1、BS ISO 19290:2016Cigarettes Determination oftobacco specific nitrosamines inmainstream cigarette smoke Method using LC-MS/MSBSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS ISO 19290:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementati
2、on of ISO 19290:2016.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 include all the necessary provi
3、sions of a contract. Users are responsible for its correct application. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 88412 2 ICS 65.160 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published
4、 under the authority of the Standards Policy and Strategy Committee on 31 December 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 19290:2016 ISO 2016Cigarettes Determination of tobacco specific nitrosamines in mainstream cigarette smoke Method using LC-MS/MSCig
5、arettes Dosage des nitrosamines spcifiques du tabac dans le courant principal de fume de cigarette Mthode par CL-SM/SMINTERNATIONAL STANDARDISO19290First edition2016-12-01Reference numberISO 19290:2016(E)BS ISO 19290:2016ISO 19290:2016(E)ii ISO 2016 All rights reservedCOPYRIGHT PROTECTED DOCUMENT IS
6、O 2016, Published in SwitzerlandAll rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permiss
7、ion. Permission can be requested from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 19290:2016ISO 19290:2
8、016(E)Foreword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 25 Apparatus . 26 Reagents 27 Preparation 37.1 Preparation of glassware 37.2 Preparation of solutions . 37.3 Preparation of standards 47.3.1 General 47.3.2 Preparation of internal standard soluti
9、ons 47.3.3 Preparation of calibration standard solutions . 48 Sampling 69 Tobacco product preparation . 610 Sample generation Smoking of cigarettes 610.1 General . 610.2 Linear smoking . 610.3 Rotary smoking 611 Sample analysis 611.1 Preparation of sample. 611.1.1 General 611.1.2 Extraction for line
10、ar smoking (44 mm pad) 611.1.3 Extraction for rotary smoking (92 mm pad) . 611.1.4 Final sample preparation . 611.2 Reversed phase high performance liquid chromatography 711.2.1 General 711.2.2 HPLC set-up parameters (Example) . 711.2.3 Mobile phase (Example) 711.2.4 Mobile phase: Gradient (Example)
11、 . 711.2.5 MS/MS Set-up parameters (Example) 711.3 Calculation 811.3.1 Calibration curve . 811.3.2 Determination of the TSNAs concentrations . 911.3.3 Sample quantification 911.3.4 Determination of mainstream smoke TSNA deliveries 912 Repeatability and reproducibility 913 Test report 11Annex A (info
12、rmative) Example of typical chromatograms .12Bibliography .14 ISO 2016 All rights reserved iiiContents PageBS ISO 19290:2016ISO 19290:2016(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of prepar
13、ing 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, governmental and non-governmental, in li
14、aison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Di
15、rectives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the possibility that
16、some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent decl
17、arations received (see www.iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information a
18、bout ISOs adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.The committee responsible for this document is ISO/TC 126, Tobacco and tobacco products.iv ISO 2016 All rights reservedBS ISO 19290:2016I
19、SO 19290:2016(E)IntroductionBetween 1999 and 2005, the CORESTA (www.coresta.org) Special Analytes Task Force studied the existing methodologies for the determination of Tobacco Specific Nitrosamines (TSNAs) in the mainstream smoke of cigarettes. Two main types of analytical methodologies had been pr
20、oposed for this determination: GC-TEA (gas chromatography with a thermal energy analyser) and LC-MS/MS (liquid chromatography- tandem mass spectrometry). The Task Force decided in the first instance to develop a method using GC-TEA, because this methodology was the most widely used in laboratories a
21、t that time.However by 2009, it was ascertained that most laboratories applied an LC-MS/MS technique to measure yields of TSNAs. The Sub-Group (changed from Task Force) then investigated an LC-MS/MS method to complement the GC-TEA technique already available as CORESTA Recommended Method N 63. Sever
