EN 14132:2003
(Main)Foodstuffs - Determination of ochratoxin A in barley and roasted coffee - HPLC method with immunoaffinity column clean-up
Foodstuffs - Determination of ochratoxin A in barley and roasted coffee - HPLC method with immunoaffinity column clean-up
This European Standard specifies a method for the determination of ochratoxin A content in barley up to 6 ug/kg and ochratoxin A content in roasted coffee up to 6 ug/kg using immunoaffinity (IA) column clean up and high performance liquid chromatography (HPLC).
Lebensmittel - Bestimmung von Ochratoxin A in Gerste und Röstkaffee - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule
Produits alimentaires - Dosage de l'ochratoxine A dans l'orge et dans le café torréfié - Méthode par purification sur colonne d'immunoaffinité suivie d'une analyse par CLHP
La présente Norme européenne spécifie une méthode de dosage de l'ochratoxine A dans l'orge et dans le café torréfié par purification sur colonne d'immunoaffinité et par chromatographie liquide haute performance (CLHP). Cette méthode a été validée pour une teneur en ochratoxine A de 0,1 mg/kg à 4,5 mg/kg dans l'orge et de 0,2 mg/kg à 5,5 mg/kg dans le café torréfié.
Živila - Določevanje ohratoksina A v ječmenu in praženi kavi - Metoda HPLC z imunoafinitetnim kolonskim čiščenjem
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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Ochratoxin A in Gerste und Röstkaffee - HPLC-Verfahren mit Reinigung an einer ImmunoaffinitätssäuleProduits alimentaires - Dosage de l'ochratoxine A dans l'orge et dans le café torréfié - Méthode par purification sur colonne d'immunoaffinité suivie d'une analyse par CLHPFoodstuffs - Determination of ochratoxin A in barley and roasted coffee - HPLC method with immunoaffinity column clean-up67.140.20Kava in kavni nadomestkiCoffee and coffee substitutesICS:Ta slovenski standard je istoveten z:EN 14132:2003SIST EN 14132:2003en01-november-2003SIST EN 14132:2003SLOVENSKI
STANDARD
SIST EN 14132:2003
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14132May 2003ICS 67.140.20English versionFoodstuffs - Determination of ochratoxin A in barley and roastedcoffee - HPLC method with immunoaffinity column clean-upProduits alimentaires - Dosage de l'ochratoxine A présentedans l'orge et dans le café torréfié - Méthode par CLHP etpar purification en colonne d'immunoaffinitéLebensmittel - Bestimmung von Ochratoxin A in Gerste undRöstkaffee - HPLC-Verfahren mit Reinigung an einerImmunoaffinitätssäuleThis European Standard was approved by CEN on 3 March 2003.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 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 Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovak Republic, Spain, Sweden, Switzerland andUnited Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2003 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14132:2003 ESIST EN 14132:2003
EN 14132:2003 (E)2ContentsForeword.31Scope.42Normative reference.43Principle.44Reagents.45Apparatus.76Procedure.87Spiking procedure.98HPLC analysis.99Calculation.1010Precision.1011Test report.11Annex A (informative)
Precision data.13Bibliography.15SIST EN 14132:2003
EN 14132:2003 (E)3ForewordThis document (EN 14132:2003) 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 orby endorsement, at the latest by November 2003, and conflicting national standards shall be withdrawn at the latestby November 2003.Annex A is informative.WARNING — Ochratoxin A is a potent nephrotoxin and liver toxin and has been reported to haveimmunosuppressant 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 flammableand 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 shallbe carried out in a fume cupboard. Operation outside the fume cupboard, such us measurement ofstandards by UV spectrophotometer, shall be performed with the standard in closed containers.Decontamination procedures for laboratory wastes have been reported by the International Agency forResearch on Cancer (IARC), see [1].According to the CEN/CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal,Slovakia, Spain, Sweden, Switzerland and the United Kingdom.SIST EN 14132:2003
