EN 14573:2004

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von 3-Monochlorpropan-1,2-diol mit GC/MSProduits alimentaires - Dosage du 3-monochloropropane-1,2-diol par CG/SMFoodstuffs - Determination of 3-monochloropropane-1,2-diol by GC/MS67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 14573:2004SIST EN 14573:2005en01-januar-2005SIST EN 14573:2005SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14573October 2004ICS 67.050 English versionFoodstuffs - Determination of 3-monochloropropane-1,2-diol byGC/MSProduits alimentaires - Dosage du 3-monochloropropane-1,2-diol par CG/SMLebensmittel - Bestimmung von 3-Monochlorpropan-1,2-diol mit GC/MSThis European Standard was approved by CEN on 29 July 2004.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 Central Secretariat 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 Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2004 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14573:2004: ESIST EN 14573:2005

Example of appropriate GC/MS operating conditions.10 Annex B (informative)
Precision data.13 Bibliography.14
2 Principle The sample is mixed with a deuterated internal standard, with sodium chloride solution and with a solid support material. The mixture is transferred to a chromatographic column and is first extracted with a mixture of n-hexane and diethyl ether for removing non-polar components. Then, 3-MCPD is eluted with diethyl ether, the eluate is concentrated and an aliquot portion is derivatized using heptafluorobutyrylimidazole. The solution is analyzed by gas chromatography using mass spectrometric detection [2]. 3 Reagents 3.1 General Unless otherwise specified, use reagents of recognized analytical quality and distilled or demineralized water. Take every precaution to avoid possible contamination of water, solvents, inorganic salts etc. by plastics and rubber materials. Use only glass containers for storage and handling of all water and reagents. WARNING — The use of this standard may involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 3.2 n-Hexane, glass distilled 3.3 Diethyl ether, glass distilled 3.4 2,2,4-Trimethylpentane 3.5 Ethyl acetate 3.6 Solvent mixture, n-hexane (3.2) and diethyl ether (3.3) 9+1 (V/V) 3.7 Sodium chloride solution, 290 g of sodium chloride dissolved in 1 l of water 3.8 Sodium sulfate, anhydrous 3.9 Heptafluorobutyrylimidazole 3.10 Solid support material, Extrelut® 1, refill packs, approx. 20 g
Extrelut® is the trade name of a suitable product available commercially from E. Merck, Darmstadt (Germany). This information is given for the convenience of the users of this European Standard and does not constitute an endorsement by CEN of this product.
Dilute 10 ml of the 3-MCPD stock solution (3.13) to the mark with ethyl acetate (3.5) in a 100 ml volumetric flask. From this dilution, transfer volumes of each 0 µl, 12,5 µl, 25 µl, 125 µl, 250 µl and 500 µl into 25 ml volumetric flasks and dilute to the mark with 2,2,4-trimethylpentane (3.4) to give 0 µg/ml, 0,05 µg/ml, 0,10 µg/ml, 0,50 µg/ml, 1,0 µg/ml, and 2,0 µg/ml 3-MCPD. 3.15 d5-3-MCPD stock solution, (d5-3-MCPD) = 1 mg/ml Weigh 25 mg of d5-3-MCPD (3.12) and dilute to the mark with ethyl acetate (3.5) in a 25 ml volumetric flask. 3.16 d5-3-MCPD internal standard solution, (d5-3-MCPD) = 10 µg/ml Dilute 1,0 ml of the d5-3-MCPD stock solution (3.15) to the mark with ethyl acetate (3.5) in a 100 ml volumetric flask. 3.17 Nitrogen 4 Apparatus 4.1 General Use usual laboratory equipment and, in particular, the following. 4.2 Ultrasonic bath 4.3 Vortex shaker 4.4 High-speed laboratory blender 4.5 Centrifuge, capable to be spun at a rotational frequency of at least 3500 min-1, with centrifuge tubes of capacity 100 ml. 4.6 Filter paper, fast flow rate 4.7 Chromatographic column, 2 cm internal diameter, 40 cm long, with a sintered glass disk and tap. 4.8 Rotary evaporator, with a water bath and with evaporation flasks of capacity 250 ml. 4.9 Syringe, 1 ml, gas-tight SIST EN 14573:2005

