1、BS EN 14133:2009ICS 67.160.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFoodstuffs Determination ofochratoxin A inwine and beer HPLC method withimmunoaffinity columnclean-upThis British Standardwas published under theauthority of the StandardsPolicy and Str
2、ategyCommittee on 30 June2009. BSI 2009ISBN 978 0 580 64237 1Amendments/corrigenda issued since publicationDate CommentsBS EN 14133:2009National forewordThis British Standard is the UK implementation of EN 14133:2009. Itsupersedes BS EN 14133:2003 which is withdrawn.The UK participation in its prepa
3、ration was entrusted to TechnicalCommittee AW/-/3, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its
4、 correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.BS EN 14133:2009EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 14133May 2009ICS 67.160.10 Supersedes EN 14133:2003 English VersionFoodstuffs - Determination of ochratoxin A in wine and beer -HPLC
5、method with immunoaffinity column clean-upProduits alimentaires - Dosage de lochratoxine 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 - HP
6、LC-Verfahren mit Reinigung an einerImmunoaffinittssuleThis European Standard was 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 alte
7、ration. Up-to-date lists and bibliographical references concerning such nationalstandards 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 trans
8、lationunder the responsibility of a CEN member into its own language and notified 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, G
9、reece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement
10、 Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14133:2009: EBS EN 14133:2009EN 14133:2009 (E) 2 Contents Foreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents . 4 5 Ap
11、paratus . 6 6 Procedure . 7 7 HPLC analysis 7 8 Calculation 8 9 Precision . 8 10 Test report 10 Annex A (informative) Precision data 11 Bibliography 13 BS EN 14133:2009EN 14133:2009 (E) 3 Foreword This document (EN 14133:2009) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Hori
12、zontal 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 November 2009, and conflicting national standards shall be withdrawn at the latest by Nove
13、mber 2009. 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 will supersede EN 14133:2003. The 2003 version has been updat
14、ed with the inclusion of the corrigendum and some minor editorial improvements. Annex A is 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)
15、as possibly carcinogenic to humans (Group 2B). Acetonitrile is hazardous. Toluene is highly 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
16、out in a fume cupboard. Operation outside the fume cupboard, such as measurement of standards 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),
17、see 1. 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, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
18、Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 14133:2009EN 14133:2009 (E) 4 1 Scope This European Standard specifies a method for the determination of ochratoxin A content in w
19、ine and beer using immunoaffinity column clean up and high performance liquid chromatography (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 anal
20、ysis for the determination of ochratoxin A in wine and beer via the analysis of naturally 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. Fo
21、r dated references, only the edition cited applies. For undated references, the latest 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
22、with a solution containing polyethylene glycol (PEG) and sodium hydrogen carbonate, 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 reco
23、veries and to reduce the number and intensity of other chromatographic peaks. 4 Reagents 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 prope
24、rties to the ones listed may be used. 4.2 Sodium chloride 4.3 Sodium hydrogen carbonate 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
25、polyethylene glycol (4.4) and 50 g sodium hydrogen carbonate (4.3) in approximately 950 ml of water and bring up to 1000 ml with water. BS EN 14133:2009EN 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 wat
26、er and bring up to 1000 ml with water. 4.11 HPLC mobile phase Mix 990 ml HPLC water (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 p
27、er volume of toluene (4.12) with 1 part per volume of glacial acetic acid (4.8). 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 approxi
28、mately 20 g/ml to 30 g/ml of ochratoxin A. 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 (5.10) and solvent mixture (4.13) as reference. Identify the wavelength for maximum absorption and calculate the m
29、ass concentration of ochratoxin A, OTA, in micrograms per millilitre, using Equation (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 oc
30、hratoxin A in the solvent mixture (4.13), (here: 544 m2/mol); b is the optical 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 so
31、lution with a mass concentration of ochratoxin A of 2 g/ml. Store this solution 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
32、the solvent under a stream of nitrogen. Redissolve in 10 ml of mobile phase (4.11). 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). BS EN
33、14133:2009EN 14133:2009 (E) 6 Table 1 Preparation of calibration solutions 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 0,0
34、6 0,20 0,60 2,00 4,00 6,00 Prepare the calibration solutions at the beginning 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 s
35、hall give a recovery of not less than 85 % when a diluted wine solution containing 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 polytetrafluoroethyle
36、ne (PTFE)-lined screw cap, or appropriate sealable screw cap. Certain types 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 t
37、wice with appropriate solvent (toluene, acetone or hexane) followed by water (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
38、, pore size 1,6 m, (e.g. Whatman GF/A1or equivalent) 5.7 Solvent evaporator 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 th
39、is European Standard and does not constitute an endorsement by CEN of these products. Equivalent products can be used if they can be shown to give equivalent results.BS EN 14133:2009EN 14133:2009 (E) 7 5.9.1 Injection system, a syringe-loading injection valve with 100 l injection loop or equivalent.
40、 5.9.2 HPLC pump, isocratic, capable of maintaining a volume flow rate 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 guar
41、d column or guard filter (0,5 m). Columns of different dimensions can 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 leas
42、t 0,02 ng of ochratoxin A shall be possible. 5.9.5 Data system 5.10 UV 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)
43、and mix vigorously. Filter through glass microfibre filter (5.6) when 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.
44、6.2 Immunoaffinity column clean-up Prepare the immunoaffinity column 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
45、solution to the reservoir and pass through the immunoaffinity column 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 r
46、ate. Dry the column by passing air through it. Remove the immunoaffinity 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
47、 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) into the
48、 chromatographic apparatus using the mobile phase (4.11) at flow rate of 1,0 ml/min. 7.2 Calibration curve Prepare a calibration curve at the beginning of every day of the analysis and whenever chromatographic conditions change. BS EN 14133:2009EN 14133:2009 (E) 8 Inject 100 l of each calibrant solu
49、tion (4.16) into the HPLC apparatus and plot peak area or peak height values of ochratoxin A calibrant solutions against the ochratoxin A masses in nanograms. If the content of ochratoxin A in the samples falls outside the calibration range, appropriate dilution shall be performed, or lower volumes can be injected; in these cases calculation shall be reconsidered accordingly. Due to the wide range of concentrations, the calibration curve should be forced through the “zero” for a more accurate quantification at low ochratoxin A
