Foodstuffs - Detection of irradiated food containing fat - Gas chromatographic/mass spectrometric analysis of 2-alkylcyclobutanones

This draft European Standard specifies a method for identification of irradia- tion treatment of food containig fat. It is based on the mass spectrometric (MS)detection of radiation-induced 2-alkylcyclobutanones after gas chromatographic (GC) separation 1 to 3. The method has been successfully tested in interlabora- tory tests on raw chicken, pork, and liquid whole egg, 4 to 6.

Lebensmittel - Nachweis von bestrahlten fetthaltigen Lebensmitteln - Gaschromatographisch / massenspektrometrische Untersuchung auf 2-Alkylcyclobutanone

Diese Europäische Norm legt ein Verfahren zum Nachweis der Behandlung von fetthaltigen Lebensmitteln mit ionisierenden Strahlen fest. Das Verfahren basiert auf einer massenspektrometrischen Detektion der strahleninduzierten 2-Alkylcyclobutanone nach gaschromatographischer (GC) Trennung [1] bis [3].
Das Verfahren wurde in Ringversuchen erfolgreich an rohem Hühnerfleisch, Schweinefleisch, flüssigem Vollei, Lachs und Camembert geprüft, [4] bis [8].
Andere Untersuchungen haben gezeigt, dass das Verfahren für viele andere Lebensmittel anwendbar ist [9] bis [21].

Produits alimentaires - Détection d'aliments ionisés contenant des lipides - Analyse par chromatographie en phase gazeuse / Spectrométrie de masse des 2-alkylcyclobutanones

La présente Norme européenne indique une méthode pour l'identification d'un traitement ionisant d'aliments contenant des lipides. Cette méthode repose sur une séparation par chromatographie en phase gazeuse (CPG) [1] a [3l des 2-alkylcyclobutanones formées par ionisation et a leur détection par spectrométrie de masse (SM).
Elle a été soumise avec succes a des essais interlaboratoires sur de la viande de poulet et de porc crues, de la coule d'?uf, du saumon et du camembert [4] a [8].
D'autres études montrent que la présente méthode peut etre appliquée a une large gamme de produits alimentaires [9] a [21].

Živila - Pepoznavanje obsevane hrane, ki vsebuje maščobo - Plinska kromatografska/masno spektrometrijska analiza 2-alkilciklobutanona

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.DDLebensmittel - Nachweis von bestrahlten fetthaltigen Lebensmitteln - Gaschromatographisch / massenspektrometrische Untersuchung auf 2-AlkylcyclobutanoneProduits alimentaires - Détection d'aliments ionisés contenant des lipides - Analyse par chromatographie en phase gazeuse / Spectrométrie de masse des 2-alkylcyclobutanonesFoodstuffs - Detection of irradiated food containing fat - Gas chromatographic/mass spectrometric analysis of 2-alkylcyclobutanones67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 1785:2003SIST EN 1785:2003en01-november-2003SIST EN 1785:2003SLOVENSKI

SIST EN 1785:2003

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 1785August 2003ICS 67.050Supersedes EN 1785:1996English versionFoodstuffs - Detection of irradiated food containing fat - Gaschromatographic/mass spectrometric analysis of 2-alkylcyclobutanonesProduits alimentaires - Détection d'aliments ioniséscontenant des lipides - Analyse par chromatographie enphase gazeuse / Spectrométrie de masse des 2-alkylcyclobutanonesLebensmittel - Nachweis von bestrahlten fetthaltigenLebensmitteln - Gaschromatographisch /massenspektrometrische Untersuchung auf 2-AlkylcyclobutanoneThis European Standard was approved by CEN on 20 June 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 1785:2003 ESIST EN 1785:2003

EN 1785:2003 (E)2ContentsPageForeword.31Scope.32Normative references.33Principle.34Reagents.45Apparatus.56Sampling technique.67Procedure.68Evaluation.79Limitations.810Validation.811Test report.9Annex A (informative)
Figures.11Bibliography.15SIST EN 1785:2003

