ISO 23921:2020

Cigarettes — Determination of tobacco specific nitrosamines in mainstream cigarette smoke with an intense smoking regime — Method using LC-MS/MS

This document specifies a method for the quantification of four tobacco specific nitrosamines (TSNAs) in the total particulate matter of cigarette mainstream smoke with the intense smoking regime specified in ISO 20778 by using reversed phase high performance liquid chromatography 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).

Cigarettes — Dosage des nitrosamines spécifiques du tabac dans le courant principal de la fumée de cigarette avec un régime de fumage intense — Méthode par CL-SM/SM

General Information

• Status: Published
• Publication Date: 29-Sep-2020
• ICS: 65.160 - Tobacco, tobacco products and related equipment
• Technical Committee: ISO/TC 126 - Tobacco and tobacco products
• Drafting Committee: ISO/TC 126/WG 10 - Intense smoking regime
• Current Stage: 6060 - International Standard published
• Start Date: 30-Sep-2020
• Due Date: 16-Aug-2020
• Completion Date: 30-Sep-2020

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 23921
First edition
2020-09
Cigarettes — Determination of
tobacco specific nitrosamines in
mainstream cigarette smoke with an
intense smoking regime — Method
using LC-MS/MS
Cigarettes — Dosage des nitrosamines spécifiques du tabac dans le
courant principal de la fumée de cigarette avec un régime de fumage
intense — Méthode par CL-SM/SM
Reference number
ISO 23921:2020(E)
©
ISO 2020

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ISO 23921:2020(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2020
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Published in Switzerland
ii © ISO 2020 – All rights reserved

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ISO 23921:2020(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Apparatus . 2
6 Reagents . 2
7 Preparation . 3
7.1 Preparation of glassware. 3
7.2 Preparation of solutions . 3
7.3 Preparation of standards. 4
7.3.1 General. 4
7.3.2 Preparation of internal standard solutions . 4
7.3.3 Preparation of calibration standard solutions . 4
8 Sampling . 6
9 Tobacco product preparation . 6
10 Sample generation — Smoking of cigarettes . 6
10.1 General . 6
10.2 Linear smoking . 6
10.3 Rotary smoking . 6
11 Sample analysis . 6
11.1 Preparation of sample. 6
11.1.1 General. 6
11.1.2 Extraction for linear smoking (44 mm pad) . 6
11.1.3 Extraction for rotary smoking (92 mm pad) . 6
11.1.4 Final sample preparation . 7
11.2 Analysis of samples by LC-MS/MS . 7
11.2.1 General. 7
11.2.2 HPLC set-up parameters (Example) . 7
11.2.3 Mobile phase (Example) . . 7
11.2.4 Mobile phase: Gradient (Example) . 7
11.2.5 MS/MS Set-up parameters (Example) . 7
11.3 Calculation . 8
11.3.1 Calibration curve . 8
11.3.2 Determination of the TSNAs concentrations . 9
11.3.3 Sample quantification . . 9
11.3.4 Determination of mainstream smoke TSNA deliveries . 9
12 Repeatability and reproducibility . 9
13 Test report .11
Annex A (informative) Example of typical chromatograms .12
Bibliography .14
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ISO 23921:2020(E)

Foreword
ISO (the International Organization 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 established 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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, 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 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 declarations 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 of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 126, Tobacco and tobacco products.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
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ISO 23921:2020(E)

