ISO 21114:2026

International
Standard
ISO 21114
First edition
Nicotine pouches — Determination
2026-10
of nicotine content using liquid
chromatography ultraviolet
spectroscopy (LC-UV)
Sachets à base de nicotine — Détermination de la teneur en
nicotine par chromatographie en phase liquide couplée à la
spectroscopie ultraviolette (LC-UV)
Reference number
© ISO 2026
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Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Apparatus . 1
6 Reagents . 3
7 Preparation . 3
7.1 Preparation of glassware .3
7.2 Preparation of solutions .3
7.2.1 Ammonium formate, 500 mmol/l (used for preparing extraction solution (7.2.2)) .3
7.2.2 Extraction solution, 100 mmol/l ammonium formate .3
7.2.3 Ammonium acetate stock buffer, 500 mmol/l (used for mobile phases) .4
7.2.4 Mobile phase A, 10 mmol/l ammonium acetate buffer + 0,025 % ammonium
hydroxide .4
7.2.5 Mobile phase B, 10 mmol/l ammonium acetate buffer in acetonitrile + 0,025 %
ammonium hydroxide .4
7.2.6 Needle wash, 40:40:20:2 of water:methanol: 2-propanol:formic acid .4
7.2.7 Seal wash, 90:10 of water:acetonitrile .4
7.3 Preparation of standards .4
7.3.1 Nicotine standard stock solution: approx. 25 mg/ml .4
7.3.2 Nicotine calibration standards .4
8 Sample preparation and extraction . 5
8.1 General .5
8.2 Sample preparation .5
8.3 Sample extraction .5
9 Sample extract analysis . . 6
9.1 General .6
9.2 Suggested UPLC parameters .6
9.3 Suggested HPLC parameters .7
9.4 System suitability .7
9.5 Calibration .7
10 Repeatability and reproducibility . 8
11 Test report . 9
Annex A (informative) Examples of UPLC chromatograms . 10
Annex B (informative) Examples of HPLC chromatograms .12
Bibliography . 14

iii
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,
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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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
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this may not represent the latest information, which may be obtained from the patent database available at
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Any trade name used in this document is information given for the convenience of users and does not
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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.

iv
Introduction
Nicotine pouches are portioned consumer products that do not contain cut, ground, powdered or leaf tobacco,
but do contain nicotine, a base filler, and material may contain flavours, sweeteners, pH adjusting agents and
other ingredients. Nicotine pouches are intended for oral use and uptake of nicotine via the oral mucosa. The
accurate determination of nicotine is an important analytical measurement for product characterization.
In 2024, ISO/TC 126 for nicotine pouches conducted a large international collaborative study with 23
participating laboratories. The method allows the use of ultra performance liquid chromatography (UPLC)
and high performance liquid chromatography (HPLC). For the collaborative study, it was agreed that the
laboratories can submit data using both HPLC and UPLC under the requirement that a separate sample
extraction was prepared for each technique. In total, 27 datasets were submitted, including 17 HPLC data
sets and 10 UPLC data sets. This study data was used as the basis for calculating the repeatability (r) and
reproducibility (R) limits estimates for this method. As agreed prior to the start of the study, the data from
both HPLC and UPLC were examined to evaluate if the two techniques gave similar results and performance.
It was concluded that both HPLC and UPLC results were similar, and that the data can be combined to
determine the r and R limits. Results from this study were evaluated in basic conformance with ISO 5725-2
to calculate repeatability and reproducibility values.

