SIST EN 14177:2004

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Patulin in klarem und trübem Apfelsaft und Apfelpüree - HPLC-Verfahren mit Reinigung durch Flüssig/Flüssig-VerteilungProduits alimentaires - Détermination de la teneur en patuline dans le jus de pommes clarifié et trouble et dans la compote de pommes - Méthode par CLHP avec purification par partage liquide/liquideFoodstuffs - Determination of patulin in clear and cloudy apple juice and puree - HPLC method with liquid/liquid partition clean-up67.080.10Sadje in sadni proizvodiFruits and derived products67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 14177:2003SIST EN 14177:2004en01-maj-2004SIST EN 14177:2004SLOVENSKI
STANDARD



SIST EN 14177:2004



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14177December 2003ICS 67.080.10; 67.160.20English versionFoodstuffs - Determination of patulin in clear and cloudy applejuice and puree - HPLC method with liquid/liquid partition clean-upProduits alimentaires - Détermination de la teneur enpatuline dans le jus de pommes et dans la compote depommes, limpides et troubles - Méthode parchromatographie en phase liquide à haute performanceavec purification par partage liquide/liquideLebensmittel - Bestimmung von Patulin in klarem undtrübem Apfelsaft und Apfelpüree - HPLC-Verfahren mitReinigung durch Flüssig/Flüssig-VerteilungThis European Standard was approved by CEN on 3 November 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 14177:2003 ESIST EN 14177:2004



EN 14177:2003 (E)2ContentsPageForeword.41Scope.52Normative references.53Principle.54Reagents.54.1General.54.2Sodium carbonate solution, mass concentration rr(Na2CO3) » 15 g/l.54.3Acetic acid, volume concentration jj(CH3COOH) » 98 %.54.4Sodium sulfate anhydrous.54.5pH 4 water.54.6Absolute ethanol, jj(CH3CH2OH) ³ 99,7 %.64.7Ethyl acetate.64.8Perchloric acid, jj(HClO4) = 60 %.64.9Acetonitrile.64.10Endogalacturonase enzyme solution, typical activity 1 400 U/g.64.11HPLC mobile phase.64.12Patulin stock solution.64.13Patulin standard solution.64.14Patulin standard solution for calibration.75Apparatus.75.1General.75.2Displacement pipettes of 5 ml, 1 ml , 200 ml and 50 ml capacity with appropriate pipettetips.75.3Analytical balance.75.4UV spectrophotometer, double beam and recording suitable for measurement at 250 nmto 350 nm.75.5Quartz cells, of optical path length 1 cm.75.6Centrifuge, capable of operating at 4 500 g.75.7Centrifuge tubes of 50 ml capacity with screw cap lids.75.8Rotary evaporator, with water bath set at 40 °C.75.9Filter paper, with pore size 20 µm to 25 µm.75.10Disposable syringe filters, of 0,2 mm pore size (optional).75.11HPLC apparatus, comprising the following.75.12Round bottom flasks, of 250 ml capacity with ground glass joint.86Procedure.86.1Preparation of test samples.86.2Extraction of patulin.86.3Removal of interfering acidic compounds.86.4Preparation of the sample test solution.97Spiking procedure.97.1Clear apple juice.97.2Cloudy apple juice.97.3Apple puree.98HPLC determination.98.1Calibration graph.98.2HPLC operating conditions.109Calculation.10SIST EN 14177:2004



EN 14177:2003 (E)310Precision.1010.1Interlaboratory test.1010.2Repeatability.1110.3Reproducibility.1110.4Test report.12Annex A (informative)
Precision data.13Bibliography.15SIST EN 14177:2004



EN 14177:2003 (E)4ForewordThis document (EN 14177:2003) 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 identicaltext or by endorsement, at the latest by June 2004, and conflicting national standards shall be withdrawn atthe latest by June 2004.Annex A is informative.WARNING — The use of this standard can involve hazardous materials, operations and equipment.This standard does not purport to address all the safety problems associated with its use. It is theresponsibility of the user of this standard to establish appropriate safety and health practices anddetermine the applicability 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.SIST EN 14177:2004



EN 14177:2003 (E)51 ScopeThis European Standard specifies a method for the determination of patulin in apple juices and apple pureeusing high performance liquid chromatography (HPLC). The method has been validated for the determinationof patulin via the analysis of naturally contaminated and spiked samples in clear apple juice at levels rangingfrom 26 mg/l up to 128 mg/l, in cloudy apple juice at levels ranging from 26 mg/l up to 106 mg/l and in applepuree at levels ranging from 23 mg/kg up to 121 mg/kg.2 Normative referencesThis 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.For dated references, subsequent amendments to or revisions of any of these publications apply to thisEuropean Standard only when incorporated in it by amendment or revision. For undated references the latestedition of the publication referred to applies (including amendments).EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987)3 PrincipleCloudy apple juice and apple puree are treated with a pectinase enzyme. Patulin is extracted from apple juiceor enzyme treated puree with ethyl acetate. The solvent extract is cleaned up by liquid-liquid extraction withaqueous sodium carbonate solution. The ethyl acetate extract is dried with anhydrous sodium sulfate. Afterevaporation of ethyl acetate, patulin is quantitatively determined by high performance liquid chromatography(HPLC) with ultra violet (UV) detection.4 Reagents4.1 GeneralDuring the analysis, unless otherwise stated, use only reagents of recognized analytical grade and onlydistilled water or water of grade 1 as defined in EN ISO 3696. Solvents shall be of quality for HPLC analysis.4.2 Sodium carbonate solution, mass concentration rr(Na2CO3) » 15 g/lDissolve 1,5 g of sodium carbonate in 100 ml of water.4.3 Acetic acid, volume concentration jj(CH3COOH) » 98 %4.4 Sodium sulfate anhydrous4.5 pH 4 waterAdjust water to pH 4 with acetic acid.SIST EN 14177:2004



EN 14177:2003 (E)64.6 Absolute ethanol, jj(CH3CH2OH) ³ 99,7 %4.7 Ethyl acetate4.8 Perchloric acid, jj(HClO4) = 60 %4.9 Acetonitrile4.10 Endogalacturonase enzyme solution, typical activity 1 400 U/gUnit definition - the amount of enzyme which catalyses the decrease in viscosity of a 1 % pectin solution by20 % in 5 min at a pH 3,4 and at 25 °C.NOTEPackage volumes of 100 ml to 200 ml are practical.4.11 HPLC mobile phaseMix 95 parts per volume of water with 5 parts per volume of acetonitrile (4.9) and 0,095 parts per volume ofperchloric acid (4.8), and degas.4.12 Patulin stock solutionWARNING - Patulin is a suspect mutagen and has been reported to have immunotoxic and neurotoxicproperties. Gloves and safety glasses should be worn at all times and all standard and samplepreparation stag
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