Cigarettes — Determination of selected carbonyls in the mainstream smoke of cigarettes with an intense smoking regime — Method using high performance liquid chromatography

This document specifies a method for the determination of selected carbonyls (formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone and n-butyraldehyde) as their 2,4-dinitrophenylhydrazones by reversed phase HPLC-UV/DAD in mainstream smoke using an intense smoking regime.

Cigarettes — Dosage de carbonyles sélectionnés dans le courant principal de la fumée de cigarette avec un régime de fumage intense — Méthode par chromatographie liquide haute performance

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Published
Publication Date
20-Sep-2020
Current Stage
6060 - International Standard published
Start Date
21-Sep-2020
Completion Date
21-Sep-2020
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ISO 23922:2020 - Cigarettes -- Determination of selected carbonyls in the mainstream smoke of cigarettes with an intense smoking regime -- Method using high performance liquid chromatography
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INTERNATIONAL ISO
STANDARD 23922
First edition
2020-09
Cigarettes — Determination of
selected carbonyls in the mainstream
smoke of cigarettes with an intense
smoking regime — Method using high
performance liquid chromatography
Cigarettes — Dosage de carbonyles sélectionnés dans le courant
principal de la fumée de cigarette avec un régime de fumage intense
— Méthode par chromatographie liquide haute performance
Reference number
ISO 23922:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 23922:2020(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
---------------------- Page: 2 ----------------------
ISO 23922:2020(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 1

5 Apparatus ..................................................................................................................................................................................................................... 2

6 Reagents ........................................................................................................................................................................................................................ 3

7 Preparation ................................................................................................................................................................................................................ 3

7.1 Preparation of glassware................................................................................................................................................................ 3

7.2 Preparation of solutions ................................................................................................................................................................. 4

7.3 Preparation of standards................................................................................................................................................................ 4

7.3.1 HPLC calibration standards and working solutions .......................................................................... 4

7.3.2 Carbonyl working standards ................................................................................................................................. 5

8 Sampling ........................................................................................................................................................................................................................ 5

9 Tobacco product preparation ................................................................................................................................................................. 5

10 Sample generation — Smoking of cigarettes .......................................................................................................................... 5

10.1 General ........................................................................................................................................................................................................... 5

10.2 Smoking machine setup .................................................................................................................................................................. 5

10.3 Smoking ......................................................................................................................................................................................................... 6

10.3.1 General...................................................................................................................................................................................... 6

10.3.2 Linear smoking .................................................................................................................................................................. 6

10.3.3 Rotary smoking ................................................................................................................................................................. 6

11 Sample analysis ...................................................................................................................................................................................................... 7

11.1 Preparation of mainstream smoke extract solution ............................................................................................... 7

11.2 Reversed phase high performance liquid chromatography ............................................................................. 7

11.2.1 Chromatographic conditions ................................................................................................................................. 7

11.2.2 Mobile phase reagents ................................................................................................................................................ 7

11.2.3 HPLC separation conditions ................................................................................................................................... 8

11.3 Calculation .................................................................................................................................................................................................. 8

11.3.1 Calibration curve ............................................................................................................................................................. 8

11.3.2 Sample quantification ........................................................................................................................................... ....... 8

12 Repeatability and reproducibility ...................................................................................................................................................... 9

12.1 General ........................................................................................................................................................................................................... 9

12.2 Results from the 2012 collaborative study ..................................................................................................................10

13 Test report ................................................................................................................................................................................................................13

Annex A (informative) Recrystallization of 2,4-dinitrophenylhydrazine ....................................................................14

Annex B (normative) Example of calibration standards preparation ............................................................................15

Annex C (informative) HPLC chromatogram of a typical combined carbonyl calibration

standard .....................................................................................................................................................................................................................16

Annex D (informative) HPLC chromatogram of carbonyls in DNPH extract of mainstream

cigarette smoke ...................................................................................................................................................................................................17

Bibliography .............................................................................................................................................................................................................................18

