ISO 21766:2021
(Main)Tobacco and tobacco products — Determination of tobacco-specific nitrosamines in tobacco products — Method using LC-MS/MS
Tobacco and tobacco products — Determination of tobacco-specific nitrosamines in tobacco products — Method using LC-MS/MS
This document specifies a method for the quantification of four tobacco specific nitrosamines (TSNAs) in tobacco and the following tobacco products: cigarettes, cigars and smokeless tobacco products using reversed phase high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). The TSNAs determined with this method are: N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).
Tabac et produits du tabac — Dosage des nitrosamines spécifiques du tabac dans les produits du tabac — Méthode par CL-SM/SM
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INTERNATIONAL ISO
STANDARD 21766
Second edition
2021-02
Tobacco and tobacco products —
Determination of tobacco-specific
nitrosamines in tobacco products —
Method using LC-MS/MS
Tabac et produits du tabac — Dosage des nitrosamines spécifiques du
tabac dans les produits du tabac — Méthode par CL-SM/SM
Reference number
ISO 21766:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO 21766:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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 permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 21766:2021(E)
Contents 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 ................................................................................................................................................................................................................ 3
7.1 Preparation of glassware................................................................................................................................................................ 3
7.2 Preparation of solutions ................................................................................................................................................................. 3
7.2.1 Extraction solution, 100 mM ammonium acetate in water ......................................................... 3
7.2.2 HPLC Mobile Phase A: Water, resistivity ≥ 18,2 MΩ·cm at 25 °C ............................................ 3
7.2.3 HPLC Mobile Phase B: 0,1 % acetic acid in methanol ...................................................................... 3
7.3 Preparation of standards................................................................................................................................................................ 3
7.3.1 General...................................................................................................................................................................................... 3
7.3.2 Preparation of internal standard solutions .............................................................................................. 4
7.3.3 Preparation of calibration standard solutions ....................................................................................... 4
8 Sampling ........................................................................................................................................................................................................................ 5
8.1 General ........................................................................................................................................................................................................... 5
8.2 Sample preparation ............................................................................................................................................................................ 5
8.3 Sample extraction ................................................................................................................................................................................. 5
9 Sample analysis ...................................................................................................................................................................................................... 6
9.1 General ........................................................................................................................................................................................................... 6
9.2 Suggested HPLC parameters ....................................................................................................................................................... 6
9.3 MS/MS parameters .............................................................................................................................................................................. 6
9.3.1 General...................................................................................................................................................................................... 6
9.3.2 Quantification and qualification transitions ............................................................................................ 7
9.4 System suitability .................................................................................................................................................................................. 7
9.5 Calibration .................................................................................................................................................................................................. 7
9.6 Calculation .................................................................................................................................................................................................. 8
10 Repeatability and reproducibility ...................................................................................................................................................... 9
11 Test report ................................................................................................................................................................................................................12
Annex A (informative) Sample clean-up using solid phase extraction (SPE) ..........................................................13
Annex B (informative) Examples of typical chromatograms ....................................................................................................15
Bibliography .............................................................................................................................................................................................................................16
© ISO 2021 – All rights reserved iii---------------------- Page: 3 ----------------------
ISO 21766:2021(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.
This second edition cancels and replaces the first edition (ISO 21766:2018), of which it constitutes a
minor revision.The main changes compared to the previous edition are as follows:
— the title and CAS number for NNK-d4 (see 5.12) have been updated;
— the nomenclature of the deuterated nitrosamines in 5.6 to 5.13 have been harmonized.
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.iv © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 21766:2021(E)
Introduction
The Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Smokeless Tobacco Sub-
Group studied various widely-used procedures for the determination of tobacco specific nitrosamines
(TSNAs) in smokeless tobacco products. A study was conducted in 2009 that evaluated several different
methodologies. This study included both liquid chromatography tandem mass spectrometry methods
(LC-MS/MS) and gas chromatography combined with nitrogen chemiluminescence detection methods.
