SIST EN 14133:2009
(Main)Foodstuffs - Determination of ochratoxin A in wine and beer - HPLC method with immunoaffinity column clean-up
Foodstuffs - Determination of ochratoxin A in wine and beer - HPLC method with immunoaffinity column clean-up
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 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 analysis 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.
Lebensmittel - Bestimmung von Ochratoxin A in Wein und Bier - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule
Diese Europäische Norm legt ein hochleistungs-flüssigchromatographisches Verfahren (HPLC) mit Reinigung
an einer Immunoaffinitätssäule (IAS) zur Bestimmung von Ochratoxin A in Wein und Bier fest, siehe [2], [3].
Dieses Verfahren wurde in einem Ringversuch nach den AOAC „Guidelines for collaborative study procedures
to validate characteristics of a method of analysis“ [4] mit natürlich kontaminierten und aufgestockten Bierund
Weinproben mit Ochratoxin A-Gehalten von 0,1 ng/ml bis 3 ng/ml validiert.
Produits alimentaires - Dosage de l'ochratoxine A dans le vin et la bière - Méthode par purification sur colonne d'immuno-affinité suivie d'une analyse par chromatographie liquide haute performance (CLHP)
La présente Norme européenne spécifie une méthode de dosage de l’ochratoxine A dans le vin et dans la bière par chromatographie liquide à haute performance (CLHP) avec purification sur colonne d’immuno-affinité, voir [2] et [3].
Cette méthode a été validée dans le cadre d’une étude interlaboratoires réalisée conformément aux directives internationales de l’AOAC [4] relatives aux modes opératoires d’études comparatives visant à valider les caractéristiques d’une méthode d’analyse, pour le dosage de l’ochratoxine A dans le vin et la bière, via l’analyse d’échantillons naturellement contaminés et dopés, dans les limites d’une gamme de concentrations comprise entre 0,1 ng/ml et 3 ng/ml.
Živila - Določevanje ohratoksina A v vinu in pivu - Metoda HPLC z imunoafinitetnim kolonskim čiščenjem
General Information
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Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Ochratoxin A in Wein und Bier - HPLC-Verfahren mit Reinigung an einer ImmunoaffinitätssäuleProduits alimentaires - Dosage de l'ochratoxine A dans le vin et la bière - Méthode par purification sur colonne d'immuno-affinité suivie d'une analyse par chromatographie liquide haute performance (CLHP)Foodstuffs - Determination of ochratoxin A in wine and beer - HPLC method with immunoaffinity column clean-up67.160.10Alcoholic beveragesICS:Ta slovenski standard je istoveten z:EN 14133:2009SIST EN 14133:2009en,fr,de01-december-2009SIST EN 14133:2009SLOVENSKI
STANDARDSIST EN 14133:2003/AC:2007SIST EN 14133:20031DGRPHãþD
SIST EN 14133:2009
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14133May 2009ICS 67.160.10Supersedes EN 14133:2003
English VersionFoodstuffs - Determination of ochratoxin A in wine and beer -HPLC method with immunoaffinity column clean-upProduits alimentaires - Dosage de l'ochratoxine A dans levin et la bière - Méthode par purification sur colonned'immuno-affinité suivie d'une analyse par chromatographieliquide haute performance (CLHP)Lebensmittel - Bestimmung von Ochratoxin A in Wein undBier - HPLC-Verfahren mit Reinigung an einerImmunoaffinitätssäuleThis 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 alteration. 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 translationunder 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, 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 STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre:
Avenue Marnix 17,
B-1000 Brussels© 2009 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14133:2009: ESIST EN 14133:2009
EN 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 . 10 Annex A (informative)
Precision data . 11 Bibliography . 13
SIST EN 14133:2009
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 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 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 updated 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) 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 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), 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, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. SIST EN 14133:2009
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 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 analysis 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. 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, 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, 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 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 properties 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, 3(CH3COOH) ≈ 99 % 4.9 Diluting solution Dissolve 10 g 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. SIST EN 14133:2009
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 water (4.7) with 990 ml acetonitrile (4.6) and 20 ml glacial acetic acid (4.8), filter through 0,45 µm 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). 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 µg/ml to 30 µg/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 mass concentration of ochratoxin A, OTA, in micrograms per millilitre, using Equation (1): bMAota×××=ερ100max (1) where Amax is 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); 0 is the molar absorption coefficient of ochratoxin 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 solution with a mass concentration of ochratoxin A of 2 µg/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 µg/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 (4.11). 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). SIST EN 14133:2009
EN 14133:2009 (E) 6
Table 1 — Preparation of calibration solutions
Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 µl of filtered mobile phase (4.11) 4970 4900 4700 4000 3000 2000 µl 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 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 shall 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 polytetrafluoroethylene (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 twice 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 fi
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