EN 1785:2003 (E)3ForewordThis document (EN 1785:2003) 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 February 2004, and conflicting national standards shall be withdrawn at the latestby February 2004.This document supersedes EN 1785:1996.This European Standard was elaborated on the basis of a protocol during a concerted action of the EuropeanCommission (DG XII C.5). Experts and laboratories from E.U. and EFTA countries, contributed jointly to thedevelopment of this protocol.The predecessor of the present standard (EN 1785:1996) has been elaborated following a mandate of theEuropean Commission.Annex A is informative.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 European Standard specifies a method for the identification of irradiation treatment of food containing fat. It isbased on the mass spectrometric (MS) detection of radiation-induced 2-alkylcyclobutanones after gaschromatographic (GC) separation [1] to [3].The method has been successfully tested in interlaboratory trials on raw chicken, pork, liquid whole egg, salmonand Camembert [4] to [8].Other studies demonstrate that the method is applicable to a wide range of foodstuffs [9] to [21].2 Normative referencesThis European Standard incorporates by dated or undated references, provisions 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 3696:1995, Water for analytical laboratory use — Specification and test methods (ISO 3696:1987).3 PrincipleDuring irradiation, the acyl-oxygen bond in triglycerides is cleaved and this reaction results in the formation of2-alkylcyclobutanones containing the same number of carbon atoms as the parent fatty acid and the alkyl group islocated in ring position 2. Thus, if the fatty acid composition is known, the 2-alkylcyclobutanones formed can bepredicted.The 2-alkylcyclobutanones which were analysed in interlaboratory studies were 2-dodecylcyclobutanone (DCB)and 2-tetradecylcyclobutanone (TCB) which are formed from palmitic and stearic acid, respectively, duringirradiation. To date, there is no evidence that the 2-alkylcyclobutanones can be detected in unirradiated foods [4],SIST EN 1785:2003

EN 1785:2003 (E)4[9] to [21]. The 2-alkylcyclobutanones are extracted using n-hexane or n-pentane along with the fat. The extract isthen fractionated using adsorption chromatography prior to separation using gas chromatography and detectionwith a mass spectrometer. Other 2-alkycyclobutanones, e.g. 2 (tetradec-5'-enyl) cyclobutanone derived from oleicacid, have also beenidentified in irradiated foodstuffs [8], [15] to [20].NOTEAs an alternative procedure for extraction and purification of the 2-alkylcyclobutanones, supercritical fluid extraction(SFE), has been successfully employed [18], [21]. Argentation chromatography has been used effectively for the detection offoods irradiated at very low doses or containing irradiated ingredients [17]. Liquid chromatography (LC)-GC-MS coupling hasbeen used successfully as an alternative procedure for purification and detection [14]. It should, however, be noted that thesealternative procedures have not been validated by interlaboratory trials.4 Reagents4.1 GeneralAll reagents and materials used shall be of recognized analytical grade the purity of which has to be testedregularly by the analysis of blank samples. Water shall be of at least grade 3 according to EN ISO 3696:1995.4.2 n-Hexane 1)4.3 Sodium sulfate, anhydrous4.4 Diethyl ether4.5 Stock standard solutionsn-hexane or isooctane may be used to prepare solutions of 2-cyclohexylcyclohexanone (5 µg/ml), and 2-dodecylcyclobutanone2) and 2-tetradecylcyclobutanone2) (100 µg/ml). Store at –20 °C.4.6 Working standard solutionsn-hexane or isooctane may be used to prepare solutions of 2-cyclohexylcyclohexanone (0,5 µg/ml) (internalstandard), 2-dodecylcyclobutanone and 2-tetradecylcyclobutanone (10 µg/ml). Store at –20 °C.4.7 Florisil®3), 150 µm to 250 µm (60 mesh to 100 mesh), pesticide residue analysis grade.Before use, activate the adsorbent by heating at 550 °C for at least 5 h or overnight. Cool in a desiccator. Keep wellsealed after cooling.Prepare deactivated Florisil® by adding 20 parts of water to 100 parts of adsorbent (m/m). Approximately 30 g ofactivated Florisil® is required to prepare sufficient deactivated adsorbent for each column. Ensure that thedeactivated Florisil® contains no lumps and that the powder flows freely. Leave to equilibrate overnight. Use withinone week.
1)n-Hexane was the solvent used to validate the method. However, it is also possible to use n-pentane on healthgrounds provided it can be shown to lead to the same result.2)Contact the National Standardization Organizations for the availability of reference standards3)Florisil® is an example for a suitable product available commercially. This information is given for the convenience ofusers of this standard and does not constitute an endorsement by CEN of this product.SIST EN 1785:2003

EN 1785:2003 (E)54.8 Nitrogen, for concentrating solutions4.9 Helium, as carrier gas5 Apparatus5.1 GeneralUsual laboratory apparatus and, in particular, the following:5.2 Electric blender5.3 Soxhlet apparatus, with suitable flask of e.g. 250 ml and extractor of e.g. 100 ml.5.4 Cellulose extraction thimbles, e. g. of length 80 mm to 100 mm, with an internal diameter of 30 mm.Extraction with n-hexane prior to use may be necessary.5.5 Cotton wool, non-absorbent, washed in n-hexane prior to use.5.6 Electric heating mantle or water-bath5.7 Chromatographic tube, made of glass, having a length of 300 mm and with an internal diameter of 20 mm,fitted with a frit, a polytetrafluoroethylene (PTFE) stopcock and a ground glass joint at the top.5.8 Separating funnel, or dropping funnel, e. g. of 250 ml, with a ground glass joint.5.9 Rotary evaporator, with evaporation flask and a water bath capable of being controlled at 45 °C (lowvacuum, approximately 25 kPa).5.10 Apparatus for concentration of solutions under nitrogen.5.11 Gas chromatograph (GC) glass vials5.12 Gas chromatograph (GC) linked to a mass spectrometer (MS).5.13 Capillary column, with suitable performance characteristics, see Annex A.SIST EN 1785:2003

