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1、DD ISO/TS 22304:2008 ICS 65.160 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW DRAFT FOR DEVELOPMENT Tobacco - Determination of tobacco specific nitrosamines - Method using alkaline dichloromethane extraction Licensed CopyChinese University of Hong Kong, 11/04/2009 09:06, Unc
2、ontrolled Copy, (c) BSI This Draft for Development was published under the authority of the Standards Policy and Strategy Committee on 31 March 2009 BSI 2009 ISBN 978 0 580 63992 0 Amendments/corrigenda issued since publication DateComments DD ISO/TS 22304:2008 National foreword This Draft for Devel
3、opment is the UK implementation of ISO/TS 22304:2008. This publication is not to be regarded as a British Standard. It is being issued in the Draft for Development series of publications and is of a provisional nature. It should be applied on this provisional basis, so that information and experienc
4、e of its practical application can be obtained. Comments arising from the use of this Draft for Development are requested so that UK experience can be reported to the international organization responsible for its conversion to an international standard. A review of this publication will be initiate
5、d not later than 3 years after its publication by the international organization so that a decision can be taken on its status. Notification of the start of the review period will be made in an announcement in the appropriate issue of Update Standards. According to the replies received by the end of
6、 the review period, the responsible BSI Committee will decide whether to support the conversion into an international Standard, to extend the life of the Technical Specification or to withdraw it. Comments should be sent to the Secretary of the responsible BSI Technical Committee at British Standard
7、s House, 389 Chiswick High Road, London W4 4AL. The UK participation in its preparation was entrusted to Technical Committee AW/40, Tobacco and tobacco products. A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not purport to in
8、clude all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard cannot confer immunity from legal obligations. Licensed CopyChinese University of Hong Kong, 11/04/2009 09:06, Uncontrolled Copy, (c) BSI DD ISO/TS 22304:2008 Refer
9、ence number ISO/TS 22304:2008(E) ISO 2008 TECHNICAL SPECIFICATION ISO/TS 22304 First edition 2008-07-15 Tobacco Determination of tobacco specific nitrosamines Method using alkaline dichloromethane extraction Tabac Dosage des nitrosamines spcifiques au tabac Mthode dextraction au dichloromthane alcal
10、in Licensed CopyChinese University of Hong Kong, 11/04/2009 09:06, Uncontrolled Copy, (c) BSI DD ISO/TS 22304:2008 ISO/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
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13、. In the unlikely event that a problem 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 mea
14、ns, electronic or mechanical, including 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 copyr
15、ightiso.org Web www.iso.org Published in Switzerland ii ISO 2008 All rights reserved Licensed CopyChinese University of Hong Kong, 11/04/2009 09:06, Uncontrolled Copy, (c) BSI DD ISO/TS 22304:2008 ISO/TS 22304:2008(E) ISO 2008 All rights reserved iii Contents Page Foreword iv Introduction v 1 Scope
16、. 1 2 Normative references. 1 3 Terms and definitions. 1 4 Principle. 1 5 Reagents 1 6 Apparatus 2 7 Preparation of solutions 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
17、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 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 Bi
18、bliography. 10 Licensed CopyChinese University of Hong Kong, 11/04/2009 09:06, Uncontrolled Copy, (c) BSI DD ISO/TS 22304:2008 ISO/TS 22304:2008(E) iv ISO 2008 All rights reserved Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
19、 (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 established has the right to be represented on that committee. International organizations,
20、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. International Standards are drafted in accordance with the rules given in the ISO/
21、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. Publication as an International Standard requires approval by at least 75 % of the me
22、mber 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 Available Specification (ISO/PAS) represents an agreement between technical experts in
23、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 the members of a technical committee and is accepted for publication if it is approv
