1、September 2009DEUTSCHE NORM English price group 10No part of this standard 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 67.160.10!$Yq“154785
2、7www.din.deDDIN EN 14133Foodstuffs Determination of ochratoxin A in wine and beer HPLC method with immunoaffinity column clean-upEnglish version of DIN EN 14133:2009-09Lebensmittel Bestimmung von Ochratoxin A in Wein und Bier HPLC-Verfahren mit Reinigung an einer ImmunoaffinittssuleEnglische Fassung
3、 DIN EN 14133:2009-09SupersedesDIN EN 14133:2003-10 andDIN EN 14133 Corrigendum1:2007-03www.beuth.deDocument comprises pages15DIN EN 14133:2009-09 National foreword This standard has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). Preli
4、minary work was done by Working Group WG 5 “Biotoxins”. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-01-03 AA Biotoxine. The DIN Standard
5、corresponding to the International Standard referred to in this document is as follows: EN ISO 3696 DIN ISO 3696 Amendments This standard differs from DIN EN 14133:2003-10 and DIN EN 14133 Corrigendum 1:2007-03 as follows: a) DIN EN 14133 Corrigendum 1:2007-03 has been incorporated, i.e. Equation (1
6、) and the key in subclause 4.14 “Ochratoxin A stock solution” have been corrected. b) The bibliography has been rendered more precise. Previous editions DIN EN 14133: 2003-10 DIN EN 14133 Corrigendum 1: 2007-03 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laborator
7、y use Specification and test methods 2 EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 14133May 2009ICS 67.160.10 Supersedes EN 14133:2003 English VersionFoodstuffs - Determination of ochratoxin A in wine and beer -HPLC method with immunoaffinity column clean-upProduits alimentaires - Dosage de lo
8、chratoxine A dans levin et la bire - Mthode par purification sur colonnedimmuno-affinit suivie dune analyse par chromatographieliquide haute performance (CLHP)Lebensmittel - Bestimmung von Ochratoxin A in Wein undBier - HPLC-Verfahren mit Reinigung an einerImmunoaffinittssuleThis European Standard w
9、as approved by CEN on 24 May 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nation
10、alstandards 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 translationunder the responsibility of a CEN member into its own language and notif
11、ied to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
12、 Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation
13、 in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14133:2009: EEN 14133:2009 (E) 2 Contents Foreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents . 4 5 Apparatus . 6 6 Procedure . 7 7 HPLC analysis 7 8 Calculation 8 9 Precision . 8 10 Test report 1
14、0 Annex A (informative) Precision data 11 Bibliography 13 DIN EN 14133:2009-09 EN 14133:2009 (E) 3 Foreword This document (EN 14133:2009) 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
15、 given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by November 2009, and conflicting national standards shall be withdrawn at the latest by November 2009. Attention is drawn to the possibility that some of the elements of this docume
16、nt 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 will supersede EN 14133:2003. The 2003 version has been updated with the inclusion of the corrigendum and some minor editorial improvements. Annex A i
17、s informative. WARNING Ochratoxin A is a potent nephrotoxin and liver toxin and has been reported to have immunosuppressant properties. It is classified by the International Agency for Research on Cancer (IARC) as possibly carcinogenic to humans (Group 2B). Acetonitrile is hazardous. Toluene is high
18、ly flammable and harmful. Observe appropriate safety precautions for handling such compounds. 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. Operation outside the fume cupboard, such as measurement of stand
19、ards by UV spectrometer, shall be performed with the standard in closed containers. Decontamination procedures for laboratory wastes have been reported by the International Agency for Research on Cancer (IARC), see 1. According to the CEN/CENELEC Internal Regulations, the national standards organiza
