Dieser europäische Normentwurf legt ein Verfahren zur Bestimmung von Deoxynivalenol in Säuglings- und Kleinkindernahrung auf Getreidebasis durch Hochleistungsflüssigkeitschromatographie (HPLC) mit Reinigung an einer Immunoaffinitätssäule und UV Detektion fest. Dieses Verfahren wurde in einem Ringversuch für Weizen, Reismehl, Hafermehl, Mais, Maisgrieß und Frühstückscerealien auf Weizenbasis validiert. Das Verfahren ist anwendbar auf Gehalte von bis zu 4 700 µg/kg.

Denrées alimentaires - Dosage du déoxynivalénol dans les céréales, les produits céréaliers, et céréales pour déjeuner en alimentation infantile - Méthode par CLHP avec purification sur colonne d'immunoaffinité et détection UV

Ce projet de Norme européenne spécifie une méthode de dosage du déoxynivalénol dans les céréales pour nourrissons et jeunes enfants par chromatographie liquide haute performance (CLHP) avec purification sur colonne d'immunoaffinité et détection UV. Cette méthode a été validée au cours d'études collaboratives sur le blé, la farine de riz, la farine d'avoine, le maïs, la polenta et les céréales à base de blé pour petit déjeuner. La méthode s'applique jusqu'à des teneurs de 4 700 g/kg.

Živila - Določevanje deoksinvalenola v žitu in žitnih proizvodih in hrani na osnovi žit za dojenčke in majhne otroke - Metoda s HPLC z imunoafinitetnim kolonskim čiščenjem in ultravijolična (UV) detekcija

Ta evropski standard določa metodo za določevanje deoksinvalenola (DON) v žitih (žita in moka), hrani na osnovi žit in hrani na osnovi žit za dojenčke in majhne otroke s tekočinsko kromatografijo visoke ločljivosti (HPLC) z imunoafinitetnim čiščenjem in ultravijolično (UV) detekcijo. Ta metoda je bila potrjena v treh medlaboratorijskih študijah. Prva študija je bila za analizo vzorca žit, riževe moke, ovsene moke, koruze, polente in žitaric za zajtrk v razponu od 85,4 μg/kg do 1768 μg/kg, druga študija je bila za žito in koruzo v razponu od 165 μg/kg do 4700 μg/kg in tretja študija je bila za hrano na osnovi žit za dojenčke in majhne otroke v razponu od 58 μg/kg do 452 μg/kg. Za nadaljnje informacije glej klavzulo 9 in dodatek B.

General Information

Status
Published
Publication Date
03-Feb-2011
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
05-Jan-2011
Due Date
12-Mar-2011
Completion Date
04-Feb-2011

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Deoxynivalenol in Getreide, Getreideerzeugnissen und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und UV-DetektionDenrées alimentaires - Dosage du déoxynivalénol dans les céréales, les produits céréaliers, et céréales pour déjeuner en alimentation infantile - Méthode par CLHP avec purification sur colonne d'immunoaffinité et détection UVFoodstuffs - Determination of deoxynivalenol in cereals, cereal products and cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup and UV detection67.230Predpakirana in pripravljena hranaPrepackaged and prepared foods67.060QMLKCereals, pulses and derived productsICS:Ta slovenski standard je istoveten z:EN 15891:2010SIST EN 15891:2011en,fr,de01-marec-2011SIST EN 15891:2011SLOVENSKI

STANDARD
SIST EN 15891:2011
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15891
September 2010 ICS 67.060; 67.230 English Version

Foodstuffs - Determination of deoxynivalenol in cereals, cereal products and cereal based foods for infants and young children -HPLC method with immunoaffinity column cleanup and UV detection

Denrées alimentaires - Dosage du déoxynivalénol dans les céréales, les produits céréaliers, et céréales pour déjeuner en alimentation infantile - Méthode par CLHP avec purification sur colonne d'immunoaffinité et détection UV

Lebensmittel - Bestimmung von Deoxynivalenol in Getreide, Getreideerzeugnissen und Säuglings- und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und UV-Detektion This European Standard was approved by CEN on 28 August 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 15891:2010: ESIST EN 15891:2011

EN 15891:2010 (E) 2 Contents Page Foreword ..............................................................................................................................................................31Scope ......................................................................................................................................................42Normative references ............................................................................................................................43Principle ..................................................................................................................................................44Reagents .................................................................................................................................................45Apparatus ...............................................................................................................................................76Procedure ...............................................................................................................................................87HPLC analysis ..................................................................................................................................... 108Calculation ........................................................................................................................................... 129Precision .............................................................................................................................................. 1210Test report ........................................................................................................................................... 14Annex A (informative)

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

Precision data ............................................................................................................. 16Bibliography ..................................................................................................................................................... 19 SIST EN 15891:2011

