1、July 2017 English price group 11No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.160.10!%h1“2691493www.din.deDIN
2、EN 16852Foodstuffs Determination of ethyl carbamate in stone fruit spirits, fruit marc spirits and other spirit drinks GCMS method;English version EN 16852:2017,English translation of DIN EN 16852:2017-07Lebensmittel Bestimmung von Ethylcarbamat in Steinobstbrnden, Obstbrnden und anderen Spirituosen
3、 GCMSVerfahren;Englische Fassung EN 16852:2017,Englische bersetzung von DIN EN 16852:2017-07Produits alimentaires Dtermination de la teneur en carbamate dthyle dans les eauxdevie de fruits noyaux, les eauxdevie de marc de fruits et les autres boissons alcoolises Mthode par CGSM;Version anglaise EN 1
4、6852:2017,Traduction anglaise de DIN EN 16852:2017-07www.beuth.deDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritative.Document comprises 19 pages 06.17 2A comma is used as the decimal marker. National foreword This document (EN 16852:201
5、7) has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products
6、), Working Committee NA 057-01-14 AA “Process contaminants”. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 5725-2 DIN ISO 5725-2 ISO/IEC 17025 DIN EN ISO/IEC 17025 National Annex NA (informative) Bibliography DIN EN ISO/IEC 17025, Gen
7、eral requirements for the competence of testing and calibration laboratories DIN ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method DIN EN 16852:2017-07 EURO
8、PEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16852 May 2017 ICS 67.160.10 English Version Foodstuffs - Determination of ethyl carbamate in stone fruit spirits, fruit marc spirits and other spirit drinks - GC-MS method Produits alimentaires - Dtermination de la teneur en carbamate dthyle dans les
9、 eaux-de-vie de fruits noyaux, les eaux-de-vie de marc de fruits et les autres boissons alcoolises - Mthode par CG-SM Lebensmittel - Bestimmung von Ethylcarbamat in Steinobstbrnden, Obstbrnden und anderen Spirituosen - GC-MS-Verfahren This European Standard was approved by CEN on 20 February 2017. C
10、EN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on ap
11、plication to the CEN-CENELEC 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 translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
12、Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, L
13、atvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre:
14、Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16852:2017 EEN 16852:2017 (E) 2 Contents Page European foreword . 3 1 Scope 4 2 Normative references 4 3 Principle . 4 4 Reagents . 4 5 Apparatu
15、s . 7 6 Procedure. 8 7 Gas chromatography and mass spectrometry (GC-MS) . 9 8 Calculation . 9 9 Precision 10 10 Test report 11 Annex A (informative) Typical chromatograms 12 Annex B (informative) Precision data . 16 Bibliography . 17 DIN EN 16852:2017-07 EN 16852:2017 (E) 3 European foreword This do
16、cument (EN 16852:2017) 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 late
17、st by November 2017, and conflicting national standards shall be withdrawn at the latest by November 2017. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN shall not be held responsible for identifying any or all such patent ri
18、ghts. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. WARNING The use of this standard can involve hazardous materials, operations and equipment. This document does not purport to address all the safety problems associa
19、ted with its use. It is the responsibility of the user of this standard to take appropriate measures for ensuring the safety and health of the personnel prior to application of the standard and to fulfil statutory requirements for this purpose. According to the CEN-CENELEC Internal Regulations, the
20、national standards organisations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
21、 Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN EN 16852:2017-07 EN 16852:2017 (E) 4 1 Scope This European Standard specifies a gas chromatographic method using mass spectrometri