20、tions of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Roman
21、ia, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 14133:2009-09 EN 14133:2009 (E) 4 1 Scope This European Standard specifies a method for the determination of ochratoxin A content in wine and beer using immunoaffinity column clean up and high performance liquid chroma
22、tography (HPLC), see 2 and 3. This method has been validated in an interlaboratory study according to AOAC International Guidelines 4 for collaborative study procedures to validate characteristics of a method of analysis for the determination of ochratoxin A in wine and beer via the analysis of natu
23、rally contaminated and spiked samples of wine and beer at levels ranging from 0,1 ng/ml to 3 ng/ml. 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 late
24、st edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) 3 Principle Wine and beer samples are diluted with a solution containing polyethylene glycol (PEG) and sodium hydrogen carbonate,
25、filtered and cleaned up by immunoaffinity column. Ochratoxin A is eluted with methanol and quantified by reversed-phase HPLC with fluorescence detection. NOTE The use of PEG is essential to increase ochratoxin A recoveries and to reduce the number and intensity of other chromatographic peaks. 4 Reag
26、ents 4.1 General Unless otherwise stated, use only reagents of recognized analytical grade and only distilled water or water of grade 1 as defined in EN ISO 3696. Commercially available reagents with equivalent properties to the ones listed may be used. 4.2 Sodium chloride 4.3 Sodium hydrogen carbon
27、ate 4.4 Polyethylene glycol, (average molecular weight of 8000) 4.5 Methanol, HPLC grade 4.6 Acetonitrile, HPLC grade 4.7 Water, HPLC grade 4.8 Glacial acetic acid, (CH3COOH) 99 % 4.9 Diluting solution Dissolve 10 g polyethylene glycol (4.4) and 50 g sodium hydrogen carbonate (4.3) in approximately
28、950 ml of water and bring up to 1000 ml with water. DIN EN 14133:2009-09 EN 14133:2009 (E) 5 4.10 Washing solution Dissolve 25 g sodium chloride (4.2) and 5 g sodium hydrogen carbonate (4.3) in approximately 950 ml of water and bring up to 1000 ml with water. 4.11 HPLC mobile phase Mix 990 ml HPLC w
29、ater (4.7) with 990 ml acetonitrile (4.6) and 20 ml glacial acetic acid (4.8), filter through 0,45 m filter and degas (e.g. with helium). 4.12 Toluene 4.13 Solvent mixture of toluene and glacial acetic acid Mix 99 parts per volume of toluene (4.12) with 1 part per volume of glacial acetic acid (4.8)
30、. 4.14 Ochratoxin A stock solution Dissolve 1 mg of ochratoxin A (in crystal form) or the contents of 1 ampoule (if ochratoxin A has been obtained as a film) in solvent mixture (4.13) to give a solution containing approximately 20 g/ml to 30 g/ml of ochratoxin A. To determine the exact concentration
31、, record the absorption curve between a wavelength of 300 nm and 370 nm in 5 nm steps in 1 cm quartz cells (5.10) and solvent mixture (4.13) as reference. Identify the wavelength for maximum absorption and calculate the mass concentration of ochratoxin A, OTA, in micrograms per millilitre, using Equ
32、ation (1): bMAota=100max(1) where Amaxis the absorption determined at the maximum of the absorption curve (here: at 333 nm); M is the molar mass of ochratoxin A (M = 403,8 g/mol); is the molar absorption coefficient of ochratoxin A in the solvent mixture (4.13), (here: 544 m2/mol); b is the optical
33、path length of the quartz cell in centimetres. This solution is stable at 18 C for at least 4 years. 4.15 Ochratoxin A standard solution Dilute the stock solution (4.14) with solvent mixture (4.13) to obtain a standard solution with a mass concentration of ochratoxin A of 2 g/ml. Store this solution
34、 in a refrigerator at approximately 4 C and check the stability. 4.16 Ochratoxin A calibration solution Pipette 0,5 ml of the 2 g/ml ochratoxin A standard solution (4.15) into a 10 ml volumetric flask (5.3) and evaporate the solvent under a stream of nitrogen. Redissolve in 10 ml of mobile phase (4.
