1、Foodstuffs Determination of ethyl carbamate in stone fruit spirits, fruit marc spirits and other spirit drinks GC-MS methodBS EN 16852:2017BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16852 May 2017 ICS 67.160.1
2、0 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 eaux-de-vie de fruits noyaux, les eaux-de-vie de marc de fruits et les autr
3、es 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. CEN members are bound to comply with the CEN/CENELEC Internal Regulations whi
4、ch 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 application to the CEN-CENELEC Management Centre or to any CEN member. This Eu
5、ropean 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 Management Centre has the same status as the official versions. CEN members
6、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, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
7、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: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any
8、 form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16852:2017 ENational forewordThis British Standard is the UK implementation of EN 16852:2017.The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods.A list
9、of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.ISBN 978 0 580 80859 3ICS 67.160.10Compliance with a British Standard
10、cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 August 2017.Amendments/corrigenda issued since publicationDate Text affectedBRITISH STANDARDBS EN 16852:2017EUROPEAN STANDARD NORME EUROPENNE EUR
11、OPISCHE 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 eaux-de-vie de fruits noyaux, le
12、s 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. CEN members are bound to comply wi
13、th 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 application to the CEN-CENELEC Mana
14、gement 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 Management Centre has the same st
15、atus 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, Latvia, Lithuania, Luxembourg, Mal
16、ta, 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: Avenue Marnix 17, B-1000 Brussels
17、 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16852:2017 EBS EN 16852:2017EN 16852:2017 (E) 2 Contents Page European foreword . 3 1 Scope 4 2 Normative references 4 3 Principle . 4 4 Reagents . 4 5 Apparatus . 7 6 Procedure
18、. 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 BS EN 16852:2017EN 16852:2017 (E) 3 European foreword This document (EN 16852:2017)
19、 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 latest by November 2017, a
20、nd 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 rights. This document ha
21、s 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 associated with its use. It i
22、s 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 national standards org
23、anisations 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, Lithuania, Luxembourg
24、, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16852:2017EN 16852:2017 (E) 4 1 Scope This European Standard specifies a gas chromatographic method using mass spectrometric detection for the determi
25、nation 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 the instrument response was p
26、roven 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 are indispensable for its
27、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 3696:1987) 3 Principle Stone fruit
28、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). Sugared brandies, liqueurs and ot
29、her 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 transformed into EC under t
30、he 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 complying with grade 1 of EN ISO
31、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 Ethanol solution, volume fraction
32、 = 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 Eluant, mixture of one part p
33、er volume of cyclohexane and one part per volume of ethyl acetate. BS EN 16852:2017EN 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)4.8 Sodium chloride. 4.9 Ethyl ca
34、rbamate, 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 ethanol (4.1). 4.13 Ethyl carbamate w
35、orking 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 (4.13) into a 50 ml volumetric fl
36、ask 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 solution, (BC) = 40 mg/l. Pipet
37、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) into a 50 ml volumetric flask and
38、 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 results. BS EN 16852:2017EN 16852:2017
39、 (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 simplified method, dry extract 70 % vol.
40、: = 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 water added in ml. Uncertainties in a
41、lcoholic 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 pure diatomaceous earth based solid ph
42、ase (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 beaker with 2 ml of ethanol (4.1) and tran
43、sfer 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 20 ml of eluant (4.5) up to a total
44、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 bath temperature. NOTE If the samples be
45、come cloudy during the SPE clean-up procedure, the concentrated eluate can be filtered through a 0,45 m PTFE syringe filter. BS EN 16852:2017EN 16852:2017 (E) 9 7 Gas chromatography and mass spectrometry (GC-MS) 7.1 GC conditions The following conditions are examples for appropriate GC settings suit
46、able 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: 20 ml/min PTV programme: 50 C with 14,5
47、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 approximately at 22 min 7.2 MS conditions The fol
48、lowing 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 line: 240 C micro scans: 3 max. ion time:
49、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 listed 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 cal