22、c detection for the determination of ethyl carbamate (EC) in stone fruit spirits, fruit marc spirits and other spirit drinks. The method has been validated in an interlaboratory study for stone fruit spirits and fruit liqueurs, at levels ranging from 0,253 mg/l to 1,11 mg/l. However, linearity of th
23、e instrument response was proven for the concentration ranges 0,10 mg/l to 4,0 mg/l (simplified method) and 0,025 mg/l to 3,0 mg/l (procedure including sample clean-up), respectively. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and
24、 are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use - Specification and test methods (ISO 3696:198
25、7) 3 Principle Stone fruit spirits with a content of total dry extract of less than 10 g/l are injected directly into the gas chromatography mass spectrometry (GC-MS) system after the adjustment of the alcoholic strength of the beverage when indicated and addition of internal standard (ISTD). Sugare
26、d brandies, liqueurs and other spirit drinks with a higher total dry extract are first transferred onto a solid phase extraction (SPE) cartridge and the ethyl carbamate is eluted with a mixture of cyclohexane and ethyl acetate. Stone fruit spirits can contain precursors of ethyl carbamate which get
27、transformed into EC under the influence of sunlight, e.g. during the shelf life of a spirit. To obtain the actual content of EC in the sample, light-protected glass ware (e.g. brown glass) shall be used during the analysis. 4 Reagents Use only reagents of recognized analytical grade and water comply
28、ing with grade 1 of EN ISO 3696:1995, unless otherwise specified. Solvents shall be of quality for HPLC (High Performance Liquid Chromatography) analysis. Ethyl carbamate has been classified by IARC as probably carcinogenic to humans (see 1). 4.1 Ethanol absolute. 4.2 Ethanol solutions. 4.2.1 Ethano
29、l solution, volume fraction = 65 %. Pipet 65 ml of ethanol (4.1) into a 100 ml volumetric flask and dilute to the mark with water. 4.2.2 Ethanol solution, = 35 %. Pipet 35 ml of ethanol (4.1) into a 100 ml volumetric flask and dilute to the mark with water. 4.3 Cyclohexane. 4.4 Ethyl acetate. 4.5 El
30、uant, mixture of one part per volume of cyclohexane and one part per volume of ethyl acetate. DIN EN 16852:2017-07 EN 16852:2017 (E) 5 4.6 n-Pentane. 4.7 Solid phase material. Approximately 20 g of a chemically inert, wide-pore and highly pure diatomaceous earth based solid phase, e.g. ExtrelutNT1)4
31、.8 Sodium chloride. 4.9 Ethyl carbamate, 99 %. 4.10 Butyl carbamate. 4.11 d5-Ethyl carbamate, 99 %. 4.12 Ethyl carbamate stock solution, mass concentration (EC) = 1 000 mg/l. Weigh, to the nearest 0,01 mg, 50 mg of ethyl carbamate (4.9) into a 50 ml volumetric flask and dilute to the mark with ethan
32、ol (4.1). 4.13 Ethyl carbamate working solution 1, (EC) = 100 mg/l. Pipet 5 ml of ethyl carbamate stock solution (4.12) into a 50 ml volumetric flask and dilute to the mark with ethanol (4.1). 4.14 Ethyl carbamate working solution 2, (EC) = 10 mg/l. Pipet 5 ml of ethyl carbamate working solution 1 (
33、4.13) into a 50 ml volumetric flask and dilute to the mark with ethanol (4.1). 4.15 Butyl carbamate stock solution, (BC) = 1 000 mg/l. Weigh, to the nearest 0,01 mg, 50 mg of butyl carbamate (4.10) into a 50 ml volumetric flask and dilute to the mark with ethanol (4.1). 4.16 Butyl carbamate working
34、solution, (BC) = 40 mg/l. Pipet 2 ml of butyl carbamate stock solution (4.15) into a 50 ml volumetric flask. Add 33 ml of ethanol (4.1) and dilute to the mark with water. 4.17 d5-Ethyl carbamate stock solution, (d5-EC) = 1 000 mg/l. Weigh, to the nearest 0,01 mg, 50 mg of d5-ethyl carbamate (4.11) i
35、nto a 50 ml volumetric flask and dilute to the mark with ethanol (4.1). 1) This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of these products. Equivalent products may be used if they can be shown to lead to the same result
36、s. DIN EN 16852:2017-07 EN 16852:2017 (E) 6 4.18 d5-Ethyl carbamate working solution, (d5-EC) = 40 mg/l. Pipet 2 ml of d5-ethyl carbamate stock solution (4.17) into a 50 ml volumetric flask. Add 33 ml of ethanol (4.1) and dilute to the mark with water. 4.19 Ethyl carbamate standard solutions for sim