Introduction
In 2009 the CORESTA (www .coresta .org) Special Analytes Sub-Group focused on the development of
a method for the determination of Tobacco Specific Nitrosamines (TSNAs) in mainstream cigarette
smoke. The Sub-Group investigated a liquid chromatography- tandem mass spectrometry (LC-MS/MS)
method to complement the gas chromatography with a thermal energy analyser (GC-TEA) technique
already available as CORESTA Recommended Method N° 63. Several LC-MS/MS methods have been
[3][4]
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 across laboratories. In
summary, mainstream cigarette smoke was collected on a glass fibre filter 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 the joint experiment.
This document was produced through a CORESTA collaborative experiment, conducted in 2011,
[5][6]
involving 20 laboratories from 12 countries . Cigarettes were smoked with the intense smoking
regime specified in Health Canada Official Method T-115 (equivalent to ISO 20778) and statistical
[1]
evaluations were made according to the recommendations provided in ISO 5725 .
No machine smoking regime can represent all human smoking behaviour.
— 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 measurements 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. Communicating differences between products in machine measurements as differences in
exposure or risk is a misuse of testing using International Standards.
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INTERNATIONAL STANDARD ISO 23921:2020(E)
Cigarettes — Determination of tobacco specific
nitrosamines in mainstream cigarette smoke with an
intense smoking regime — Method using LC-MS/MS
WARNING — 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 and determine the applicability of any other restrictions prior to use.
1 Scope
This document specifies a method for the quantification of four tobacco specific nitrosamines (TSNAs) in
the total particulate matter of cigarette mainstream smoke with the intense smoking regime specified
in ISO 20778 by using reversed phase high performance liquid chromatography 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 references
The following documents are referred to in the text in such a way that 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 3402, Tobacco and tobacco products — Atmosphere for conditioning and testing
ISO 8243, Cigarettes — Sampling
ISO 20778, Cigarettes — Routine analytical cigarette smoking machine — Definitions and standard
conditions with an intense smoking regime
ISO 20779, Cigarettes — Generation and collection of total particulate matter using a routine analytical
smoking machine with an intense smoking regime
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
tobacco specific 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)
[SOURCE: ISO 22303:2008, 3.1]
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ISO 23921:2020(E)

4 Principle
Cigarettes are smoked on a routine analytical cigarette smoking machine according to ISO 20778. 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 acetate
solution using a shaker.
The extract is syringe filtered through a 0,45 μm PTFE syringe filter directly into an autosampler vial.
The samples are quantitated by LC-MS/MS.
5 Apparatus
In addition to the list provided below, usual laboratory apparatus and equipment are needed for
preparation of samples and standards. All glassware shall be cleaned before use to avoid any
contamination.
5.1 Analytical balance, capable of measuring to at least four decimal places.
5.2 Extraction container, of approximately 50 ml.
5.3 Dispenser, of capacity 20 ml.
5.4 Gas-tight syringes, of capacity 250 μl.
5.5 Mechanical volumetric pipette.
5.6 Shaker.
5.7 High performance liquid chromatograph coupled to tandem mass spectrometer (LC-MS/
MS), consisting of:
5.7.1 Binary pump.
5.7.2 Autosampler.
5.7.3 Tandem mass spectrometer.
5.7.4 Data collection system.
®1)
5.7.5 LC column: Waters XBridge BEH C18 , 2,5 μm, 2,1 mm × 50 mm or equivalent.
6 Reagents
Use only reagents of recognized analytical reagent grade.
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 % (mass fraction).
1) Waters XBridge BEH 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 of this product.
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ISO 23921:2020(E)

6.3 N-Nitrosoanabasine, (NAB, CAS-No: 1133-64-8), w ≥ 98 % (mass fraction).
6.4 4-(N-Methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK) CAS-No: 64091-91-4), w ≥ 98 %.
6.5 Deuterated (N-Nitrosonornicotine), (NNN-d4) CAS-No: 66148-19-4, w ≥ 98 %, isotopic purity
w ≥ 99 %.
6.6 Deuterated (N-Nitrosoanatabine), (NAT-d4) CAS-No: 1020719-69-0, w ≥ 98 %, isotopic purity
w ≥ 99 %.
6.7 Deuterated (N-Nitrosoanabasine), (NAB-d4) CAS-No: 1020719-68-9, w ≥ 98 %, isotopic purity
w ≥ 99 %.
6.8 Deuterated 4-(N-Methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK-d4) CAS-No
...