1、May 2010 Translation by DIN-Sprachendienst.English price group 12No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67
2、.060; 67.230!$bW=“1635226www.din.deDDIN EN 15835Foodstuffs Determination of ochratoxin A in cereal based foods for infants andyoung children HPLC method with immunoaffinity column clean up and fluorescencedetectionEnglish translation of DIN EN 15835:2010-05Lebensmittel Bestimmung von Ochratoxin A in
3、 Suglings- und Kleinkindernahrung auf Getreidebasis HPLC-Verfahren mit Reinigung an einer Immunoaffinittssule und FluoreszenzdetektionEnglische bersetzung von DIN EN 15835:2010-05Produits alimentaires Dosage de lochratoxine A dans les aliments base de crales pour nourrissons etjeunes enfants Mthode
4、CLHP avec purification sur colonne dimmuno-affinit et dtection parfluorescenceTraduction anglaise de DIN EN 15835:2010-05www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritative.2005.10 DIN EN 15835:2010-05 National foreword This standard h
5、as been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). Preliminary work was done by Working Group WG 5 “Biotoxins”. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkt
6、e (Food and Agricultural Products Standards Committee), Working Committee NA 057-01-03 AA Biotoxine. Mycotoxins are extremely toxic secondary metabolites produced by fungi. Food that is grown, harvested or stored under humid conditions can be infected by fungi whose metabolites invade the food. Beca
7、use some mycotoxins are extremely toxic for humans, their reliable detection is especially important for consumer health and protection. In Germany the regulation “Mykotoxin-Hchstmengenverordnung” governs the maximum allowable content of mycotoxins in food. Since 2004 this regulation has contained s
8、pecifications not only for aflatoxins, but also for ochratoxin A, fumonisins, deoxynivalenol (DON), and zearalenone. National regulations have been supplemented by EU-wide regulations regarding the maximum content of contaminants in food since 2001. The maximum content of mycotoxins in certain foods
9、 is also laid down in various other regulations. The DIN Standard corresponding to the International Standard referred to in this document is as follows: EN ISO 3696 DIN ISO 3696 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test met
10、hods 2 A comma is used as the decimal marker. EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15835 February 2010 ICS 67.050; 67.230 English Version Foodstuffs Determination of ochratoxin A in cereal based foods for infants and young children HPLC method with immunoaffinity column cleanup and f
11、luorescence detection Produits alimentaires Dosage de lochratoxine A dans les aliments base de crales pour nourrissons et jeunes enfants Mthode CLHP avec purification sur colonne dimmuno-affinit et dtection par fluorescence Lebensmittel Bestimmung von Ochratoxin A in Suglings- und Kleinkindernahrung
12、 auf Getreidebasis HPLC-Verfahren mit Reinigung an einer Immunoaffinittssule und Fluoreszenzdetektion This European Standard was approved by CEN on 25 December 2009. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Stan
13、dard 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, Frenc
14、h, 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,
15、 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 STANDARDIZATI
16、ON COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR 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 15835:2010: EEN 15835:2010 (E) 2 Contents Page Foreword 31 S
17、cope 42 Normative references 43 Principle 44 Reagents .45 Apparatus .76 Procedure .97 HPLC analysis . 108 Calculation . 119 Precision 1210 Test report . 13Annex A (informative) Typical chromatograms 14Annex B (informative) Precision data . 15Annex C (informative) Data on the in-house study . 16Bibli
18、ography . 18DIN EN 15835:2010-05 EN 15835:2010 (E) 3 Foreword This document (EN 15835:2010) 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
19、 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 C
20、ENELEC 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. Annexes A, B and C are informative. According to the CEN/CENELEC Internal Regulations,
21、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, Neth
22、erlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15835:2010-05 EN 15835:2010 (E) 4 1 Scope This European Standard specifies a method for the determination of ochratoxin A in cereal based foods for infants and young children by
23、high performance liquid chromatography (HPLC) with immunoaffinity column 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 0,050 g/kg to 0,217 g/kg. For further information on
24、 the validation see Clause 8 and Annex B. Additional studies have shown that this method is applicable to cereal based baby foods containing 8 different types of cereals, honey and cocoa, at levels up to 3,540 g/kg, see Annex C and 6. WARNING The use of this standard can involve hazardous materials,
25、 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 N
26、ormative 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 a
27、nalytical laboratory use Specification and test methods (ISO 3696:1987) 3 Principle A test portion is extracted with tert-butyl methyl ether after addition of 0,5 mol/l phosphoric acid / 2 mol/l sodium chloride solution. The extract is evaporated and redissolved in methanol and phosphate buffered sa
