1、DD ISO/TS22304:2008ICS 65.160NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWDRAFT FOR DEVELOPMENTTobacco -Determination oftobacco specificnitrosamines -Method using alkalinedichloromethaneextractionThis Draft for Developmentwas published under theauthority of the StandardsPoli
2、cy and StrategyCommittee on 31 March2009 BSI 2009ISBN 978 0 580 63992 0Amendments/corrigenda issued since publicationDate CommentsDD ISO/TS 22304:2008National forewordThis Draft for Development is the UK implementation of ISO/TS22304:2008.This publication is not to be regarded as a British Standard.
3、It is being issued in the Draft for Development series of publications andis of a provisional nature. It should be applied on this provisional basis,so that information and experience of its practical application can beobtained.Comments arising from the use of this Draft for Development arerequested
4、 so that UK experience can be reported to the internationalorganization responsible for its conversion to an international standard.A review of this publication will be initiated not later than 3 years afterits publication by the international organization so that a decision can betaken on its statu
5、s. Notification of the start of the review period will bemade in an announcement in the appropriate issue of Update Standards.According to the replies received by the end of the review period,the responsible BSI Committee will decide whether to support theconversion into an international Standard, t
6、o extend the life of theTechnical Specification or to withdraw it. Comments should be sent tothe Secretary of the responsible BSI Technical Committee at BritishStandards House, 389 Chiswick High Road, London W4 4AL.The UK participation in its preparation was entrusted to TechnicalCommittee AW/40, To
7、bacco and tobacco products.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a British Standard can
8、not confer immunityfrom legal obligations.DD ISO/TS 22304:2008Reference numberISO/TS 22304:2008(E)ISO 2008TECHNICAL SPECIFICATION ISO/TS22304First edition2008-07-15Tobacco Determination of tobacco specific nitrosamines Method using alkaline dichloromethane extraction Tabac Dosage des nitrosamines sp
9、cifiques au tabac Mthode dextraction au dichloromthane alcalin DD ISO/TS 22304:2008ISO/TS 22304:2008(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are e
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12、roblem relating to it is found, please inform the Central Secretariat at the address given below. COPYRIGHT PROTECTED DOCUMENT ISO 2008 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, in
13、cluding photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Publ
14、ished in Switzerland ii ISO 2008 All rights reservedDD ISO/TS 22304:2008ISO/TS 22304:2008(E) ISO 2008 All rights reserved iiiContents Page Foreword iv Introduction v 1 Scope . 1 2 Normative references . 1 3 Terms and definitions. 1 4 Principle. 1 5 Reagents 1 6 Apparatus 2 7 Preparation of solutions
15、 and standards. 3 7.1 Preparation of sodium hydroxyde solution; w = 10 % (mass fraction) . 3 7.2 Preparation of internal standard . 3 7.3 Preparation of calibration standards 3 7.4 Setting-up of gas chromatograph and detector 4 7.5 Tobacco extraction and isolation of TSNAs 5 7.6 Sample extraction. 5
16、 8 Data analysis and calculation of results 6 9 Repeatability and reproducibility 6 10 Test report . 7 Annex A (informative) Typical chromatograms and calibration curves . 8 Bibliography . 10 DD ISO/TS 22304:2008ISO/TS 22304:2008(E) iv ISO 2008 All rights reservedForeword ISO (the International Orga
17、nization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been establis
18、hed has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization
19、. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. P
20、ublication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. In other circumstances, particularly when there is an urgent market requirement for such documents, a technical committee may decide to publish other types of document: an ISO Publicly Ava