EN 14132:2003 (E)41 ScopeThis European Standard specifies a method for the determination of ochratoxin A content in barley and roastedcoffee using immunoaffinity column clean up and high performance liquid chromatography (HPLC). This methodhas been validated for ochratoxin A contents in barley in the range from 0,1 µg/kg up to 4,5 µg/kg and for roastedcoffee in the range from 0,2 µg/kg up to 5,5 µg/kg.2 Normative referenceThis European Standard incorporates by dated or undated reference, provision from other publications. Thesenormative references are cited at the appropriate places in the text and the publications are listed hereafter. Fordated references, subsequent amendments to or revisions of any of these publications apply to this EuropeanStandard only when incorporated in it by amendment or revision. For undated references the latest edition of thepublication referred to applies (including amendments).EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987)3 PrincipleOchratoxin A is extracted from barley by blending with aqueous acetonitrile. The extract is purified by passingthrough an immunoaffinity column. Ochratoxin A is extracted from ground roasted coffee by blending with methanoland sodium hydrogen carbonate. The extract is cleaned up by passing first through a phenyl silane column andthen through an immunoaffinity column. Ochratoxin A is separated by reverse-phase HPLC and determined byfluorescence.4 Reagents4.1 GeneralDuring the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only distilledwater or water of grade 1 as defined in EN ISO 3696. Solvents shall be of quality for HPLC analysis.Commercially available reagents with equivalent properties to the ones listed may be used.4.2 Sodium chloride4.3 Disodium hydrogen phosphate4.4 Potassium dihydrogen phosphate4.5 Potassium chloride4.6 Sodium hydroxide solution, (NaOH) = 8,0 g/lDissolve 8 g of sodium hydroxide in 900 ml of water, then dilute to 1 l with water.SIST EN 14132:2003
EN 14132:2003 (E)54.7 Phosphate buffered saline (PBS)Dissolve 8 g of sodium chloride (4.2), 1,2 g of disodium hydrogen phosphate (4.3), 0,2 g of potassium dihydrogenphosphate (4.4) and 0,2 g of potassium chloride (4.5) in 900 ml of distilled water. Adjust the pH to 7,4 with sodiumhydroxide solution (4.6) then dilute to 1 l with water.Commercially available phosphate buffered saline tablets with equivalent properties may be used.4.8 Sodium hydrogen carbonate solution, (NaHCO3) = 30 g/lIn a 1-l-volumetric flask dissolve 30 g sodium hydrogen carbonate in 900 ml of water. Dilute to volume with water.4.9 Glacial acetic acid, (CH3COOH) = 98 %4.10 Methanol4.11 Acetonitrile4.12 Toluene4.13 Solvent mixture of toluene and glacial acetic acidMix 99 parts per volume of toluene (4.12) with 1 part per volume of glacial acetic acid (4.9).4.14 Barley extraction solvent mixtureMix 6 parts per volume acetonitrile (4.11) with 4 parts per volume of water.4.15 Roasted coffee extraction solvent mixtureMix 1 part per volume of methanol (4.10) with 1 part per volume of sodium hydrogen carbonate solution (4.8).4.16 Injection solventMix 30 parts per volume of methanol (4.10) with 70 parts per volume of water and 1 part per volume of glacialacetic acid (4.9).4.17 Mobile phaseMix 102 parts per volume of water with 96 parts per volume of acetonitrile (4.11) and 2 parts per volume of glacialacetic acid (4.9), filter through a 0,2 m filter (5.12) and degas with for example helium before use.4.18 Phenyl silane column wash reagent 1Mix 20 parts per volume of methanol (4.10) with 80 parts per volume of sodium hydrogen carbonate solution (4.8).4.19 Phenyl silane column wash reagent 2, (NaHCO3) = 1 g/100 mlIn a 100 ml volumetric flask dissolve 1 g of sodium hydrogen carbonate in 90 ml water. Dilute to volume with water.4.20 Phenyl silane column elution reagentMix 7 parts per volume methanol (4.10) with 93 parts per volume of water.SIST EN 14132:2003