EN 15891:2010 (E) 3 Foreword This document (EN 15891:2010) 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 March 2011, and conflicting national standards shall be withdrawn at the latest by March 2011. 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. Annexes A and B are informative. 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 15891:2011

EN 15891:2010 (E) 4 1 Scope This European Standard specifies a method for the determination of deoxynivalenol (DON) in cereals (grain and flour), cereal based foods and cereal based foods for infants and young children by high performance liquid chromatography (HPLC) with immunoaffinity cleanup and UV detection. This method has been validated in three interlaboratory studies. The first study was for the analysis of samples of wheat, rice flour, oat flour, maize, polenta, and wheat based breakfast cereal ranging from 85,4 µg/kg to 1 768 µg/kg, the second study was for wheat and maize ranging from 165 µg/kg to 4 700 µg/kg and the third study was for cereal based foods for infants and young children ranging from 58 µg/kg to 452 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 Deoxynivalenol is extracted from the sample using water. The aqueous extract is cleaned up on an immunoaffinity column to remove impurities from the sample. Deoxynivalenol is then quantitatively determined by HPLC and UV 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 the reagents listed may be used. 4.2 Disodium hydrogen phosphate, 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. SIST EN 15891:2011

EN 15891:2010 (E) 5 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. 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 Polyethylene glycol (PEG), with a molecular mass of approximately 8 000 g/mol. 4.13 Methanol. 4.14 Acetic acid, with a mass fraction of 96 % or glacial acetic acid, with a mass fraction of 100 %. 4.15 Diluent for HPLC analysis. Mix 9,5 parts per volume of methanol (4.13) with 90,5 parts per volume of water. 4.16 HPLC mobile phase. Mix 15 parts per volume of methanol (4.13) with 85 parts per volume of water. Add 0,1 parts per volume of acetic acid (4.14).The exact amount of methanol used and the use of acetic acid will depend on the HPLC column chosen for analysis and shall be adjusted if necessary. Degas this solution before use. 4.17 Wash solvent. Mix 50 parts per volume of methanol (4.13) with 50 parts per volume of water. 4.18 Immunoaffinity (IA) column. The immunoaffinity column shall contain antibodies raised against DON. The column shall have a capacity of not less than 1 000 ng of DON and shall have a recovery of not less than 80 % when 500 ng of DON are applied in 1 ml to 2 ml of water. SIST EN 15891:2011

EN 15891:2010 (E) 6 4.19 Deoxynivalenol, purity not less than 97 % mass fraction. WARNING — Deoxynivalenol is highly toxic. 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. 4.20 Deoxynivalenol stock solution 1, mass concentration

≈ 1,25 mg/ml. Add 4,0 ml of acetonitrile (4.11) to approximately 5 mg of deoxynivalenol (4.19) to form a solution with a concentration of approximately 1,25 mg/ml. Alternatively, available commercial solutions with equivalent properties can be used. Store this solution in a freezer at approximately - 18 °C. A solution stored in this way is stable for 12 months. Confirm the concentration of the solution if it is older than six months. 4.21 Deoxynivalenol stock solution 2,

≈ 250 µg/ml. Dilute 800 µl of stock solution 1 (4.20) to 4 ml with acetonitrile (4.11) to form a solution with a concentration of approximately 250 µg/ml. Store this solution in a freezer at approximately - 18 °C. A solution stored in this way is stable for 12 months. Confirm the concentration of the solution if it is older than six months. 4.22 Deoxynivalenol standard solution A. Dilute 200 µl of stock solution 2 (4.21) to 2,0 ml with acetonitrile (4.11) to form a solution with a concentration of approximately 25 µg/ml. To determine the exact mass concentration, record the absorption curve between a wavelength of 200 nm to 270 nm, e.g. in 5 nm steps; in the spectrometer (5.16) against acetonitrile as reference. Identify the wavelength for maximum absorption and calculate the mass concentration of deoxynivalenol, DON, in micrograms per millilitre using Equation (1):

bMA×××=ερ100maxDON (1) where Amax is the absorption determined at the maximum of the absorption curve (here: at 220 nm); M is the molar mass, in grams per mole, of deoxynivalenol (M = 296,3 g/mol); 0 is the molar absorption coefficient, in square metres per mole, of deoxynivalenol in acetonitrile (4.11) (here: 681 m2/mol, see [1]); b is the optical path length, in centimetres, of the quartz cell. Calculate the mass concentration of the stock solution 2 (4.21), DON2, in micrograms per millilitre using Equation (2):

10DONDON2×=ρρ (2) Store this solution in a freezer at approximately - 18 °C. A solution stored in this way is stable for 12 months. Confirm the concentration of the solution if it is older than six months. NOTE Preparation of standard solutions can be carried out gravimetrically by accurately weighing the deoxynivalenol standard material and the solvent used to dissolve it.

