SIST EN 14123:2003
(Main)Foodstuffs - Determination of aflatoxin B1 and the sum of aflatoxin B1, B2, G1 and G2 in peanuts, pistachios, figs, and paprika powder - High performance liquid chromatographic method with postcolumn derivatization and immunoaffinity column clean-up
Foodstuffs - Determination of aflatoxin B1 and the sum of aflatoxin B1, B2, G1 and G2 in peanuts, pistachios, figs, and paprika powder - High performance liquid chromatographic method with postcolumn derivatization and immunoaffinity column clean-up
This draft European Standard is applicable to the determination of aflatoxins B1, B2, G1 and G2 in figs, pistachios, peanuts and paprika powder. The limit of quantification of the method is 0,8 ng/g for each aflatoxin or better (value derived from in-house and collaborative study), depending on the equipment used.
Lebensmittel - Bestimmung von Aflatoxin B1 und der Summe von Aflatoxin B1, B2, G1 und G2 in Erdnüssen, Pistazien, Feigen und Paprikapulver - Hochleistungsflüssigchromatographisches Verfahren mit Immunoaffinitätssäulen-Reinigung und Nachsäulenderivatisierung
Diese Europäische Norm legt ein Verfahren zur Bestimmung des Gehaltes von Aflatoxin B1, B2, G1 und G2 in Erdnüssen, Feigen, Pistazien und Paprikapulver fest. Die Bestimmungsgrenze liegt bei 0,8 ng/g für jedes der Aflatoxine oder besser (der Wert stammt aus einer hausinternen Untersuchung und einem Ringversuch) je nach verwendetem Untersuchungsgerät.
Produits alimentaires - Dosage de l'aflatoxine B1 et de la somme des aflatoxines B1, B2, G1 et G2 présentes dans les cacahuetes, les pistaches, les figues et le paprika en poudre - Méthode de chromatographie liquide haute performance avec dérivation post-colonne et purification sur colonne d'immunoaffinité
Le présent projet de Norme européenne peut etre appliqué pour le dosage des aflatoxines B1, B2, G1 et G2 présentes dans les figues, les pistaches, les cacahuetes et le paprika en poudre. La limite de quantification de la méthode est d'au moins 0,8 ng/g pour chaque aflatoxine (valeur obtenue a partir d'une étude interne et d'une étude interlaboratoires) selon l'équipement utilisé.
Živila - Določevanje aflatoksina B1 in vsote aflatoksinov B1, B2, G1 in G2 v arašidih, pistacijah, figah in papriki v prahu - Tekočinska kromatografija visoke ločljivosti s postkolonsko derivatizacijo in imunoafinitetnim kolonskim čiščenjem
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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Aflatoxin B1 und der Summe von Aflatoxin B1, B2, G1 und G2 in Erdnüssen, Pistazien, Feigen und Paprikapulver - Hochleistungsflüssigchromatographisches Verfahren mit Immunoaffinitätssäulen-Reinigung und NachsäulenderivatisierungProduits alimentaires - Dosage de l'aflatoxine B1 et de la somme des aflatoxines B1, B2, G1 et G2 présentes dans les cacahuetes, les pistaches, les figues et le paprika en poudre - Méthode de chromatographie liquide haute performance avec dérivation post-colonne et purification sur colonne d'immunoaffinitéFoodstuffs - Determination of aflatoxin B1 and the sum of aflatoxin B1, B2, G1 and G2 in peanuts, pistachios, figs, and paprika powder - High performance liquid chromatographic method with postcolumn derivatization and immunoaffinity column clean-up67.080.01Sadje, zelenjava in njuni proizvodi na splošnoFruits, vegetables and derived products in general67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 14123:2003SIST EN 14123:2003en01-november-2003SIST EN 14123:2003SLOVENSKI
STANDARD
SIST EN 14123:2003