EN 14132:2003 (E)64.21 Ochratoxin A stock solutionDissolve 1 mg of the ochratoxin A (in crystal form) or the contents of 1 ampoule (if ochratoxin A has been obtainedas 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, record the absorption curve between a wavelength of 300 nm and 370 nm in5 nm steps in 1 cm quartz cells (5.14) and solvent mixture (4.13) as reference. Identify the wavelength formaximum absorption and calculate the mass concentration of ochratoxin A, ota, in micrograms per millilitre, usingequation (1):1000maxMAota(1)where:Amaxis the absorption determined at the maximum of the absorption curve (here: at 333 nm)Mis the relative molecular mass of ochratoxin A (M = 403,8 g/mol);is the relative molar absorption coefficient of ochratoxin A in the solvent mixture (4.13), (here: 5440 m2/mol);is the path length of the quartz cell in centimetres.This solution is stable at –18°C for at least four years.4.22 Ochratoxin A standard solutionDilute the stock solution (4.21) with the solvent mixture (4.13) to obtain a standard solution with a massconcentration of ochratoxin A of 10 g/ml. Store this solution in a refrigerator at approximately 4C and check itsstability.4.23 Ochratoxin A calibration solutionPipette 200 µl of the 10 µg/ml ochratoxin A standard solution (4.22) into a glass vial and dilute to 1 ml with 800 µl ofsolvent mixture (4.13). This gives 2 µg/ml ochratoxin A solution. Pipette 100 µl of the 2 µg/ml ochratoxin A solutioninto a glass vial (5.1). Evaporate the solvent under a stream of nitrogen. Redissolve in 10 ml injection solvent (4.16)which has been filtered through a 0,2 µm filter (5.12). This gives a calibration solution containing 20 ng/ml.Prepare the calibration solutions at the beginning of every day of the analysis.4.24 Spiking solutionPipette 100 µl of the 10 µg/ml ochratoxin A standard solution (4.22) into a glass vial. Dilute to 2 ml with 1,9 ml of themixture of toluene and acetic acid (4.13). This gives a mass concentration of 500 ng/ml ochratoxin A.4.25 Immunoaffinity columnThe immunoaffinity column contains antibodies raised against ochratoxin A. The column shall have a total bindingcapacity of not less than 100 ng of ochratoxin A. The performance of the column should be checked by applying asolution of 100 ng ochratoxin A in a solvent mixture of the same composition as the sample extract (6.1.3) to beapplied. This shall give a recovery of not less than 85 %.4.26 Phenyl silane solid phase extraction columns500 mg sorbent weight and 3 ml reservoir volume (to ensure adequate column bed depth and prevent analytebreakthrough). The column should have a total capacity of not less than 100 ng ochratoxin A and should give arecovery of not less than 85 % when applied in a standard solution of ochratoxin A in the roasted coffee extractionsolvent (4.15) containing 100 ng of ochratoxin A.SIST EN 14132:2003
EN 14132:2003 (E)75 ApparatusUsual laboratory equipment and, in particular, the following:5.1 Analytical balance, accurate to 0,01 mg5.2 Glass vials, of at least 10 mlCertain types of vials might lead to losses of ochratoxin A during evaporation. To avoid this, silanization could beapplied. Prepare vials by filling them with silanizing reagent and leave this reagent in the vial for one minute. Rinsethe vial twice with appropriate solvent (toluene, acetone or hexane) followed by water (twice) and dry the vial.5.3 Blender, explosion proofWith 1 l capacity jar and cover and with a high speed of approximately 20 000 min-15.4 Displacement pipettes of 5 ml, 1 ml and 200 l capacity with appropriate pipettes tips5.5
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