The results generated with a supplied LC-MS/MS method proved to be the most consistent and was
[7]used as the basis for CORESTA Recommended Method N° 72 . Nine laboratories provided data using
this method. This study included nine commercial smokeless tobacco products covering eight different
product styles. CORESTA Recommended Method N° 72 was updated in 2016 to include repeatability
and reproducibility for the four CORESTA reference products.CORESTA Recommended Method N° 72 was used as the basis for this document. However, the scope of
this document was broadened to include ground tobacco, cigarette fillers and cigar fillers in addition
to smokeless tobacco products. The respective values for repeatability (r) and reproducibility (R)
for ground tobacco, cigarette fillers and cigar fillers have been determined through an international
collaborative study that was conducted in 2017 and involved 18 laboratories.© ISO 2021 – All rights reserved v
---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 21766:2021(E)
Tobacco and tobacco products — Determination of
tobacco-specific nitrosamines in tobacco products —
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 ScopeThis document specifies a method for the quantification of four tobacco specific nitrosamines (TSNAs)
in tobacco and the following tobacco products: cigarettes, cigars and smokeless tobacco products using
reversed phase high performance liquid chromatography with tandem mass spectrometry (LC-MS/
MS). The TSNAs determined with this method are: N-nitrosonornicotine (NNN), N-nitrosoanatabine
(NAT), N-nitrosoanabasine (NAB) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).
2 Normative referencesThere are no normative references in this document.
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-(N-Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)
[SOURCE: ISO 22303:2008, 3.1]4 Principle
Deuterium-labelled (d4) internal standards are added to the tobacco sample and subsequently extracted
with an aqueous buffer. The sample extracts are filtered and then analysed by reversed phase high
performance liquid chromatography (HPLC) and quantified by tandem mass spectrometry (MS/MS).
The amounts of TSNAs in the tobacco products are reported as ng/g, as is wet mass.
5 ReagentsUse only reagents of recognized analytical grade during the analysis. Solvents shall be of HPLC-grade
or better.5.1 Water, deionized, resistivity ≥ 18,2 MΩ·cm at 25 °C.
© ISO 2021 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO 21766:2021(E)
5.2 Acetonitrile, HPLC grade or better.
5.3 Methanol, HPLC grade or better.
5.4 Ammonium acetate, w ≥ 98 % (mass fraction).
5.5 Acetic acid, w ≥ 98 % (mass fraction).
5.6 N-Nitrosoanabasine, (NAB) CAS-No: 1133-64-8), w ≥ 98 % (mass fraction).
5.7 N-Nitrosoanatabine, (NAT) CAS-No: 71267-22-6, w ≥ 98 % (mass fraction).
5.8 4-(N-Methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK, CAS-No: 64091-91-4), w ≥ 98 %
(mass fraction).5.9 N-Nitrosonornicotine, (NNN, CAS-No: 80508-23-2), w ≥ 98 % (mass fraction).
5.10 Deuterated (N-Nitrosoanabasine), (NAB-d4, CAS-No: 1020719-68-9), w ≥ 98 %, isotopic purity
w ≥ 99 %.5.11 Deuterated (N-Nitrosoanatabine), (NAT-d4, CAS-No: 1020719-69-0), w ≥ 98 %, isotopic purity
w ≥ 99 %.5.12 Deuterated 4-(N-Methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK-d4, CAS-No: 764661-
24-7), w ≥ 98 %, isotopic purity w ≥ 99 %.5.13 Deuterated (N-Nitrosonornicotine), (NNN-d4) CAS-No: 66148-19-4, w ≥ 98 %, isotopic purity
w ≥ 99 %.6 Apparatus
Usual laboratory apparatus and supplies, and in particular the following items. All glassware shall be
cleaned before use to avoid any contamination.6.1 High performance liquid chromatograph tandem mass spectrometer (LC-MS/MS) with
electrospray ion source (ESI), consisting of the following.6.1.1 Binary pump.
6.1.2 Autosampler.
6.1.3 Column oven.
6.1.4 Data collection system.
6.2 HPLC column: reversed-phase C18 , 2,5 μm particle size, 2,1 mm × 50 mm, or equivalent.
6.3 Orbital shaker, wrist action shaker, or similar.1) Waters XTerra® MS C18, 2,5 μm, 2,1 × 50 mm has been shown to be a suitable column. This information is
given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.