v
International Standard ISO 21114:2026(en)
Nicotine pouches — Determination of nicotine content using
liquid chromatography ultraviolet spectroscopy (LC-UV)
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 determination of nicotine content of nicotine pouches and nicotine
pouch filler using HPLC/UPLC with UV spectroscopy.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
the 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 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
nicotine pouch
portioned consumer product that does not contain cut, ground, powdered, or leaf tobacco but does contain
nicotine, a base filler material, and may contain flavours, sweeteners, and pH adjusting agents, and other
ingredients
Note 1 to entry: Nicotine pouches are intended for oral use and uptake of nicotine via the oral mucosa.
4 Principle
Nicotine is extracted from nicotine pouch/nicotine pouch filler sample with a buffer solution (pH 3). The
extracts are filtered and analysed by HPLC/UPLC with UV spectroscopy. Nicotine is reported for pouch as
mg/pouch or mg/g and for filler as mg/g, on an as is basis.
The method calibration range is (0,025 to 0,5) mg/ml or (2 to 40) mg/g.
5 Apparatus
Usual laboratory apparatus, and particularly the following items.

5.1 High performance liquid chromatography (HPLC) or ultra performance liquid chromatography
(UPLC) system, consisting of a binary pump, autosampler with appropriate sampling loop, UV detector,
column oven, data collection system.
1)
5.2 HPLC column, reversed-phase C18 , 3,5 µm particle size, 4,6 mm × 150 mm, or equivalent.
2)
5.3 UPLC column, reversed-phase C18 , 1,7 – 2,1 µm particle size, 2,1 mm × 100 mm, or equivalent.
5.4 Extraction vessel, polypropylene centrifuge tubes (50 ml) with tapered inner-surface caps, or glass
Erlenmeyer flask (100 ml) and stopper or equivalent. The sealed tubes should not leak on vigorous shaking.
5.5 Amber glass bottles with PTFE lined cap, in range of sizes.
5.6 Glass bottles with screw caps, in range of sizes.
5.7 Graduated cylinder, glass, of capacities 10 ml, 100 ml, 1 000 ml and 2 000 ml.
5.8 Amber volumetric flasks, class A, in range of sizes.
5.9 Analytical balance, suitable for measuring to the nearest 0,1 mg.
5.10 Shaker, orbital shaker, wrist action shaker, or similar.
5.11 Glass volumetric pipettes, class A, and/or certified glass disposable micro capillary pipettes,
and/or positive-displacement pipettes, in range of sizes.
5.12 Bottle-top dispenser, to repeatably dispense 40 ml.
5.13 Amber autosampler vials with caps and PTFE lined septa, glass.
5.14 Syringe filter, 0,2 µm PTFE, 25 mm diameter, or equivalent.
5.15 Polypropylene Luer lock syringe, of appropriate size for filtering samples.
5.16 Glass Pasteur pipettes, 150 mm to 250 mm length.
5.17 Filter equipment, including 0,45 μm polypropylene membrane filter.
5.18 Autosampler vials with caps and PTFE lined septa, glass or polypropylene. Autosampler vials with
0,2 µm PTFE in-line filters may also be used. ®
1) Waters XBridge Premier BEH C18 column, 3,5 µm , 4,6 mm x 150 mm has been shown to provide suitable results.
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 provide equivalent results, i.e., the analyte is
sufficiently resolved from matrix interferences. ®
2) Waters Acquity C18 column, 1,7 µm , 2,1 mm × 100 mm have been shown to provide suitable results.
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 provide equivalent results, i.e., the analyte is
sufficiently resolved from matrix interferences.

6 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified. Solvents shall be of HPLC-
grade or better.
6.1 Water, water complying with grade 2 as specified in ISO 3696, or better.
3)
6.2 Acetonitrile (CAS RN® 75-05-8), HPLC gradient grade or better.
6.3 Methanol (CAS RN® 67-56-1), HPLC gradient grade or better.
6.4 2-propanol (CAS RN® 67-63-0), HPLC gradient grade or better.
6.5 Nicotine (CAS RN® 54-11-5), w ≥ 99 % (mass fraction).
6.6 Formic acid (CAS RN® 64-18-6), w ≥ 98 %.
6.7 Ammonium hydroxide (CAS RN® 1336-21-6), 25 %, p.a. grade.
6.8 Ammonium acetate (CAS RN® 631-61-8), w ≥ 98 % (mass fraction).
6.9 Ammonium formate (CAS RN® 540-69-2),
...