22、al such methods have been described in the literature and are referenced herein. A joint experiment was carried out in which 14 laboratories participated, using their in-house LC-MS/MS methodologies. The reproducibility data was better for LC-MS/MS than for GC-TEA and methodology was very similar ac
23、ross laboratories. In summary, cigarette mainstream smoke was collected on a Cambridge filter (CF) pad, an internal standard solution was added and, after extraction, an aliquot was separated and quantitatively analysed by LC-MS/MS. A general methodology was agreed, incorporating key learnings from
24、the joint experiment.This document was produced through a final collaborative experiment involving 20 laboratories from 12 countries. The method includes some notes to inform other laboratories that might wish to adopt it about some of the main features that need to be well controlled to provide dat
25、a as robust and consistent as the repeatability and reproducibility data provided. Cigarettes were smoked using the smoking regime parameters given in ISO 3308 and statistical evaluations were made according to ISO 5725 recommendations.No machine smoking regime can represent all human smoking behavi
26、our: it is recommended that cigarettes also be tested under conditions of a different intensity of machine smoking than those specified in this document; machine smoking testing is useful to characterize cigarette emissions for design and regulatory purposes, but communication of machine measurement
27、s to smokers can result in misunderstandings about differences in exposure and risk across brands; smoke emission data from machine measurements may be used as inputs for product hazard assessment, but they are not intended to be nor are they valid as measures of human exposure or risks. Communicati
28、ng differences between products in machine measurements as differences in exposure or risk is a misuse of testing using ISO standards. ISO 2016 All rights reserved vBS ISO 19290:2016BS ISO 19290:2016Cigarettes Determination of tobacco specific nitrosamines in mainstream cigarette smoke Method using
29、LC-MS/MSWARNING The use of this document can involve hazardous materials, operations and equipment. This document does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this document to establish appropriate safety and health practices an
30、d determine the applicability of regulatory limitations prior to use.1 ScopeThis document specifies a method for the quantification of four tobacco specific nitrosamines (TSNAs) in the total particulate matter of mainstream cigarette smoke by using reversed phase high performance liquid chromatograp
31、hy with tandem mass spectrometry (LC-MS/MS). The quantified TSNAs are: N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).2 Normative referencesThe following documents are referred to in the text in such a way that
32、some or all of their content constitutes requirements 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 3308, Routine analytical cigarette-smoking machine Definitions an
33、d standard conditionsISO 3402, Tobacco and tobacco products Atmosphere for conditioning and testingISO 4387, Cigarettes Determination of total and nicotine-free dry particulate matter using a routine analytical smoking machineISO 8243, Cigarettes Sampling3 Terms and definitionsFor the purposes of th
34、is document, the following terms and definitions apply.ISO and IEC maintain terminological databases for use in standardization at the following addresses: IEC Electropedia: available at http:/www.electropedia.org/ ISO Online browsing platform: available at http:/www.iso.org/obp3.1tobacco specific n
35、itrosaminesTSNAsfour nitrosamines found predominantly in tobacco: N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)SOURCE: ISO 22303:2008, 3.1INTERNATIONAL STANDARD ISO 19290:2016(E) ISO 2016 All rights reserved 1B
36、S ISO 19290:2016ISO 19290:2016(E)4 PrincipleCigarettes are smoked on a standard smoking machine. The mainstream smoke is trapped on a glass-fibre filter pad. After addition of an internal standard, the total particulate matter collected on the glass-fibre filter pad is extracted with 100 mM ammonium
