ISO 23921:2020
(Main)Cigarettes — Determination of tobacco specific nitrosamines in mainstream cigarette smoke with an intense smoking regime — Method using LC-MS/MS
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
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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 2020
© ISO 2020
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ii © ISO 2020 – All rights reserved
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
Foreword
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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).
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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
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iv © ISO 2020 – All rights reserved
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.
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
spectrom
...
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