If analysis cannot proceed immediately, store all samples in air-tight containers, frozen if necessary. 5.2 Extraction 5.2.1 Hydrolyzed vegetable protein, soy sauce, soup powder and malt extract Weigh 5 g of soup powder, 8 g of hydrolyzed vegetable protein or soy sauce or 10 g of malt extract each to the nearest 0,01 g and add 100 µl of the d5-3-MCPD internal standard solution (3.16). Add sodium chloride solution (3.7) to give a total weight (sample plus sodium chloride solution) of 20 g. Blend all components to obtain a homogenous mixture, using a spatula for crushing all small lumps. Allow the mixture to stand for 10 min in an ultrasonic bath (4.2). 5.2.2 Flour, starch, cereals and bread Weigh 10 g of the sample to the nearest 0,01 g and add 100 µl of the d5-3-MCPD internal standard solution (3.16). Add sodium chloride solution (3.7) to give a total weight (sample plus sodium chloride solution) of 40 g. Blend all components to obtain a homogenous mixture, using a spatula for crushing all small lumps. Allow the mixture to stand for 10 min in an ultrasonic bath (4.2). Cover the mixture with a watch glass and leave it to soak overnight. 5.2.3 Salami and cheese Weigh 20 g of the sample to the nearest 0,01 g and add 100 µl of the d5-3-MCPD internal standard solution (3.16). Add sodium chloride solution (3.7) to give a total weight (sample plus sodium chloride solution) of 70 g. Blend all components to obtain a homogenous mixture, using a further 10 g of sodium chloride solution if necessary. Transfer the mixture to a centrifuge tube (4.5) and centrifuge at 3500 min-1 for 20 min. Decant the supernatant layer into a beaker, avoiding the transfer of solid material and visible fat. Weigh a 20 g portion of the supernatant into a 250 ml beaker. 5.2.4 Cream, butter, margarine and other yellow fats Weigh 20 g of the sample to the nearest 0,01 g and add 100 µl of the d5-3-MCPD internal standard solution (3.16). Add sodium chloride solution (3.7) to give a total weight (sample plus sodium chloride solution) of 50 g. Heat the mixture at 45 °C until the fat has melted. Blend it for 2 min in a high-speed blender (4.4) and allow it to stand in a refrigerator for 1 h or until the fat layer has solidified. Decant the supernatant sodium chloride solution and weigh a 20 g portion of the supernatant into a 250 ml beaker. 5.3 Column chromatography Take 20 g of the mixture derived from 5.2.1 or 5.2.2 or the solution derived from 5.2.3 or 5.2.4, add the contents of an Extrelut refill pack (3.10) and mix all components thoroughly with a spatula. Add the mixture to the chromatographic column (4.7), agitate the column filling by hand briefly to compact the contents, top it with SIST EN 14573:2005

Extract non-polar components with 80 ml of solvent mixture (3.6) with unrestricted flow, except for powdered soup samples where the flow is restricted to approximately 8 ml/min to 10 ml/min. Close the tap of the column when the solvent mixture reaches the sodium sulfate layer and discard the collected washings. Next, elute the column with 250 ml diethyl ether (3.3) at a flow rate of approximately 8 ml/min and collect the eluate in a 250 ml volumetric flask. Dilute the eluate to the mark with diethyl ether, add 15 g sodium sulfate (3.8) to the flask and allow the mixture to stand for 10 min to 15 min.
Filter the dried solution through a filter paper (4.6) into a 250 ml round-bottomed flask. Concentrate the filtrate to approximately 5 ml in a rotary evaporator (4.8) with approximately 35 °C water bath temperature. Do not allow the solution to run dry. Transfer the concentrate to a 10 ml volumetric flask, rinsing the round-bottomed flask with diethyl ether, and dilute the solution up to the mark with diethyl ether. Add a small quantity (spatula tip) of sodium sulfate, shake well and allow to stand for 5 min to 10 min.
5.4 Derivatization 5.4.1 Derivatization of the sample solution Using a gas-tight syringe (4.9), transfer 1 ml of the solution derived from 5.3 to a 4 ml vial (4.11) and evaporate it just to dryness using a gentle stream of nitrogen (3.17). NOTE It has been shown by some users of the method that the use of 2,2,4-trimethylpentane (3.4) as a keeper can avoid losses. To the residue, immediately add 1 ml of 2,2,4-trimethylpentane (3.4) and 0,05 ml of heptafluorobutyrylimidazole (3.9) and seal the vial. Shake the vial for a few seconds using a vortex shaker (4.3) and heat it for 20 min in an aluminium block heater (4.10) at 70 °C. Allow the mixture to cool to below 40 °C. Add 1 ml of distilled water, shake the vial on a vortex shaker for 30 s, allow the phases to separate and repeat shaking. Remove the upper 2,2,4-trimethylpentane phase to a 2 ml vial (4.11), add a small quantity (spatula tip) of sodium sulfate, shake, and allow to stand for 2 min to 5 min. Transfer the solution to a new vial (2 ml) for gas-chromatographic analysis. 5.4.2 Derivatization of standard solutions
To a set of six 4-ml vials (4.11), transfer 100 µl each of the 3-MCPD standard solutions (3.14), 10 µl of the d5-3-MCPD internal standard solution (3.16) and 0,9 ml of 2,2,4-trimethylpentane (3.4). Add 0,05 ml of heptafluorobutyrylimidazole and seal the vial. Shake the vial for a few seconds using a vortex shaker (4.3) and heat it for 20 min in an aluminium block heater (4.10) at 70 °C. Allow the mixture to cool
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