SIST EN 15891:2011
EN 15891:2010 (E) 7 4.23 Deoxynivalenol spiking solution,

= 100 µg/ml. Pipette an aliquot of the stock solution 2 (4.21) equivalent to 500 µg of deoxynivalenol in a 5 ml volumetric flask. Dilute to the mark with acetonitrile (4.11). Store this solution in a freezer at approximately - 18 °C. A solution stored in this way is stable for 12 months. Confirm the concentration of the solution if it is older than six months. 4.24 Deoxynivalenol standard solution B,

= 10 µg/ml. Pipette 500 µl of the spiking solution (4.23) in a 5 ml volumetric flask. Dilute to the mark with acetonitrile (4.11). Store this solution in a freezer at approximately - 18 °C. A solution stored in this way is 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 Analytical balance, capable of weighing to 0,000 1 g. 5.3 Laboratory balance, capable of weighing to 0,1 g. 5.4 High speed blender or homogenizer. 5.5 Laboratory shaker or magnetic stirrer, speed adjustable to approximately 500 min-1. 5.6 Vortex mixer, or equivalent. 5.7 Centrifuge, capable of a centrifugal force of 2 500 g. 5.8 Centrifuge tube, of 250 ml capacity. 5.9 Filter paper, qualitative, strong, fast flow, pre-folded and with a diameter of 18,5 cm. 5.10 Glass fibre filter, fast flow, fine porosity, retention size 1,6 µm or smaller. 5.11 Pipettes, e.g. of 10 ml, 5 ml, 1 ml, and 25 µl to 250 µl capacity. 5.12 Reservoirs for immunoaffinity columns, of for example 20 ml capacity, with appropriate adaptors. 5.13 Glass vials or assay tubes, of various size. 5.14 Heating block or thermostatic waterbath, capable of maintaining approximately 50 °C. 5.15 HPLC apparatus, comprising the following: SIST EN 15891:2011

EN 15891:2010 (E) 8 5.15.1 Injection system, capable of injecting e.g. 100 µl to 300 µl. 5.15.2 Mobile phase pump, pulse free, capable of maintaining a volume flow rate of 1 ml/min. 5.15.3 Analytical reverse-phase HPLC separating column, for example C18 octadecylsilane (ODS), length of 15 cm to 25 cm, inner diameter of 4,6 mm and a particle size of 5 µm, which ensures resolution of deoxynivalenol from all other peaks.

The maximum overlapping of peaks shall be less than 10 %. It can be necessary to adjust the mobile phase for a sufficient baseline resolution. A suitable corresponding reverse-phase guard column should be used. 5.15.4 UV detector, set at 220 nm. 5.15.5 Recorder, integrator or computer based data processing system. 5.15.6 Mobile phase switching unit, or second HPLC pump, if necessary. 5.16 UV spectrometer. 6 Procedure 6.1 General This method has been validated in three interlaboratory studies. These studies were performed by different laboratories at different times. This is the reason that slightly different procedures were used for extraction and immunoaffinity column cleanup for wheat, rice flour, oat flour, maize, polenta and wheat based breakfast cereal (described in 6.2 and 6.4) and for cereal based food for infants and young children (described in 6.3 and 6.5). The procedures described here are similar to the ones described in the original interlaboratory studies. 6.2 Extraction for wheat, rice flour, oat flour, maize, polenta and wheat based breakfast cereal Weigh, to the nearest 0,1 g, a 25 g (ms) test portion and 5 g of PEG (4.12) into a centrifuge tube (5.8). Add 200 ml (V1) of water (or another volume as specified by the IA column manufacturer) and homogenize at high speed for 3 min using a homogenizer (5.4). Alternative extraction procedures have been shown to give equivalent results.

Either shake the sample and the extraction solvent on a wrist action shaker for 2 h or add a magnetic stirrer bar to the flask, cap it and place it on a magnetic stirrer (5.5) to mix at medium-high speed for 30 min.

In both cases, shake the sample and reagents together thoroughly by hand to ensure they are well mixed before placing on shaker. Centrifuge the homogenized sample for 15 min at 2 500 g. After centrifugation filter the sample with a glass fibre filter (5.10). NOTE It has been shown during the validation study that for some matrices (maize), samples that have been extracted on a magnetic stirrer do not require centrifugation. 6.3 Extraction for cereal based food for infants and young children Weigh, to the nearest 0,1 g, a 25 g (ms) test portion into a 250 ml or 500 ml conical flask. Add 200 ml (V1) of water, cap and shake for 1 h on a laboratory shaker (5.5). Allow the sample to settle after shaking and transfer 50 ml of supernatant to a centrifuge tube (5.8) and centrifuge for 15 min at 2 500 g.

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