21、ilable Specification (ISO/PAS) represents an agreement between technical experts in an ISO working group and is accepted for publication if it is approved by more than 50 % of the members of the parent committee casting a vote; an ISO Technical Specification (ISO/TS) represents an agreement between
22、the members of a technical committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting a vote. An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a further three years, revised to become an Internation
23、al Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an International Standard or be withdrawn. Attention is drawn to the possibility that some of the elements of this document may be the
24、subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO/TS 22304 was prepared by Technical Committee ISO/TC 126, Tobacco and tobacco products. DD ISO/TS 22304:2008ISO/TS 22304:2008(E) ISO 2008 All rights reserved vIntroduction During the develop
25、ment of this Technical Specification, interlaboratory tests were carried out using two different methods for the determination of tobacco specific nitrosamines. These comprise the method discussed in this Technical Specification, using alkaline dichloromethane extraction, and the method using buffer
26、 extraction (see References 2, 3). These studies show that no differences occur between the results obtained by the two different methods (see Reference 4). The method described in this Technical Specification could be preferable for those preferring a faster analysis using less solvent. The method
27、using buffer extraction is described in ISO 22303 (voir Reference 1). DD ISO/TS 22304:2008DD ISO/TS 22304:2008TECHNICAL SPECIFICATION ISO/TS 22304:2008(E) ISO 2008 All rights reserved 1Tobacco Determination of tobacco specific nitrosamines Method using alkaline dichloromethane extraction 1 Scope Thi
28、s Technical Specification specifies the procedure for the determination of the tobacco specific nitrosamines (TSNAs): N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in ground leaf tobacco, manufactured tobacco a
29、nd tobacco products. The determination is by means of gas chromatography. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenc
30、ed document (including any amendments) applies. ISO 1042, Laboratory glassware One-mark volumetric flasks ISO 3696, Water for analytical laboratory Specification and test methods 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 tobacco specifi
31、c nitrosamines TSNAs four nitrosamines found predominantly in tobacco: N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) 4 Principle TSNAs are extracted from ground tobacco samples with dichloromethane containing s
32、odium hydroxide. The extract is eluted through a mixed bed of magnesium sulphate and sodium sulphate using dichloromethane,evaporated to near dryness and reconstituted in chloroform. The individual nitrosamines are then separated and quantified by gas chromatography with chemiluminescent detection.
33、Quantification is performed by an internal standard technique. 5 Reagents Use only reagents of analytical reagent grade. SAFETY PRECAUTIONS Nitrosamines are suspected carcinogens; therefore, appropriate safety precautions should be taken when preparing standards. Always wear laboratory gloves when h
34、andling standard solutions and making dilutions. DD ISO/TS 22304:2008ISO/TS 22304:2008(E) 2 ISO 2008 All rights reserved5.1 N-nitrosonornicotine, (NNN, CAS1): 53759-22-1), w W 98 % (mass fraction). 5.2 N-nitrosoanatabine, (NAT, CAS: 71267-22-6), w W 98 %. 5.3 N-nitrosoanabasine, (NAB, CAS: 1133-64-8
35、), w W 98 %. 5.4 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK, CAS: 64091-91-4), w W 98 %. 5.5 N-nitrosodi-n-hexylamine, (NDHA, CAS: 6949-28-6) (internal standard), w W 98 %. 5.6 Magnesium sulphate (MgSO4), anhydrous (CAS: 7487-88-9), (powder), w W 98,0 %. 5.7 Sodium sulphate (Na2SO4), anhyd
36、rous (CAS: 7757-82-6), (granular), w W 99,0 %, 2 mm (10 mesh) to 0,25 mm (60 mesh). 5.8 Sodium hydroxide (NaOH), pellets (CAS: 1310-73-2), w W 97,0 %. 5.9 Dichloromethane (CAS: 75-09-02), w W 99,9 %. 5.10 Chloroform (CAS: 67-66-3), w W 99,9 %. 5.11 Nicotine (CAS: 54-11-5), w W 99,0 %. 5.12 Water, (C
