1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 15891:2010Foodstuffs Determinationof deoxynivalenol in cereals,cereal products and cerealbased foods for infants andyoung children HPLCmethod with immunoaffinitycolumn clea
2、nup and UVdetectionBS EN 15891:2010 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 15891:2010.The UK participation in its preparation was entrusted to TechnicalCommittee AW/-/3, Food analysis - Horizontal methods.A list of organizations represented on this comm
3、ittee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2010ISBN 978 0 580 63013 2ICS 67.060; 67.230Compliance with a British Standard cannot confer immunity fromleg
4、al obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 October 2010.Amendments issued since publicationDate Text affectedBS EN 15891:2010EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15891 September 2010 ICS 67.060; 67.230 En
5、glish 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 Denres alimentaires - Dosage du doxynivalnol dans les crales, les produits craliers, et crales
6、 pour djeuner en alimentation infantile - Mthode par CLHP avec purification sur colonne dimmunoaffinit et dtection UV Lebensmittel - Bestimmung von Deoxynivalenol in Getreide, Getreideerzeugnissen und Suglings- und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaf
7、finittssule 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 l
8、ists 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 r
9、esponsibility 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
10、, 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 COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management
11、 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: EBS EN 15891:2010EN 15891:2010 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 A
12、pparatus .76 Procedure .87 HPLC analysis . 108 Calculation . 129 Precision 1210 Test report . 14Annex A (informative) Typical chromatogram 15Annex B (informative) Precision data . 16Bibliography . 19BS EN 15891:2010 EN 15891:2010 (E) 3 Foreword This document (EN 15891:2010) has been prepared by Tech
13、nical 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 stand
14、ards 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.
15、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, B
16、elgium, 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. BS EN
17、15891:2010EN 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 cl
18、eanup 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 g/kg to 1 768 g/kg, the second study was for wheat and maize ra
19、nging from 165 g/kg to 4 700 g/kg and the third study was for cereal based foods for infants and young children ranging from 58 g/kg to 452 g/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 equi
20、pment. 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
21、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
22、 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 Re
23、agents 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 Di
24、sodium hydrogen phosphate, anhydrous or Na2HPO412 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. BS EN 15891:2010EN 15891:2010 (E) 5 4.8 Hydroch
25、loric 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) =
26、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 Na2HPO412 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.
27、 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. WARNI
28、NG 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
29、 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 volu
30、me 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
31、) 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 m
32、l of water. BS EN 15891:2010EN 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 Deox
33、ynivalenol 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. Stor
34、e 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 g/ml. Dilute 800 l of stock solution 1 (4.20) to 4 ml with acetonitrile (4.
35、11) to form a solution with a concentration of approximately 250 g/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. D
36、ilute 200 l of stock solution 2 (4.21) to 2,0 ml with acetonitrile (4.11) to form a solution with a concentration of approximately 25 g/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)
37、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=100maxDON(1) where Amax is the absorption determined at the maximum of the absorption curve (here: at 22
38、0 nm); M is the molar mass, in grams per mole, of deoxynivalenol (M = 296,3 g/mol); 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
39、 concentration of the stock solution 2 (4.21), DON2, in micrograms per millilitre using Equation (2): 10DONDON2= (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 mont
40、hs. NOTE Preparation of standard solutions can be carried out gravimetrically by accurately weighing the deoxynivalenol standard material and the solvent used to dissolve it. BS EN 15891:2010EN 15891:2010 (E) 7 4.23 Deoxynivalenol spiking solution, = 100 g/ml. Pipette an aliquot of the stock solutio
41、n 2 (4.21) equivalent to 500 g 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
42、 months. 4.24 Deoxynivalenol standard solution B, = 10 g/ml. Pipette 500 l 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
43、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
44、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 an
45、d with a diameter of 18,5 cm. 5.10 Glass fibre filter, fast flow, fine porosity, retention size 1,6 m or smaller. 5.11 Pipettes, e.g. of 10 ml, 5 ml, 1 ml, and 25 l to 250 l capacity. 5.12 Reservoirs for immunoaffinity columns, of for example 20 ml capacity, with appropriate adaptors. 5.13 Glass via
46、ls or assay tubes, of various size. 5.14 Heating block or thermostatic waterbath, capable of maintaining approximately 50C. 5.15 HPLC apparatus, comprising the following: BS EN 15891:2010EN 15891:2010 (E) 8 5.15.1 Injection system, capable of injecting e.g. 100 l to 300 l. 5.15.2 Mobile phase pump,
47、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 m, which ensures resolution of deoxynivalenol from all other p
48、eaks. 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 da
49、ta 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 base