© ISO 2020 – All rights reserved iii
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ISO 23922:2020(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.
iv © ISO 2020 – All rights reserved
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ISO 23922:2020(E)
Introduction

At the outset of this work, discussions in the CORESTA (www .coresta .org) Special Analytes Sub-Group

(since 2017 the Sub-Group changed its name to Smoke Analytes) determined that most laboratories used

a method involving derivatization of carbonyls with 2,4-dinitrophenylhydrazine (DNPH) because they

considered it the most suitable. This was chosen as the basis of the CORESTA Recommended Method

(CRM). The CRM comprised smoke collection in impinger traps, derivatization of carbonyls with DNPH

followed by their determination using reversed phase High Performance Liquid Chromatography with

Ultra Violet or Diode Array Detection (HPLC-UV or HPLC-DAD).

This document was produced from a 2012 collaborative study involving 19 laboratories from 11 countries

[1][2]

and included 10 samples with different tar yields . This method includes recommendations about

critical steps that should be controlled to provide data as robust and consistent as the repeatability and

reproducibility data provided. Cigarettes were smoked using the intense smoking regime parameters

given in Health Canada Official Method T-115. At the time the collaborative study was conducted, the

study protocol stipulated the use of Health Canada Official Method (T-115) for intense conditions as

there was not an International Standard that defined intense smoking conditions. ISO 20778 was

published in 2018 and is equivalent to Health Canada Intense conditions. Statistical evaluations were

[3] [4]
carried out according to ISO 5725-1 and ISO 5725-2 .
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.
© ISO 2020 – All rights reserved v
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INTERNATIONAL STANDARD ISO 23922:2020(E)
Cigarettes — Determination of selected carbonyls in
the mainstream smoke of cigarettes with an intense
smoking regime — Method using high performance liquid
chromatography

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 selected carbonyls (formaldehyde,

acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone and n-butyraldehyde) as

their 2,4-dinitrophenylhydrazones by reversed phase HPLC-UV/DAD in mainstream smoke using an

intense smoking regime.
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
3 Terms and definitions
No terms and definitions are listed in this document.

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/
4 Principle

Cigarettes are smoked on a standard smoking machine as specified in ISO 20778 that has been fitted

with impingers, but without a glass fibre filter pad as described in ISO 20778 [e.g. Cambridge filter pad

(CFP), for example of equivalent product] and the filter pad holder, with the ISO 20778 smoking regime.

The carbonyls in mainstream tobacco smoke are trapped by passing each puff through an impinger

device containing an acidified solution of 2,4-dinitrophenylhydrazine (DNPH) in 1:1 acetonitrile: water.

1) Cambridge filter pad (CFP) 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.

© ISO 2020 – All rights reserved 1
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ISO 23922:2020(E)

An aliquot of the smoke extract is then syringe-filtered and diluted with tris-(hydroxymethyl)-

aminomethane dilution solution.
The samples are subjected to analysis using reversed phase HPLC-UV or HPLC-DAD.
5 Apparatus

The usual laboratory apparatus and equipment for use in preparation of samples and standards is

needed in addition to the list provided below.
5.1 Equipment for conditioning of tobacco products.
5.2 Equipment for butt length marking.
5.3 Equipment for smoking of tobacco products, complying with ISO 20778.
5.4 Impingers for trapping mainstream smoke.

5.5 Polyvinylchloride (PVC) tubing, suitable for connection of the trapping system.

5.6 Analytical balance, capable of measuring to four decimal places.
5.7 Amber glass flasks, of capacities 10 ml, 25 ml, 200 ml, 1 l and 2 l.
5.8 Pipettes, of appropriate volumes.
5.9 Glass graduated measuring cylinders, of capacities 25 ml, 50 ml and 100 ml.
5.10 Dispenser, capable of delivering 35 ml.
5.11 Hot plate/stirrer.
5.12 Syringe filter, 0,45 µm PVDF or equivalent.
5.13 Disposable syringes, 5 ml.
5.14 Disposable glass Pasteur pipettes.
5.15 Amber Autosampler vials, caps and PTFE faced septa.
5.16 HPLC system, consisting of:
— tertiary gradient pump;
— auto-sampler with appropriate sampling loop;
— UV and/or DAD detector;
— data collection system;