Equivalent columns may be used if they can be shown to lead to the same results, i.e. that the analytes and internal
standards are sufficiently resolved from interferences.2 © ISO 2021 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 21766:2021(E)
6.4 Autosampler vials (amber).
6.5 Disposable syringes, of appropriate size for filtering samples.
6.6 Syringe filter, of diameter 25 mm and pore size 0,45 μm, made of polytetrafluoroethylene (PTFE)
or equivalent.NOTE Various filter materials were evaluated during the collaborative study and PTFE had the highest
recovery from those verified.Other filter materials may also be suitable, however, they should be evaluated before routine use.
6.7 Extraction containers, glass, of capacity 50 ml to 100 ml.6.8 Amber volumetric flasks, class A, in a range of sizes.
6.9 Glass volumetric pipettes, class A, and/or positive-displacement pipettes, in a range of sizes.
6.10 Analytical balance, capable of measuring to at least four decimal places (gram).
7 Preparation7.1 Preparation of glassware
Glassware 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 may interfere with the analytical
process are removed from the work area.Standard solutions and sample extracts shall be protected from light.
7.2 Preparation of solutions
7.2.1 Extraction solution, 100 mM ammonium acetate in water
Weigh 15,4 g ± 0,05 g of ammonium acetate. Quantitatively transfer into a 2 000 ml volumetric flask and
dilute to the mark with deionized water.7.2.2 HPLC Mobile Phase A: Water, resistivity ≥ 18,2 MΩ·cm at 25 °C
7.2.3 HPLC Mobile Phase B: 0,1 % acetic acid in methanol
Add 1 ml of acetic acid into a 1 000 ml volumetric flask and dilute to the mark with methanol.
Stability studies should be performed by the laboratory to determine the shelf life of these solutions.
7.3 Preparation of standards7.3.1 General
All standard solutions shall be prepared in amber, or light protected glassware and stored at about
−20 °C, except the calibration standards which shall be stored in a refrigerator. Produce a series of
calibration standards to cover the range of expected results to be found in the test samples, as in 7.3.3.4.
Stability studies should be performed by the laboratory to determine the shelf life of these solutions.
© ISO 2021 – All rights reserved 3---------------------- Page: 8 ----------------------
ISO 21766:2021(E)
7.3.2 Preparation of internal standard solutions
7.3.2.1 Stock solution
Weigh, to the nearest 0,1 mg, approximately 10 mg each of NNN-d4, NAT-d4, NAB-d4 and NNK-d4.
Quantitatively transfer 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 μg/ml.
7.3.2.2 Combined secondary internal standard solutionTransfer 4,00 ml of each of the four internal standard stock solutions into a 100 ml volumetric flask and
dilute to volume with acetonitrile. Mix well. The concentration is approximately 40 µg/ml of NNN-d4,
NNK-d4, NAT-d4 and NAB-d4.7.3.2.3 Internal standard spiking solution
Transfer 5,00 ml of mixed internal standard solution into a 100 ml volumetric flask and dilute to volume
with acetonitrile. Mix well. The concentration is approximately 2 000 ng/ml of NNN-d4, NNK-d4, NAT-d4
and NAB-d4.7.3.3 Preparation of calibration standard solutions
7.3.3.1 Stock solution
Weigh, to the nearest 0,1 mg, approximately 10 mg each of NNN, NAT, NAB and NNK. Quantitatively
transfer 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 μg/ml.7.3.3.2 Mixed TSNA standard solution (I)
Transfer 4,00 ml of each of the three TSNA stock solutions NNN, NNK, NAT and 1,00 ml of the TSNA
stock solution NAB into a 100 ml volumetric flask and dilute to volume with acetonitrile. Mix well. The
concentration will be approximately 40 µg/ml of NNN, NNK, NAT and 10 µg/ml of NAB.
7.3.3.3 Mixed TSNA standard solution (II)Transfer 2,50 ml of the mixed TSNA standard solution (I) into a 250 ml volumetric flask and dilute to
volume with acetonitrile/deionized water (30 %/70 %). Mix well. The concentration of NNN, NNK and
NAT will be approximately 400 ng/ml, and the concentration of NAB will be approximately 100 ng/ml.