37、AS: 7732-18-5), complying with grade 2 of ISO 3696 or better. 5.13 Nitrogen, (CAS: 7727-37-9), for eluent evaporator, w W 99,995 %. 5.14 Helium, (CAS: 7440-59-7), for carrier gas, w W 99,995 %. 5.15 Oxygen, (CAS: 7782-44-7), for generating ozone in the detector, w W 99,6 %. 6 Apparatus Usual laborat
38、ory apparatus and, in particular, the following items. 6.1 Gas chromatograph (GC), with a chemiluminescence detector and autosampler (optional). 6.2 Eluent evaporator, for concentration of sample extract. 6.3 Mixer, vortex type, for sample extraction/mixing. 6.4 GC column, fused silica capillary col
39、umn of length 30 m and internal diameter 0,53 mm, coated with a 1,5 m film of 5 % phenyl methylpolysiloxane. NOTE Other columns can be used provided that a satisfactory separation is achieved. 6.5 Chromatography data acquisition system, for measuring peak areas electronically. 6.6 One-mark volumetri
40、c flasks, complying with class A of ISO 1042. 6.7 Disposable glass transfer pipettes, length 229 mm. 1) CAS: Chemical Abstract Service. DD ISO/TS 22304:2008ISO/TS 22304:2008(E) ISO 2008 All rights reserved 36.8 Glass tilting repeating dispensers, constant volume, 10 ml. 6.9 Glass extraction test tub
41、es, 20 mm ID 150 mm. 6.10 Test tube caps for glass extraction test tubes, 18 thread to 415 thread. 6.11 Glass concentration test tubes, 16 mm ID 100 mm. 6.12 Blank extraction column, reservoir capacity 14 ml. 6.13 Frits for blank extraction column, outer diameter 1,4 cm, pore size 20 m. 6.14 Sample
42、containers, borosilicate glass autosampler vials, capacity 2 ml, with PTFE-lined septum screwcap closures. 6.15 Refrigerating unit, for storing standards at 20 C. 7 Preparation of solutions and standards 7.1 Preparation of sodium hydroxyde solution; w = 10 % (mass fraction) Weigh about 10 g of NaOH
43、pellets (5.8) in a 100 ml beaker. Add water to a total mass of 100 g and stir until dissolved. 7.2 Preparation of internal standard Prepare the internal standard stock solution by dissolving NDHA (5.5) in dichloromethane (5.9) to yield a concentration of about 200 g ml1 NDHA. Further dilute this sto
44、ck solution such that the concentration of NDHA is 0,40 g ml1. This diluted solution in dichloromethane is used as part of the extractant (see Table 1). Store all solutions of the internal standard at about 20 C when not in use. 7.3 Preparation of calibration standards Table 1 lists typical concentr
45、ations of standards to be used for the analysis of tobacco.When not in use, store standard solutions at about 20 C. At least four levels of the TSNAs should be used for calibration. Table 1 Standard concentrations in chloroform (gml1) Level NDHA NNN NAT NAB NNK 1 5,00 0,05 0,05 0,05 0,05 2 5,00 0,30
46、 0,30 0,15 0,15 3 5,00 1,50 1,50 0,30 2,88 4 5,00 5,00 5,00 0,50 5,00 5 5,00 8,50 9,00 0,75 7,20 NOTE Depending on the final volume of the standard, 600 l of nicotine should be added per 10 ml of chloroform, e.g. a 50 ml volume of standard should contain 3 ml of nicotine. DD ISO/TS 22304:2008ISO/TS
47、22304:2008(E) 4 ISO 2008 All rights reserved7.4 Setting-up of gas chromatograph and detector The GC (6.1) is set up for splitless injections and configured to operate with a chemiluminescence detector. A 4 mm internal diameter tube with a glass wool split liner is used in the split/splitless injecti
48、on port. The GC column is connected to the ceramic pyrolyser tube of the chemiluminescence detector. Approximately 13 cm of the capillary GC column are inserted into the pyrolyser. Table 2 lists typical temperatures and ramp rates used to separate the TSNAs. Table 3 gives recommended settings for th
49、e analysis. These conditions may be varied at the analysts discretion to achieve better resolution, decrease run time or improve detection limit. The injection liner and septum should be changed after each autosampler run (about 100 injections), followed by two solvent (chloroform) injection runs. Figure A.1 is a chromatogram of a standard analysed under the conditions given in Tables 2 and 3. Table 2 GC oven programme Column oven initial temperature (C) 100 Column oven initial time (min) 1,00
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