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14123June 2003ICS 67.050English versionFoodstuffs - Determination of aflatoxin B1 and the sum ofaflatoxin B1, B2, G1 and G2 in peanuts, pistachios, figs, andpaprika powder - High performance liquid chromatographicmethod with postcolumn derivatization and immunoaffinitycolumn clean-upProduits alimentaires - Dosage de l'aflatoxine B1 et de lasomme des aflatoxines B1, B2, G1 et G2 dans lescacahuètes, les pistaches, les figues et le paprika enpoudre - Méthode par purification sur colonne d'immuno-affinité suivie d'une chromatographie liquide à hauteperformance avec dérivation post-colonneLebensmittel - Bestimmung von Aflatoxin B1 und derSumme von Aflatoxin B1, B2, G1 und G2 in Erdnüssen,Pistazien, Feigen und Paprikapulver -Hochleistungsflüssigchromatographisches Verfahren mitImmunoaffinitätssäulen-Reinigung undNachsäulenderivatisierungThis European Standard was approved by CEN on 21 April 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, Slovakia, Spain, Sweden, Switzerland and UnitedKingdom.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 14123:2003 ESIST EN 14123:2003
EN 14123:2003 (E)2ContentspageForeword.31Scope.32Normative reference.33Principle.34Reagents.45Apparatus.76Procedures.97Precision.128Test report.16Annex A (informative)
Typical chromatograms.17Annex B (informative)
Precision data.20Bibliography.24SIST EN 14123:2003
EN 14123:2003 (E)3ForewordThis document (EN 14123:2003) 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 December 2003, and conflicting national standards shall be withdrawn at the latestby December 2003.Annexes A and B are informative.WARNING — The use of this standard can involve hazardous materials, operations and equipment. Thisstandard does not purport to address all the safety problems associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and determine theapplicability of regulatory limitations prior to use.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.1 ScopeThis draft European Standard is applicable to the determination of aflatoxins B1, B2, G1 and G2 in figs, pistachios,peanuts and paprika powder. The limit of quantification of the method is 0,8 ng/g for each aflatoxin or better (valuederived from in-house and collaborative study), depending on the equipment used.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 PrincipleA test portion is either extracted with a solvent solution (methanol/water) or the solvent solution plus hexane (orcyclohexane). The sample extract is filtered, diluted with phosphate buffered saline (PBS) and applied to animmunoaffinity column (IAC) containing antibodies specific to aflatoxins B1, B2, G1 and G2. The aflatoxins are elutedfrom the immunoaffinity column with methanol. Aflatoxins are quantified by reverse-phase high performance liquidchromatography (RP-HPLC) with post column derivatization (PCD) involving bromination followed by fluorescencedetection. The PCD is achieved with either electrochemically generated bromine or with pyridinium hydrobromideperbromide (PBPB).SIST EN 14123:2003
EN 14123:2003 (E)44 Reagents4.1 GeneralUse only reagents of recognized analytical grade and water complying with grade 3 of EN ISO 3696, unlessotherwise specified.4.2 Phosphate buffered saline (PBS)Dissolve 0,20 g of potassium chloride, 0,20 g of potassium dihydrogen phosphate, 1,16 g of disodium hydrogenorthophosphate (or 2,92 g of hydrogenphosphate·12 H20) and 8,00 g of sodium chloride in 0,9 l of water. Afterdissolution, adjust the pH to 7,4 with HCl (0,1 mol/l) or NaOH (0,1 mol/l) as appropriate. Dilute to 1 l with water.Commercially available phosphate buffered saline tablets with equivalent properties may be used.4.3 Sodium chloride4.4 Pyridinium hydrobromide perbromide (PBPB), [CAS: 39416-48-3]4.5 Potassium bromide4.6 Acetonitrile, HPLC grade4.7 Methanol, HPLC grade4.8 Methanol, technical grade4.9 Toluene4.10 Extraction solvent mixture of methanol and waterMix 8 parts per volume of methanol (4.8) with 2 parts per volume of water.4.11 n-Hexane, cyclohexane, technical grade4.12 Nitric acid, c(HNO3) = 4 mol/l4.13 Immunoaffinity columnThe affinity column contains antibodies raised against aflatoxins B1, B2, G1 and G2. The column shall have amaximum capacity of not less than 100 ng of aflatoxin B1 and shall give a recovery of not less than 80 % foraflatoxins B1, B2, G1 and not less than 60 % for aflatoxin G2 when applied as an aqueous standard solution (10 %of methanol) containing 5 ng of each toxin. The maximum solvent concentration of solutions that can be applied onthe column shall not exceed 12 % of methanol.4.14 HPLC mobile phase solvent (A), for use with PBPBMix 6 parts per volume of water with 2 parts per volume of acetonitrile (4.6) and 3 parts per volume of methanol(4.7). Degas the solution before use. The mobile phase shall be free of particles and should be filtered prior use.4.15 HPLC mobile phase solvent (B), for use with electochemically generated bromineMix 6 parts per volume of water with 2 parts per volume of acetonitrile (4.6) and 3 parts per volume of methanol(4.7). Add 120 mg of potassium bromide (4.5) and 350 l of nitric acid (4.12) per litre of mobile phase. Degas thesolution before use.SIST EN 14123:2003