35、11). This gives a mass concentration of 100 ng/ml ochratoxin A. Prepare six HPLC calibrants in separate 5 ml volumetric flasks (5.3) according to Table 1. Dilute each solution to 5 ml with HPLC mobile phase (4.11). DIN EN 14133:2009-09 EN 14133:2009 (E) 6 Table 1 Preparation of calibration solutions
36、 Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 l of filtered mobile phase (4.11) 4970 4900 4700 4000 3000 2000 l of 100 ng/ml OTA solution 30 100 300 1000 2000 3000 OTA mass concentration (ng/ml) 0,6 2,0 6,0 20 40 60 injected ng OTA 0,06 0,20 0,60 2,00 4,00 6,00 Prepare the calibration solutions at the beginn
37、ing of every day of the analysis. 4.17 Immunoaffinity columns The immunoaffinity column shall contain antibodies raised against ochratoxin A. The column shall have a total capacity of not less than 100 ng of ochratoxin A and shall give a recovery of not less than 85 % when a diluted wine solution co
38、ntaining 100 ng of ochratoxin A is applied. 5 Apparatus Usual laboratory equipment and, in particular, the following: 5.1 Microbalance, capable to measure 0,01 mg 5.2 Glass vials, approximately 4 ml, with polytetrafluoroethylene (PTFE)-lined screw cap, or appropriate sealable screw cap. Certain type
39、s of vials can lead to losses of ochratoxin A during evaporation. To avoid this, silanization can be applied. Prepare vials by filling them with silanizing reagent and leave this reagent in the vial for 1 min. Rinse the vial twice with appropriate solvent (toluene, acetone or hexane) followed by wat
40、er (twice) and dry the vial. 5.3 Volumetric flasks, 5 ml and 10 ml volume 5.4 Vacuum manifold, to accommodate immunoaffinity columns 5.5 Reservoirs and attachments, to fit to immunoaffinity columns 5.6 Glass microfibre filters, pore size 1,6 m, (e.g. Whatman GF/A1or equivalent) 5.7 Solvent evaporato
41、r 5.8 Calibrated microliter syringe(s) or microliter pipette(s) 5.9 HPLC apparatus consisting of 1Whatman is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of thes
42、e products. Equivalent products can be used if they can be shown to give equivalent results.DIN EN 14133:2009-09 EN 14133:2009 (E) 7 5.9.1 Injection system, a syringe-loading injection valve with 100 l injection loop or equivalent. 5.9.2 HPLC pump, isocratic, capable of maintaining a volume flow rat
43、e of 1,0 ml/min. 5.9.3 Analytical reverse phase separating column, for example stainless steel (150 mm length, 4,6 mm inner diameter) packed with 5 m C18reverse-phase material preceded by a suitable corresponding reverse-phase guard column or guard filter (0,5 m). Columns of different dimensions can
44、 be used provided that they ensure a baseline resolution of the ochratoxin A peak from all other peaks. 5.9.4 Fluorescence detector, fitted with a flow cell and set at 333 nm (excitation) and 460 nm (emission). Detection of at least 0,02 ng of ochratoxin A shall be possible. 5.9.5 Data system 5.10 U
45、V spectrometer, with suitable quarz cells 6 Procedure 6.1 Sample preparation Degas sparkling wine and beer prior to dilution. Pour 10 ml of wine (beer) into a sealable 100 ml conical flask. Add 10 ml of the diluting solution (4.9) and mix vigorously. Filter through glass microfibre filter (5.6) when
46、 cloudy solutions or solid residues are formed after dilution. If there is a problem with degassing, degassing should be performed by sonicating samples for 1 h, previously cooled at +4 C for 30 min to prevent fast foam formation. 6.2 Immunoaffinity column clean-up Prepare the immunoaffinity column
47、according to the suppliers instructions. Connect the immunoaffinity column (4.17) to the vacuum manifold (5.4) and attach the reservoir (5.5) to the immunoaffinity column. Add 10 ml (equivalent to 5 ml wine or beer) of the diluted solution to the reservoir and pass through the immunoaffinity column
48、at a flow rate of about 1 drop per second. The immunoaffinity column shall not be allowed to run dry. Wash the immunoaffinity column with 5 ml of washing solution (4.10) and then with 5 ml of water at 1 to 2 drops per second flow rate. Dry the column by passing air through it. Remove the immunoaffin
49、ity column from the vacuum manifold and place it over a vial (5.2). 6.3 Preparation of the sample test solution Elute ochratoxin A into the vial by passing 2 ml of methanol (4.5) at 1 drop per second flow rate. Evaporate the eluate to dryness under a stream of nitrogen e.g. at approximately 50 C. Redissolve in 250 l HPLC mobile phase (4.11) and store at 4 C until HPLC analysis. This is the sample test solution. 7 HPLC analysis 7.1 Sample analysis Inject 100 l of reconstituted extract (equivalent to 2 ml wine or beer)