37、 acetate solution using a shaker.The extract is syringe filtered through a 0,45 m PTFE column directly into an auto sampler vial.The samples are subjected to reversed phase high performance liquid chromatography (HPLC) and quantified via tandem mass spectrometry (MS/MS).5 ApparatusUsual laboratory a
38、pparatus and equipment for use in preparation of samples and standards and in particular the following items. All glassware shall be cleaned before use to avoid any contamination.5.1 Equipment needed to perform conditioning of cigarettes, in accordance with ISO 3402.5.2 Equipment needed to perform m
39、arking for butt length of cigarettes.5.3 Equipment needed to perform smoking of cigarettes, complying with ISO 3308.5.4 Analytical balance, capable of measuring to at least four decimal places.5.5 Centrifuge tubes, 50 ml.5.6 Dispenser, of capacity 20 ml for extracting solutions.5.7 Gas-tight syringe
40、s, of capacity 250 l.5.8 Automated volumetric pipette.5.9 Shaker.5.10 High performance liquid chromatograph coupled to tandem mass spectrometer (LC-MS/MS), consisting of:5.10.1 Binary pump.5.10.2 Autosampler.5.10.3 Tandem mass spectrometer.5.10.4 Data collection system.5.10.5 LC column: XTerra MS C1
41、81), 2,5 m, 2,1 mm 50 mm or equivalent.6 ReagentsUse only reagents of recognized analytical reagent grade.1) XTerra MS C18 is an example of a suitable product available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO
42、 of this product.2 ISO 2016 All rights reservedBS ISO 19290:2016ISO 19290:2016(E)6.1 N-Nitrosonornicotine, (NNN) CAS-No: 80508-23-2, w 98 % (mass fraction).6.2 N-Nitrosoanatabine, (NAT) CAS-No: 71267-22-6, w 98 %.6.3 N-Nitrosoanabasine, (NAB) CAS-No: 1133-64-8, w 96 %.6.4 4-(N-Nitrosomethylamino)-1-
43、(3-pyridyl)-1-butanone, (NNK) CAS-No: 64091-91-4, w 97 %.6.5 N-Nitrosonornicotine-2,4,5,6-d4, (NNN-d4) CAS-No: 66148-19-4, w 98 %.6.6 N-Nitrosoanatabine-2,4,5,6-d4, (NAT-d4) CAS-No: 1426174-82-4, w 98 %.6.7 N-Nitrosoanabasine-2,4,5,6-d4, (NAB-d4) CAS-No: 1020719-68-9, w 98 %.6.8 4-(N-Nitrosomethylam
44、ino)-1-(3-pyridyl)-1-butanone-2,4,5,6-d4,(NNK-d4), CAS-No: 76661-24-7, w 98 %.6.9 Ammonium acetate, w 97 %.6.10 Acetonitrile, HPLC grade.6.11 Methanol, HPLC grade.6.12 Acetic acid, w 99,77 %.6.13 De-ionized water, 18,8 M.6.14 Syringe filter, 0,45 m polytetrafluoroethylene (PTFE) or equivalent.6.15 D
45、isposable syringes, 5 ml.6.16 Autosampler vials (amber), caps and PTFE faced septa.7 Preparation7.1 Preparation of glasswareGlassware shall be cleaned and dried in such a manner to ensure that contamination does not occur.It is important that all possible sources of contamination which could interfe
46、re with the analytical process are removed from the work area.7.2 Preparation of solutions7.2.1 Extraction solution, 100 mM ammonium acetate solution.Weigh 15,4 g 0,05 g of ammonium acetate. Put into a 2 000 ml volumetric flask and dilute to the mark with de-ionized water.7.2.2 HPLC mobile phase A,
47、0,1 % acetic acid solution in water.Add 1 ml of acetic acid into a 1 000 ml volumetric flask and dilute to the mark with de-ionized water. ISO 2016 All rights reserved 3BS ISO 19290:2016ISO 19290:2016(E)7.2.3 HPLC Mobile Phase B, 0,1 % acetic acid solution in methanol.Add 1 ml of acetic acid into a
48、1 000 ml volumetric flask and dilute to the mark with methanol.NOTE Extraction solution and mobile phases are stable for up to three months at room temperature.7.3 Preparation of standards7.3.1 GeneralFor the preparation of standard solutions volumetric pipettes should be used.7.3.2 Preparation of i
49、nternal standard solutions7.3.2.1 Primary solutionWeigh, to the nearest 0,1 mg, approximately 10 mg each of NNN-d4, NAT-d4, NAB-d4 and NNK-d4.Put into individual 10 ml volumetric flasks and dilute each flask to the mark with acetonitrile and mix well.The concentration in each solution is approximately 1 000 g/ml.7.3.2.2 Combined secondary solutionTransfer 5 ml of each primary solution of NNN-d4, NAT-d4 and NNK-d4 and 1 ml of NAB-d4 into a 100 ml volumetric flask. Dilute to the mark with ace