EN 14123:2003 (E)54.16 Post-column reagentDissolve 50 mg of PBPB (4.4) in 1 l of water. The solution may be used up to four days if stored in a dark place atroom temperature.4.17 Mixture of toluene and acetonitrileMix 98 parts per volume of toluene (4.9) with 2 parts per volume of acetonitrile (4.6).4.18 Aflatoxins, either in form of crystals or film in ampoules or in form of commercially available aflatoxinsolutions.WARNING 1 — Decontamination procedures for laboratory wastes of aflatoxins were developed by theInternational Agency for Research on Cancer (IARC) [1], [2].WARNING 2 — Aflatoxins are subject to light degradation. Protect the laboratory, where the analyses aredone, adequately from daylight. This can be achieved effectively by using Ultraviolet (UV) absorbing foil onthe windows in combination with subdued light (no direct sunlight) or curtains or blinds in combination withartificial light (fluorescent tubes are acceptable).Protect aflatoxin containing solutions from light as much as possible (keep in the dark, use aluminium foil or amber-coloured glassware) and store at the temperature recommended by the manufacturer (e.g -18 oC).4.19 Aflatoxins stock solutionDissolve aflatoxin B1, B2, G1 and G2 separately in the mixture of toluene and acetonitrile (4.17) to give separatesolutions with a concentration of 10 g/ml for each aflatoxin. Wrap the flasks tightly in aluminium foil and store themat less than 4 °C.To determine the exact concentration of aflatoxins in each stock solution, record the absorption curve between awavelength of 330 nm and 370 nm in 1 cm quartz glass cells in a spectrometer with the mixture of toluene andacetonitrile (4.17) in the reference cell. Calculate the mass concentration of each aflatoxin, i, in micrograms permillilitre, using equation (1):d100MAmaxiii(1)where:Amaxis the absorbance determined at the maximum of the absorption curve;Miis the molar mass of each aflatoxin, in grams per mol;iis the molar absorptivity of each aflatoxin in toluene and acetonitrile (4.17), in square metres per mol;dis the optical path length of the cell, in centimetres.Mi and i of aflatoxins B1, B2, G1 and G2 are given in Table 1.SIST EN 14123:2003
EN 14123:2003 (E)6Table 1 — Molar mass and molar absorptivity of aflatoxins B1, B2, G1 and G2(In mixture of toluene and acetonitrile (4.17))AflatoxinMi, (g/mol)i, (m2/mol)B13121930B23142040G13281660G233017904.20 Mixed aflatoxins stock solutionsPrepare a mixed aflatoxins stock solution containing 1000 ng/ml of aflatoxin B1 and G1, 200 ng/ml of aflatoxin B2and G2 in the toluene and acetonitrile mixture (4.17) by appropriate dilution of aflatoxins (B1, B2, G1 and G2) stocksolutions (4.19).Pipette exactly 2,0 ml of this solution into a 20 ml calibrated volumetric flask (5.10), fill to the mark with the tolueneand acetonitrile mixture (4.17) and mix well to give a diluted mixed aflatoxins stock solution containing 100 ng/ml ofaflatoxin B1 and G1, 20 ng/ml of aflatoxin B2 and G2.Wrap the flask tightly in aluminium foil and store it at less than 4 °C. Before use, do not open the flask until thecontents have reached room temperature to avoid incorporation of water by condensation.4.21 Mixed aflatoxins standard solutionsUse the diluted mixed aflatoxins stock solution containing 100 ng/ml of aflatoxin B1 and G1, 20 ng/ml of aflatoxin B2and G2 (see 4.20) for pipetting the volumes as given in Table 2 into a set of 10 ml volumetric flasks (5.10).Evaporate the toluene/acetonitrile solution just to