24、ed 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 International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed, it is reviewed aga
25、in 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 subject of patent rights. ISO shall not be held responsible for identifying any or a
26、ll such patent rights. ISO/TS 22304 was prepared by Technical Committee ISO/TC 126, Tobacco and tobacco products. Licensed CopyChinese University of Hong Kong, 11/04/2009 09:06, Uncontrolled Copy, (c) BSI DD ISO/TS 22304:2008 ISO/TS 22304:2008(E) ISO 2008 All rights reserved v Introduction During th
27、e development 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 usi
28、ng buffer 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. Th
29、e method using buffer extraction is described in ISO 22303 (voir Reference 1). Licensed CopyChinese University of Hong Kong, 11/04/2009 09:06, Uncontrolled Copy, (c) BSI DD ISO/TS 22304:2008 Licensed CopyChinese University of Hong Kong, 11/04/2009 09:06, Uncontrolled Copy, (c) BSI DD ISO/TS 22304:20
30、08 TECHNICAL SPECIFICATION ISO/TS 22304:2008(E) ISO 2008 All rights reserved 1 Tobacco Determination of tobacco specific nitrosamines Method using alkaline dichloromethane extraction 1 Scope This Technical Specification specifies the procedure for the determination of the tobacco specific nitrosamin
31、es (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 and tobacco products. The determination is by means of gas chromatography. 2 Normative references The follo
32、wing 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 referenced document (including any amendments) applies. ISO 1042, Laboratory glassware One-mark volumetric flasks
33、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 specific nitrosamines TSNAs four nitrosamines found predominantly in tobacco: N-nitrosonornicotine (NNN), N-nitro
34、soanatabine (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 sodium hydroxide. The extract is eluted through a mixed bed of magnesium sulphate and sodium sulphate using
35、 dichloromethane, evaporated to near dryness and reconstituted in chloroform. The individual nitrosamines are then separated and quantified by gas chromatography with chemiluminescent detection. Quantification is performed by an internal standard technique. 5 Reagents Use only reagents of analytical
36、 reagent grade. SAFETY PRECAUTIONS Nitrosamines are suspected carcinogens; therefore, appropriate safety precautions should be taken when preparing standards. Always wear laboratory gloves when handling standard solutions and making dilutions. Licensed CopyChinese University of Hong Kong, 11/04/2009
37、 09:06, Uncontrolled Copy, (c) BSI DD ISO/TS 22304:2008 ISO/TS 22304:2008(E) 2 ISO 2008 All rights reserved 5.1 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), w W 98 %.
38、 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), anhydrous (CAS: 7
39、757-82-6), (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, (CAS: 7732-18-
40、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 laboratory apparatu
41、s 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 column of lengt
42、h 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 volumetric flasks, co
43、mplying with class A of ISO 1042. 6.7 Disposable glass transfer pipettes, length 229 mm. 1) CAS: Chemical Abstract Service. Licensed CopyChinese University of Hong Kong, 11/04/2009 09:06, Uncontrolled Copy, (c) BSI DD ISO/TS 22304:2008 ISO/TS 22304:2008(E) ISO 2008 All rights reserved 3 6.8 Glass ti
44、lting repeating dispensers, constant volume, 10 ml. 6.9 Glass extraction test tubes, 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
45、 for blank extraction column, outer diameter 1,4 cm, pore size 20 m. 6.14 Sample 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
46、 of sodium hydroxyde solution; w = 10 % (mass fraction) Weigh about 10 g of NaOH 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 dichloromethan
47、e (5.9) to yield a concentration of about 200 g ml1 NDHA. Further dilute this stock 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 no
48、t in use. 7.3 Preparation of calibration standards Table 1 lists typical concentrations 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 c
49、hloroform (g ml 1) Level NDHA NNN NAT NAB NNK 1 5,00 0,05 0,05 0,05 0,05 2 5,00 0,30 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. Licensed CopyChinese University of Hong Kong, 11/04/2009 09:06, Uncontrolled Copy, (c) BSI DD ISO/TS 22304:2008 ISO/TS 22304:2008(E) 4 ISO 200