EN 1785:2003 (E)65.14 Laboratory oven, capable of being maintained at 100° C6 Sampling techniqueWhen taking samples, give preference to those parts of the food which have a high fat content (e. g. chicken skin).Keep the sample in a sealable glass vessel or in a fat-free metal foil.7 Procedure7.1 Sample preparationCoarsely chop the samples of food and then, with the exception of Camembert, homogenize in an electric blender(5.2). Camembert should be cut into small cubes prior to extraction. For liquid whole egg, ensure that the sample isthoroughly mixed prior to sampling.7.2 Fat extractionWeigh 20 g of anhydrous sodium sulfate (4.3) and 20 g of well mixed homogenized sample into an extractionthimble (5.4), mix and plug with cotton wool (5.5). Extra sodium sulfate may be used if necessary. For foodstuffshaving a low fat content, it may be necessary to increase the size of the test sample and quantity of anhydroussodium sulfate accordingly. It is recommended that liquid egg is dried at 100 °C for 12 h prior to extraction. A thinfilm of egg partially dried (2 h at 100 °C) has given comparable results. Alternative drying procedures, e. g. freeze-drying, may be used provided recovery of 2-alkylcyclobutanones is checked (see 7.6).Pour 100 ml of n-hexane (4.2) into a suitable flask (5.3) and place extractor on top. Place extraction thimble in theextractor and add 40 ml of n-hexane. Place the flask on the heating mantle (5.6) and condenser on top of theextractor. Reflux and extract gently for 6 h. The solvent should siphon over four times in approximately 1 h.Remove the flask from the heat and dispose of the thimble and the n-hexane in the extractor. Transfer the lipidextract from the flask to a 100 ml-stoppered glass measuring cylinder and adjust the volume to 100 ml with moresolvent. Add 5 g to 10 g of anhydrous sodium sulfate, stopper, mix and leave overnight.Alternative fat extraction procedures may be used if they can be shown to lead to the same results.7.3 Preparation of the lipid extract7.3.1 Determination of lipid content – method IDry duplicate flasks for at least 4 h or overnight at 100 °C. Cool and weigh. Pipette an aliquot of lipid extract (7.2)into each flask and rotary evaporate (5.9) to dryness. Dry for at least 4 h or overnight at 100 °C and reweigh.Alternatively, to provide a more rapid measurement of lipid content, pipette an aliquot of lipid extract (7.2) into apre-weighed glass vial. Evaporate the solvent under a stream of nitrogen (5.10) to constant weight. Calculate thevolume of extract required to provide approximately 200 mg of lipid.7.3.2 Determination of lipid concent – method IIAlternatively to 7.3.1, concentrate the whole lipid extract (7.2) to a few millilitres (2 ml to 3 ml) using a rotaryevaporator (5.9). Transfer the concentrated lipid extract to a small (e. g. 10 ml) sealable pre-weighed glass vial. Drythe sample under a stream of nitrogen (5.10) to constant weight.7.4 Florisil® column chromatographyPrepare a Florisil® column (20 cm to 21 cm) using a chromatographic tube (5.7), deactivated Florisil® (4.7) and n-hexane (4.2). Allow the n-hexane level to drop to just above the top of the Florisil®.Take a volume of the extract (7.3.1) which provides approximately 200 mg of lipid and concentrate if necessary.The final volume should not exceed 5 ml. Alternatively, dissolve 200 mg of the lipid obtained in 7.3.2 in not morethan 5 ml of n-hexane.Apply the lipid extract quantitatively to the column (record exact weight of lipid applied) and allow the n-hexanelevel to drop to just above the top of the Florisil® and add 5 ml to 10 ml of n-hexane. Place the remaining n-hexane(150 ml in total) in a separating funnel (5.8) on top of the column, elute at 2 ml/min to 5 ml/min and collect theeluent in a suitable flask e. g. 250 ml.SIST EN 1785:2003

EN 1785:2003 (E)7When the funnel is empty (take care that the column does not run dry), change the collection flasks and elute with150 ml of 1 % diethyl ether (4.4) in n-hexane. Rotary evaporate (5.9) the 1 % diethyl ether fraction at 40 °C, usingminimum vacuum, t

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