dryness under a stream of nitrogen at room temperature. To eachflask, add 4 ml of methanol, let aflatoxins dissolve, dilute to 10 ml with water, and shake well.Methanol and water are subject to volume contraction when mixed, so adjust the volume again to the givenvolume.Table 2 — Preparation of mixed aflatoxins standard solutionsStandardsolutionMass concentration of standard solution, in ng/mlTaken fromdilutedstock solution(4.20) (l)B1B2G1G21400,4000,0800,4000,08021201,2000,2401,2000,24032002,0000,4002,0000,40042802,8000,5602,8000,56053603,6000,7203,6000,7204.22 Spiking solutionPrepare a spiking solution by pipetting 2 ml of the mixed aflatoxins stock solution (containing 1000 ng/ml ofaflatoxin B1 and G1, 200 ng/ml of aflatoxin B2 and G2, see 4.20) into a 10 ml calibrated volumetric flask. Evaporatethe toluene/acetonitrile solution just to dryness under a stream of nitrogen at room temperature. Dilute to the markwith methanol and shake well. The concentration of this spiking solution is 200 ng/ml of aflatoxin B1 and G1, and40 ng/ml of aflatoxin B2 and G2.SIST EN 14123:2003
EN 14123:2003 (E)7Wrap the flask tightly in aluminium foil and store it at less than 4 °C. Before use, do not open the flask until thecontents have reached room temperature to avoid incorporation of water by condensation.5 Apparatus5.1 GeneralAll glassware coming into contact with aqueous solutions of aflatoxins shall be washed with acid solution beforeuse. Many laboratory washing machines do this as part of the washing program. Otherwise soak such laboratoryglassware in sulfuric acid (2 mol/l) for several hours (e.g. 15 h overnight), then rinse well (e.g. three times) withwater to remove all traces of acid. Check the absence of acid with pH paper.This treatment is necessary, because the use of non-acid washed glassware may cause losses of aflatoxins. Inpractice, the treatment is necessary for round bottomed flasks, volumetric flasks, measuring cylinders, vials ortubes used for calibration solutions and final extracts (particularly autosampler vials), and Pasteur pipettes, if theseare used to transfer calibration solutions or extracts.5.2 Usual laboratory apparatus and, in particular, the following5.3 Laboratory millor explosion proof High speed blender1) , necessary
for the production and extraction of pastes from peanuts,pistachios and figs, with suitable blender jar.5.4 Adjustable vertical or horizontal shaker, needed for the analysis of paprika powder5.5 Paper filter, e.g. 24 cm diameter, prefolded5.6 Conical flask, with screw top or glass stopper5.7 Glass microfiber filter paper, retention size 1,6 m or smaller5.8 Reservoir, 75 ml with luer tip connector for immunoaffinity column (IAC)5.9 Hand pump, 20 ml syringe with luer lock or rubber stopper for IAC5.10 Volumetric glassware, flasks of e.g. 5 ml, 10 ml and 20 ml, with an accuracy of at least 0,5 %5.11 HPLC system, consisting of5.11.1 HPLC pump, suitable for flow rate at 1,0 ml/min5.11.2 Injection system, capable for total loop injection. A valve with a 200 l loop is recommended.In the case that a different loop size than recommended is used it shall be guaranteed that the limit of detection(LOD) for the system is
0,2 ng/g (signal-to-noise-ratio = 3) and the limit of quantification (LOQ) is
0,5 ng/g(signal-to-noise-ratio = 6) for each aflatoxin (using the standard solutions).5.11.3 RP-HPLC column, e.g. C18 or ODS-2 (length of 25 cm, inner diameter of 4,6 mm and particle size of 5which ensures a baseline resolution of the aflatoxin B1, B2, G1 and G2 peaks from all other peaks. The maximum
1)Contact your National Standardization institute for appropriate high speed blenders.SIST EN 14123:2003
EN 14123:2003 (E)8overlapping of peaks shall be less than 10 % (it could be necessary to adjust the mobile phase for a sufficientbaseline resolution). A suitable pre-column should be used.5.11.4 Post-column derivatization system, with PBPB (only to be used with mobile phase A (4.14)Consisting of an HPLC pulseless pump, zero-dead volume T-piece, reaction tubing min. 45 cm x 0,5 mm internaldiameter PTFE.5.11.5 System for derivatization with electrochemically generated bromine, e.g. KOBRA cell® 2) (only to beused with mobile phase B (4.15)5.11.6 Fluorescence detector, with a wavelength of