— LC column: 250 mm × 4 mm, 100 Å, Reversed Phase (RP) C18 endcapped (5 µm), or equivalent;

— disposable guard column: 4 mm × 4 mm RP C18 endcapped (5 µm), or equivalent;
— vacuum filter;
2 © ISO 2020 – All rights reserved
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ISO 23922:2020(E)
— amber glass bottles 1 l and 4 l;
— desiccator.
6 Reagents
6.1 Acetonitrile, MeCN, HPLC grade.
6.2 Isopropanol, IPA, HPLC grade.
6.3 Ethyl acetate, HPLC grade.
6.4 Tetrahydrofuran, THF, HPLC grade.
6.5 Ethanol, HPLC grade.
6.6 Phosphoric acid, 85 %.
6.7 Deionized water, resistivity >18,0 MΩ.cm at 25 °C.
6.8 Formaldehyde-DNPH, min. 99 %.
6.9 Acetaldehyde-DNPH, min. 99 %.
6.10 Acetone-DNPH, min. 99 %.
6.11 Acrolein-DNPH, min. 99 %.
6.12 Propionaldehyde-DNPH, min. 98 %.
6.13 Crotonaldehyde-DNPH, min. 99 %.
6.14 2-Butanone-DNPH, min. 98 %; methyl ethyl ketone-DNPH derivative.
6.15 n-Butyraldehyde-DNPH, min. 99 %.
6.16 Tris-(hydroxymethyl)-aminomethane, ACS reagent grade .
6.17 2,4-Dinitrophenylhydrazine (DNPH) (approximately 30 % water).
7 Preparation
7.1 Preparation of glassware

Glassware shall be cleaned and dried in such a manner as to ensure that contamination from glassware

does not occur.

All possible sources of contamination shall be removed from the work area (e.g. acetone solvent wash

bottles).

2) A reagent that meets the requirements of the American Chemical Society (ACS) Committee on Analytical

Reagents.
© ISO 2020 – All rights reserved 3
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ISO 23922:2020(E)
7.2 Preparation of solutions
7.2.1 DNPH solution (using phosphoric acid)

Add approximately 150 ml deionized water to a 200 ml volumetric flask, then carefully add 28 ml of

85 % phosphoric acid (approximately 2,05 mol/l) and mix the solution.
Make up the solution to volume with deionized water.

Weigh approximately 6,8 g (24,0 mmol should be achieved) of DNPH (approximately 30 % water) into

a 2 l volumetric flask and add 1 l of acetonitrile. Dissolve DNPH by alternately gently swirling the flask.

Make sure there are no crystals remaining.
WARNING — Do not sonicate as a precipitation of DNPH may occur.

If using re-crystallized DNPH, weigh 4,8 g to achieve the same molarity (see Annex A).

After the DNPH is dissolved, add 58 ml of the diluted phosphoric acid solution while gently mixing.

Dilute to volume with deionized water. The colour of the solution will become bright orange upon

addition of the deionized water.

The addition of acid or water will cool the solution and may initiate the precipitation of the DNPH. Add

the acid or water slowly. Gentle swirling may be required to maintain the solution at room temperature

and to prevent the precipitation of DNPH. If crystals appear, do not sonicate.

Store the solution in an appropriately sized amber bottle at room temperature. This solution has been

shown to be stable for one week. Stability shall be assessed by each laboratory.

7.2.2 Tris-(hydroxymethyl)-aminomethane dilution solution, 80:20 (volume fraction), MeCN:

aqueous solution.

Dissolve 2,00 g of tris-(hydroxymethyl)-aminomethane in 200 ml of deionized water in a 1 l volumetric

flask. Dilute to volume with acetonitrile.

Store in a 1 l amber bottle with PTFE-lined cap or equivalent at ambient temperature.

7.3 Preparation of standards
7.3.1 HPLC calibration standards and working solutions
The cali
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