28、line (PBS) solution. After removal of lypophilic compounds with hexane, the extract is applied to an immunoaffinity column containing antibodies specific to ochratoxin A. The toxin is eluted from the column with methanol. Ochratoxin A is determined by HPLC with enhanced fluorescence detection involv
29、ing post column reaction with ammonia. NOTE Some investigations indicate that HPLC can be also performed without the use of ammonia although this results in at least a two-fold decrease of the response for ochratoxin A. In this case, the fluorescence detection conditions need to be changed (excitati
30、on wavelength = 333 nm, emission wavelength = 460 nm). 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 equiv
31、alent 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 4. 4.2 Helium purified compr
32、essed gas 4.3 Nitrogen DIN EN 15835:2010-05 EN 15835:2010 (E) 5 4.4 Disodium hydrogen phosphate, Na2HPO4 anhydrous or Na2HPO412 H2O 4.5 Potassium chloride 4.6 Potassium dihydrogen phosphate 4.7 Sodium chloride 4.8 Sodium hydroxide 4.9 Ammonium hydroxide solution, the mass fraction w(NH4OH) = 25 % in
33、 water (post column reagent) Degas the solution with a degasser (5.21.7). 4.10 Hydrochloric acid solution, w(HCl) is 37 % (acidimetric) 4.11 Phosphoric acid solution, w(H3PO4) = 85 % 4.12 Hydrochloric acid solution, c(HCl) = 0,1 mol/l Dilute 8,28 ml of hydrochloric acid solution (4.10) to 1 l of wat
34、er. 4.13 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l Dissolve 4 g of sodium hydroxide (4.8) in 1 l of water. 4.14 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 (4.7), 1,2 g of anhydr
35、ous disodium hydrogen phosphate or 2,9 g of Na2HPO412 H2O (4.4), 0,2 g of potassium dihydrogen phosphate (4.6) and 0,2 g of potassium chloride (4.5) in 900 ml of water. After dissolution, adjust the pH to 7,4 with hydrochloric acid solution (4.12) or sodium hydroxide solution (4.13) as appropriate,
36、then dilute to 1 l with water. Alternatively, a PBS solution with equivalent properties can be prepared from commercially available PBS material. 4.15 Mixture of phosphoric acid solution and sodium chloride solution, c(H3PO4) = 0,5 mol/l, c(NaCl) = 2 mol/l Dissolve 118 g of sodium chloride (4.7) in
37、approximately 900 ml of water. Add 33 ml of phosphoric acid (4.11) and make up to 1,0 l with water. 4.16 Glacial acetic acid, the mass fraction 99,7 % 4.17 Acetic acid solution, the volume fraction is 9 % Add 90 ml of glacial acetic acid (4.16) and 910 ml of water. 4.18 Hexane WARNING Hexane is high
38、ly flammable. Operations involving this solvent shall be performed in a fume cupboard. Serious health problems can be derived from prolonged exposure to this reagent. DIN EN 15835:2010-05 EN 15835:2010 (E) 6 4.19 Methanol, gradient grade 4.20 Toluene 4.21 Mixture of methanol and acetic acid solution
39、 Mix 72 parts per volume of methanol (4.19) with 28 parts per volume of acetic acid solution (4.17). 4.22 Tert-butyl methyl ether WARNING Tert-butyl methyl ether is hazardous and samples shall be blended using an explosion proof blender which is housed within a fume cupboard. Centrifugation of extra
40、cts shall be performed at cool temperature (4 C to 8 C). 4.23 Mixture of toluene and glacial acetic acid Mix 99 parts per volume of toluene (4.20) with one part per volume of glacial acetic acid (4.16). 4.24 HPLC mobile phase A Acetic acid solution (4.17). 4.25 HPLC mobile phase B Methanol (4.19). D
41、egas the mobile phases A and B with for example helium (4.2). Helium can be pumped into the reservoirs of both mobile phases A and B. The pumping rate should be 50 ml/min to 100 ml/min. The use of a degasser is also an acceptable option. 4.26 Immunoaffinity columns The immunoaffinity column contain
42、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 85 % when applied as a standard solution of ochratoxin A in a mixture of 15 parts per volume of methanol (4.19) and 85 parts per volume of PBS s
43、olution (4.14) containing 3 ng of ochratoxin A. 4.27 Ochratoxin A, in crystal form or as a film in ampoules 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
44、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. 4.28 Ochratoxin A stock solution Prepare a stock solution of ochratoxin A in the mixture o
45、f toluene and glacial acetic acid (4.23) with a nominal concentration of 10 g/ml. To determine the exact concentration, record the absorption curve between a wavelength of 300 nm and 370 nm in 5 nm steps in 1 cm quartz cells (5.22) in a spectrometer with the solvent mixture (4.23) as reference. Iden
46、tify the wavelength for maximum absorption and calculate the mass concentration of ochratoxin A, ota, in micrograms per millilitre, using Equation (1): bMA=100maxota(1) DIN EN 15835:2010-05 EN 15835:2010 (E) 7 where Amax is the absorption determined at the maximum of the absorption curve (here: at 3
47、33 nm); M is the molar mass, in grams per mole, of ochratoxin A (M = 403,8 g/mol); is the molar absorption coefficient, in square metres per mole, of ochratoxin A in the solvent mixture (4.23), (here: 544 m2/mol); b is the path length, in centimetres, of the quartz cell. Store this solution in a fre
48、ezer 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. 4.29 Ochratoxin A standard solution Pipette a volume of ochratoxin A stock solution (4.28) containing exactly 400 ng ochratoxin A into a 10 ml calibrated volumetric flask (5.13) and dilute to 10 ml with the mixture of
