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 and roasted coffee using immunoaffinity column clean up and high performance liquid chromatography (HPLC). This method has been validated for ochratoxin A contents in barley in the range from 0,1 g/kg up to 4,5 g/kg and for roasted coffee in the range from 0,2 g/kg up to 5,5 g/kg.

Lebensmittel - Bestimmung von Ochratoxin A in Gerste und Röstkaffee - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule

Diese Europäische Norm legt ein Verfahren zur Bestimmung von Ochratoxin A in Gerste und Röstkaffee mit Reinigung an einer Immunoaffinitätssäule und Hochleistungs Flüssigchromatographie (HPLC) fest. Das Verfahren wurde für Ochratoxin A Gehalte in Gerste von 0,1 µg/kg bis 4,5 µg/kg und in Röstkaffee von 0,2 µg/kg bis 5,5 µg/kg validiert.

Produits alimentaires - Dosage de l'ochratoxine A dans l'orge et le café torréfié - 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 l’orge et dans le
café torréfié par chromatographie liquide à haute performance (CLHP) avec purification sur colonne
d’immuno-affinité. Cette méthode a été validée pour une teneur en ochratoxine A de 0,1 µg/kg à 4,5 µg/kg
dans l’orge et de 0,2 µg/kg à 5,5 µg/kg dans le café torréfié.

Živila - Določevanje ohratoksina A v ječmenu in praženi kavi - Metoda HPLC z imunoafinitetnim kolonskim čiščenjem

General Information

Status
Published
Publication Date
26-May-2009
Current Stage
9093 - Decision to confirm - Review Enquiry
Completion Date
28-Oct-2020

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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 le café torréfié - Méthode par purification sur colonne d'immuno-affinité suivie d'une analyse par chromatographie liquide haute performance (CLHP)Foodstuffs - 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 14132:2009SIST EN 14132:2009en,fr,de01-september-2009SIST EN 14132:2009SLOVENSKI

STANDARDSIST EN 14132:2003/AC:2007SIST EN 14132:20031DGRPHãþD
SIST EN 14132:2009

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14132May 2009ICS 67.140.20Supersedes EN 14132:2003

English VersionFoodstuffs - Determination of ochratoxin A in barley and roastedcoffee - HPLC method with immunoaffinity column clean-upProduits alimentaires - Dosage de l'ochratoxine A dansl'orge et le café torréfié - Méthode par purification surcolonne d'immuno-affinité suivie d'une analyse parchromatographie liquide haute performance (CLHP)Lebensmittel - Bestimmung von Ochratoxin A in Gerste undRöstkaffee - HPLC-Verfahren mit Reinigung an einerImmunoaffinitätssäuleThis European Standard was approved by CEN on 24 March 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 14132:2009: ESIST EN 14132:2009

EN 14132:2009 (E) 2 Contents Foreword ...................................................................................................................................................................... 3 1 Scope .............................................................................................................................................................. 4 2 Normative reference ...................................................................................................................................... 4 3 Principle .......................................................................................................................................................... 4 4 Reagents ......................................................................................................................................................... 4 5 Apparatus ....................................................................................................................................................... 6 6 Procedure ....................................................................................................................................................... 7 7 Spiking procedure ......................................................................................................................................... 8 8 HPLC analysis ................................................................................................................................................ 9 9 Calculation .................................................................................................................................................... 10 10 Precision ....................................................................................................................................................... 10 11 Test report .................................................................................................................................................... 11 Annex A (informative)

Precision data ...................................................................................................................... 12 Bibliography .............................................................................................................................................................. 14

SIST EN 14132:2009

EN 14132:2009 (E) 3 Foreword This document (EN 14132:2009) 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 14132:2003 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 us measurement of standards by UV spectrophotometer, 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 14132:2009

EN 14132:2009 (E) 4 1 Scope This European Standard specifies a method for the determination of ochratoxin A content in barley and roasted coffee using immunoaffinity column clean up and high performance liquid chromatography (HPLC). This method has been validated for ochratoxin A contents in barley in the range from 0,1 µg/kg up to 4,5 µg/kg and for roasted coffee in the range from 0,2 µg/kg up to 5,5 µg/kg. 2 Normative reference 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 Ochratoxin A is extracted from barley by blending with aqueous acetonitrile. The extract is purified by passing it through an immunoaffinity column. Ochratoxin A is extracted from ground roasted coffee by blending with methanol and sodium hydrogen carbonate. The extract is cleaned up by passing it first through a phenyl silane column and then through an immunoaffinity column. Ochratoxin A is separated by reverse-phase HPLC and determined by fluorescence. 4 Reagents 4.1 General During the analysis, 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. Solvents shall be of quality for HPLC analysis. Commercially available reagents with equivalent properties to the ones listed may be used.