= 360 nm excitation filter and a wavelength of
> 420 nmcut-off emission filter, or equivalent (e.g. a detector with an adjustable monochromator).Recommended settings for adjustable detectors are 365 nm (excitation wavelength), 435 nm (emissionwavelength) and a bandwidth of 18 nm.5.12 Disposable filter unit,
of pore size 0,45 mPrior to usage, verify that no aflatoxin losses occur during filtration (recovery testing).NOTE
There is a possibility that various filter materials can retain aflatoxins.5.13 Pipettes, 2 ml and 10 ml capacity, with an accuracy of at least 0,5 %5.14 Analytical balance, capable of weighing to 0,1 mg5.15 Laboratory balance, capable of weighing to 0,01 g5.16 Calibrated microliter syringe(s) or microliter pipette(s), 25 l to 500 l5.17 Vacuum manifold, optional6 Procedures6.1 Sample preparationHomogenize a suitable amount (e.g. 10 kg, see European legislation, [3]) of pistacios, peanuts and figsappropriately to give a paste, e.g. using a high speed blender (5.3). Information on sample sizes and sampling canbe seen in [3].6.2 Conditioning of immunoaffinity columnsAllow the immunoaffinity columns (4.13) to reach room temperature prior to conditioning. Connect theimmunoaffinity column to the vacuum manifold (5.17) and attach the reservoir (5.8) to the immunoaffinity column.For conditioning transfer 10 ml of PBS (4.2) on the top of the column and let it pass at a speed of 2 ml/min to3 ml/min through the column (e.g. by gravity). Make sure that a small portion (0,5 ml) of the PBS remains on thecolumn until the sample solution is applied.Different conditioning procedures shall be considered in accordance with the manufacturer’s instructions.
2)KOBRA cell® is the trade name of a suitable product available commercially. This information is given for the convenienceof users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent productsmay be used if they can be shown to lead to the same results.SIST EN 14123:2003
EN 14123:2003 (E)96.3 Extraction6.3.1 GeneralFor the extraction of fig paste, peanut butter and pistachio paste a high speed blender shall be used, since the fattycommodities (peanut butter and pistachio paste) need to form an emulsion to break the fatty layers and allow asufficient extraction. In addition, fig paste needs to break down in the solvent, which cannot be guaranteed if ashaker is used, due to its consistency. Paprika powder can be extracted by shaking (provided that the powder isground sufficiently to a particle size up to 500 µm) to process several samples simultaneously and reduces the riskof cross contamination.6.3.2 FigsWeigh, to the nearest 0,1 g, approximately 50 g of the homogenized (6.1) test portion into a 500 ml conical flask(5.6) or blender jar. Add 5 g of sodium chloride (4.3) and 300 ml of extraction solvent (4.10). Blend for 3 min with ahigh speed blender (5.3).Filter the extract using a paper filter (5.5). Pipette 10,0 ml of the clear filtrate into a 100 ml glass beaker (or similar)and dilute with 60 ml of PBS (4.2). Add the diluted sample extract to the reservoir connected to the conditionedimmunoaffinity column (4.13) and proceed as described in 6.4.Slurries or larger test portions may be used, provided that ratios (sample-to-extraction solvent as well as theextraction solvent composition for slurries) are maintained.6.3.3 PeanutsWeigh, to the nearest 0,1 g, approximately 50 g of the homogenized (6.1) test portion into a 500 ml conical flask(5.6) or blender jar. Add 5 g of sodium chloride (4.3), 200 ml of extraction solvent (4.10) and 100 ml of n-hexane orcyclohexane (4.11). Blend for 3 min with a high speed blender (5.3).Filter the extract using a paper filter (5.5). In case of a solvent layer separation carry on with the lower phase.Pipette 10,0 ml of the clear filtrate into a 100 ml glass beaker (or similar) and dilute with 60 ml of PBS (4.2). A
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