7.3.3.4 TSNA calibration standardsPrepare seven working standard solutions that cover the concentration range of interest. Table 1
provides an example of calibration standard preparation.The TSNA calibration standards are prepared in seven separate 100 ml volumetric flasks, each
containing 10 ml of 100 mM ammonium acetate solution. Transfer 1,00 ml of the internal standard
spiking solution (2 000 ng/ml) to each of the seven volumetric flasks. Next, add the appropriate volume
of the mixed TSNA standard solution (II), given in Table 1. Then add the volume of acetonitrile, given in
Table 1. Finally, dilute each of the seven flasks to volume with 100 mM ammonium acetate and mix well.
Calculate the exact con...
FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 21766
ISO/TC 126
Tobacco and tobacco products —
Secretariat: DIN
Determination of tobacco-specific
Voting begins on:
20201029 nitrosamines in tobacco products —
Method using LC-MS/MS
Voting terminates on:
20201224
Tabac et produits du tabac — Dosage des nitrosamines spécifiques du
tabac dans les produits du tabac — Méthode par CL-SM/SM
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO
ISO/FDIS 21766:2020(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. ISO 2020
---------------------- Page: 1 ----------------------
ISO/FDIS 21766:2020(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
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 permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 21766:2020(E)
Contents 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 ................................................................................................................................................................................................................ 3
7.1 Preparation of glassware................................................................................................................................................................ 3
7.2 Preparation of solutions ................................................................................................................................................................. 3
7.2.1 Extraction solution, 100 mM ammonium acetate in water ......................................................... 3
7.2.2 HPLC Mobile Phase A: Water, resistivity ≥ 18,2 MΩ·cm at 25 °C ............................................ 3
7.2.3 HPLC Mobile Phase B: 0,1 % acetic acid in methanol ...................................................................... 3
7.3 Preparation of standards................................................................................................................................................................ 3
7.3.1 General...................................................................................................................................................................................... 3
7.3.2 Preparation of internal standard solutions .............................................................................................. 4
7.3.3 Preparation of calibration standard solutions ....................................................................................... 4
8 Sampling ........................................................................................................................................................................................................................ 5
8.1 General ........................................................................................................................................................................................................... 5
8.2 Sample preparation ............................................................................................................................................................................ 5
8.3 Sample extraction ................................................................................................................................................................................. 5
9 Sample analysis ...................................................................................................................................................................................................... 6
9.1 General ........................................................................................................................................................................................................... 6
9.2 Suggested HPLC parameters ....................................................................................................................................................... 6
9.3 MS/MS parameters .............................................................................................................................................................................. 6
9.3.1 General...................................................................................................................................................................................... 6
9.3.2 Quantification and qualification transitions ............................................................................................ 7
9.4 System suitability .................................................................................................................................................................................. 7
9.5 Calibration .................................................................................................................................................................................................. 7
9.6 Calculation .................................................................................................................................................................................................. 8
10 Repeatability and reproducibility ...................................................................................................................................................... 9
11 Test report ................................................................................................................................................................................................................12
Annex A (informative) Sample clean-up using solid phase extraction (SPE) ..........................................................13
Annex B (informative) Examples of typical chromatograms ....................................................................................................15
Bibliography .............................................................................................................................................................................................................................16
© ISO 2020 – All rights reserved iii---------------------- Page: 3 ----------------------
ISO/FDIS 21766:2020(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 nongovernmental, 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.
This second edition cancels and replaces the first edition (ISO 21766:2018), of which it constitutes a
minor revision.The main changes compared to the previous edition are as follows:
— the title and CAS number for NNKd4 (see 5.12) have been updated;
— the nomenclature of the deuterated nitrosamines in 5.6 to 5.13 have been harmonized.
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.iv © ISO 2020 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/FDIS 21766:2020(E)
Introduction
The Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Smokeless Tobacco Sub-
Group studied various widely-used procedures for the determination of tobacco specific nitrosamines
(TSNAs) in smokeless tobacco products. A study was conducted in 2009 that evaluated several different
methodologies. This study included both liquid chromatography tandem mass spectrometry methods
(LC-MS/MS) and gas chromatography combined with nitrogen chemiluminescence detection methods.