4.2 Sodium chloride 4.3 Disodium hydrogen phosphate 4.4 Potassium dihydrogen phosphate 4.5 Potassium chloride 4.6 Sodium hydroxide solution, ρ(NaOH) = 8,0 g/l Dissolve 8 g of sodium hydroxide in 900 ml of water, then dilute to 1 l with water. 4.7 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 dihydrogen phosphate (4.4) and 0,2 g of potassium chloride (4.5) in 900 ml of water. Adjust the pH to 7,4 with sodium hydroxide solution (4.6) then dilute to 1 l with water. Commercially available phosphate buffered saline tablets with equivalent properties may be used. SIST EN 14132:2009

EN 14132:2009 (E) 5 4.8 Sodium hydrogen carbonate solution, ρ(NaHCO3) = 30 g/l In 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, ϕ(CH3COOH) = 98 % 4.10 Methanol 4.11 Acetonitrile 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 (4.9). 4.14 Barley extraction solvent mixture Mix 6 parts per volume acetonitrile (4.11) with 4 parts per volume of water. 4.15 Roasted coffee extraction solvent mixture Mix 1 part per volume of methanol (4.10) with 1 part per volume of sodium hydrogen carbonate solution (4.8). 4.16 Injection solvent Mix 30 parts per volume of methanol (4.10) with 70 parts per volume of water and 1 part per volume of glacial acetic acid (4.9). 4.17 Mobile phase Mix 102 parts per volume of water with 96 parts per volume of acetonitrile (4.11) and 2 parts per volume of glacial acetic acid (4.9), filter through a 0,2 µm filter (5.12) and degas with for example helium before use. 4.18 Phenyl silane column wash reagent 1 Mix 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, ρ(NaHCO3) = 1 g/100 ml In 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 reagent Mix 7 parts per volume methanol (4.10) with 93 parts per volume of water. 4.21 Ochratoxin A stock solution Dissolve 1 mg of the 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 (5.14) and solvent mixture (4.13) as reference. Identify the wavelength for maximum absorption and calculate the mass concentration of ochratoxin A, ρota, in micrograms per millilitre, using Equation (1): bMAota×××=ερ100max (1) SIST EN 14132:2009

EN 14132:2009 (E) 6 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. 4.22 Ochratoxin A standard solution

Dilute the stock solution (4.21) with the solvent mixture (4.13) to obtain a standard solution with a mass concentration of ochratoxin A of 10 µg/ml. Store this solution in a refrigerator at approximately 4°C and check its stability. 4.23 Ochratoxin A calibration solution Pipette 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 of solvent mixture (4.13). This gives 2 µg/ml ochratoxin A solution. Pipette 100 µl of the 2 µg/ml ochratoxin A solution into a glass vial (5.2). 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 solution Pipette 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 the mixture of toluene and acetic acid (4.13). This gives a mass concentration of 500 ng/ml ochratoxin A. 4.25 Immunoaffinity column The immunoaffinity column contains antibodies raised against ochratoxin A. The column shall have a total capacity of not less than 100 ng of ochratoxin A. The performance of the column should be checked by applying a solution of 100 ng ochratoxin A in a solvent mixture of the same composition as the sample extract (6.1.3) to be applied. This shall give a recovery of not less than 85 %. 4.26 Phenyl silane solid phase extraction columns

500 mg sorbent weight and 3 ml reservoir volume (to ensure adequate column bed depth and prevent analyte breakthrough). The column should have a total capacity of not less than 100 ng ochratoxin A and should give a recovery of not less than 85 % when applied in a standard solution of ochratoxin A in the roasted coffee extraction solvent (4.15) containing 100 ng of ochratoxin A. 5 Apparatus Usual laboratory equipment and, in particular, the following: 5.1 Analytical balance, accurate to 0,01 mg 5.2 Glass vials, of at least 10 ml Certain types of vials might lead to losses of ochratoxin A during evaporation. To avoid this, silanization could 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. SIST EN 14132:2009

EN 14132:2009 (E) 7 5.3 Blender, explosion proof With 1 l capacity jar and cover and with a high speed of approximately 20 000 min-1. 5.4 Displacement pipettes of 5 ml, 1 ml and 200 µl capacity with

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