37、plified method, dry extract 70 % vol.: = 107yw (1) alcoholic strength 40 % vol.: = 40010wy(3) alcoholic strength 40 % vol.: ( )( )=400 10100yey(4) where y is the original alcoholic strength of the beverage in % vol. (volume fraction); e is the amount of ethanol (4.1) added in ml; w is the amount of
38、water added in ml. Uncertainties in alcoholic strength resulting from volume contraction when mixing alcohol with water do not have a significant influence on the measured amount of EC. 6.2 Preparation of samples for method using sample clean up Mix 20 g of a chemically inert, wide-pore and highly p
39、ure diatomaceous earth based solid phase (4.7) with 10 g of sodium chloride (4.8) and transfer into a solid phase extraction column. Mix 20 ml of sample with 100 l of d5-ethyl carbamate working solution (4.18) in a beaker. Transfer the sample onto the solid phase extraction material, rinse the beake
40、r with 2 ml of ethanol (4.1) and transfer the solution onto the solid phase extraction material. Allow the sample to permeate into the material for 15 min. Wash the extraction material with 2 20 ml of n-pentane (4.6), discard the eluate. Elute the ethyl carbamate in several portions of approximately
41、 20 ml of eluant (4.5) up to a total of 90 ml. Concentrate the eluate to approximately 1 ml and transfer into a GC vial. When using a rotary evaporator for the concentration of the eluate, the following conditions have proven to be suitable: 25 kPa to 30 kPa (250 mbar to 300 mbar) and 40 C water bat
42、h temperature. NOTE If the samples become cloudy during the SPE clean-up procedure, the concentrated eluate can be filtered through a 0,45 m PTFE syringe filter. DIN EN 16852:2017-07 EN 16852:2017 (E) 9 7 Gas chromatography and mass spectrometry (GC-MS) 7.1 GC conditions The following conditions are
43、 examples for appropriate GC settings suitable for analysing ethyl carbamate using a PTV injector. As stated in 5.4, a SSL injector may also be used. Column: DB-WAX, 30 m 0,25 mm internal diameter 0,25 m film thickness (see 5.5) Injection progamme: PTV, splitless Splitless time: 1,00 min, split flow
44、: 20 ml/min PTV programme: 50 C with 14,5 C/s to 250 C, hold for 5 min. Carrier gas: Helium, 1,2 ml/min constant flow Injection volume: 1 l Oven programme: 50 C (1 min) with 5 C/min to 150 C, and with 20 C/min to 220 C, hold for 10,5 min Retention times: EC, d5-EC approximately at 18 min, BC approxi
45、mately at 22 min 7.2 MS conditions The following conditions are an example for appropriate MS settings suitable for analysing ethyl carbamate. Ionization: Electron impact, positive mode (EI+), single ion monitoring (SIM) Source temperature: 230 C Electron ionization energy: 70 eV Ion trap: Transfer
46、line: 240 C micro scans: 3 max. ion time: 20 ms mass defect: 0 Quadrupole: transfer line: 250 C dwell time: 100 ms Selected ions: m/z 44 and m/z 62 for EC and BC m/z 64 for d5-EC Examples for typical chromatograms are given in Annex A. 8 Calculation Inject the ethyl carbamate standard solutions list
47、ed in 4.19 or 4.20, respectively, at the beginning of every sequence. Bring all solutions to room temperature before injection. Inject the standards from the lower to the higher mass concentration to reduce the risk of cross contamination. Construct a calibration graph using the analytical software
48、of the respective GC-MS system or other suitable calculating programme (e.g. spreadsheet) by plotting the peak area ratios of EC and ISTD against the mass concentration of EC in the standard solutions. Calculate the mass concentration of EC in the samples using the obtained calibration function. Exp
49、ress the mass concentration with two significant figures, expressed usually in mg/l. If the peak area ratios of the sample lie outside of the calibrated range, dilute the samples appropriately. DIN EN 16852:2017-07 EN 16852:2017 (E) 10 9 Precision 9.1 General Details of the interlaboratory test of the precision of the method are summarized in Annex B. The values derived from the interlaboratory test may not be applicable