The results generated with a supplied LCMS/MS method proved to be the most consistent and was
[7]used as the basis for CORESTA Recommended Method N° 72 . Nine laboratories provided data using
this method. This study included nine commercial smokeless tobacco products covering eight different
product styles. CORESTA Recommended Method N° 72 was updated in 2016 to include repeatability
and reproducibility for the four CORESTA reference products.CORESTA Recommended Method N° 72 was used as the basis for this document. However, the scope of
this document was broadened to include ground tobacco, cigarette fillers and cigar fillers in addition
to smokeless tobacco products. The respective values for repeatability (r) and reproducibility (R)
for ground tobacco, cigarette fillers and cigar fillers have been determined through an international
collaborative study that was conducted in 2017 and involved 18 laboratories.© ISO 2020 – All rights reserved v
---------------------- Page: 5 ----------------------
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 21766:2020(E)
Tobacco and tobacco products — Determination of
tobacco-specific nitrosamines in tobacco products —
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 ScopeThis document specifies a method for the quantification of four tobacco specific nitrosamines (TSNAs)
in tobacco and the following tobacco products: cigarettes, cigars and smokeless tobacco products using
reversed phase high performance liquid chromatography with tandem mass spectrometry (LC-MS/
MS). The TSNAs determined with this method are: Nnitrosonornicotine (NNN), Nnitrosoanatabine
(NAT), N-nitrosoanabasine (NAB) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).
2 Normative referencesThere are no normative references in this document.
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]4 Principle
Deuterium-labelled (d4) internal standards are added to the tobacco sample and subsequently extracted
with an aqueous buffer. The sample extracts are filtered and then analysed by reversed phase high
performance liquid chromatography (HPLC) and quantified by tandem mass spectrometry (MS/MS).
The amounts of TSNAs in the tobacco products are reported as ng/g, as is wet mass.
5 ReagentsUse only reagents of recognized analytical grade during the analysis. Solvents shall be of HPLC-grade
or better.5.1 Water, deionized, resistivity ≥ 18,2 MΩ·cm at 25 °C.
© ISO 2020 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO/FDIS 21766:2020(E)
5.2 Acetonitrile, HPLC grade or better.
5.3 Methanol, HPLC grade or better.
5.4 Ammonium acetate, w ≥ 98 % (mass fraction).
5.5 Acetic acid, w ≥ 98 %.
5.6 N-Nitrosoanabasine, (NAB) CASNo: 1133648), w ≥ 98 % (mass fraction).
5.7 N-Nitrosoanatabine, (NAT) CASNo: 71267226, w ≥ 98 % (mass fraction).
5.8 4-(N-Methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK, CASNo: 64091914), w ≥ 98 %
(mass fraction).5.9 N-Nitrosonornicotine, (NNN, CAS-No: 80508-23-2), w ≥ 98 % (mass fraction).
5.10 Deuterated (N-Nitrosoanabasine), (NABd4, CASNo: 1020719689), w ≥ 98 %, isotopic purity
w ≥ 99 %.5.11 Deuterated (N-Nitrosoanatabine), (NATd4, CASNo: 1020719690), w ≥ 98 %, isotopic purity
w ≥ 99 %.5.12 Deuterated 4-(N-Methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNKd4, CASNo: 764661
247), w ≥ 98 %, isotopic purity w ≥ 99 %.5.13 Deuterated (N-Nitrosonornicotine), (NNNd4) CASNo: 66148194, w ≥ 98 %, isotopic purity
w ≥ 99 %.6 Apparatus
Usual laboratory apparatus and supplies, and in particular the following items. All glassware shall be
cleaned before use to avoid any contamination.6.1 High performance liquid chromatograph tandem mass spectrometer (LC-MS/MS) with
electrospray ion source (ESI), consisting of the following.6.1.1 Binary pump.
6.1.2 Autosampler.
6.1.3 Column oven.
6.1.4 Data collection system.
6.2 HPLC column: reversedphase C18 , 2,5 μm particle size, 2,1 mm × 50 mm, or equivalent.
6.3 Orbital shaker, wrist action shaker, or similar.1) Waters XTerra® MS C18, 2,5 μm, 2,1 × 50 mm has been shown to be a suitable column. This information is
given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.
Equivalent columns may be used if they can be shown to lead to the same results, i.e. that the analytes and internal
standards are sufficiently resolved from interferences.2 © ISO 2020 – All rights reserved
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ISO/FDIS 21766:2020(E)
6.4 Autosampler vials.
6.5 Disposable syringes, of appropriate size for filtering samples.
6.6 Syringe filter, of diameter 25 mm and pore size 0,45 μm, made of polytetrafluoroethylene (PTFE)
or equivalent.NOTE Various filter materials were evaluated during the collaborative study and PTFE had the highest
recovery from those verified.Other filter materials may also be suitable, however, they should be evaluated before routine use.
6.7 Extraction containers, glass, of capacity 50 ml to 100 ml.6.8 Amber volumetric flasks, class A, in a range of sizes.
6.9 Glass volumetric pipettes, class A, and/or positive-displacement pipettes, in a range of sizes.
6.10 Analytical balance, capable of measuring to at least four decimal places (gram).
7 Preparation7.1 Preparation of glassware
Glassware 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 may interfere with the analytical
process are removed from the work area.Standard solutions and sample extracts shall be protected from light.
7.2 Preparation of solutions
7.2.1 Extraction solution, 100 mM ammonium acetate in water
Weigh 15,4 g ± 0,05 g of ammonium acetate. Quantitatively transfer into a 2 000 ml volumetric flask and
dilute to the mark with deionized water.7.2.2 HPLC Mobile Phase A: Water, resistivity ≥ 18,2 MΩ·cm at 25 °C
7.2.3 HPLC Mobile Phase B: 0,1 % acetic acid in methanol
Add 1 ml of acetic acid into a 1 000 ml volumetric flask and dilute to the mark with methanol.
Stability studies should be performed by the laboratory to determine the shelf life of these solutions.
7.3 Preparation of standards7.3.1 General
All standard solutions shall be prepared in amber, or light protected glassware and stored at about
−20 °C, except the calibration standards which shall be stored in a refrigerator. Produce a series of
calibration standards to cover the range of expected results to be found in the test samples, as in 7.3.3.4.
Determine the shelflife of the standard and internal standard solutions.© ISO 2020 – All rights reserved 3
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ISO/FDIS 21766:2020(E)
7.3.2 Preparation of internal standard solutions
7.3.2.1 Stock solution
Weigh, to the nearest 0,1 mg, approximately 10 mg each of NNN-d4, NAT-d4, NAB-d4 and NNK-d4.
Quantitatively transfer 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 μg/ml.
7.3.2.2 Combined secondary internal standard solutionTransfer 4,00 ml of each of the four internal standard stock solutions into a 100 ml volumetric flask and
dilute to volume with acetonitrile. Mix well. The concentration is approximately 40 µg/ml of NNN-d4,
NNKd4, NATd4 and NABd4.7.3.2.3 Internal standard spiking solution
Transfer 5,00 ml of mixed internal standard solution into a 100 ml volumetric flask and dilute to volume
with acetonitrile. Mix well. The concentration is approximately 2 000 ng/ml of NNN-d4, NNK-d4, NAT-d4
and NABd4.7.3.3 Preparation of calibration standard solutions
7.3.3.1 Stock solution
Weigh, to the nearest 0,1 mg, approximately 10 mg each of NNN, NAT, NAB and NNK. Quantitatively
transfer 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 μg/ml.7.3.3.2 Mixed TSNA standard solution (I)
Transfer 4,00 ml of each of the three TSNA stock solutions NNN, NNK, NAT and 1,00 ml of the TSNA
stock solution NAB into a 100 ml volumetric flask and dilute to volume with acetonitrile. Mix well. The
concentration will be approximately 40 µg/ml of NNN, NNK, NAT and 10 µg/ml of NAB.
7.3.3.3 Mixed TSNA standard solution (II)Transfer 2,50 ml of the mixed TSNA standard solution (I) into a 250 ml volumetric flask and dilute to
volume with acetonitrile/deionized water (30 %/70 %). Mix well. The concentration of NNN, NNK and
NAT will be approximately 400 ng/ml, and the concentration of NAB will be approximately 100 ng/ml.
7.3.3.4 TSNA calibration standardsPrepare seven working standard solutions that cover the concentration range of interest. Table 1
provides an example of calibration standard preparation.The TSNA calibration standards are prepared in seven separate 100 ml volumetric flasks, each
containing 10 ml of 100 mM ammonium acetate solution. Transfer 1,00 ml of the internal standard
spiking solution (2 000 ng/ml) to each of the seven volume...
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