Organisation mondiale de la santé (OMS) · Publications

Standard operating procedure for determination of benzo[a]pyrene in mainstream cigarette smoke under ISO and intense smoking conditions: WHO TobLabNet Official Method SOP 05

3 juin 2015 Organisation mondiale de la santé

Le texte intégral est hébergé par l’organisation qui le publie. lawenc.com indexe les métadonnées et renvoie vers la source officielle.

Texte intégral

WHO TobLabNet Official Method

SOP 05

Standard Operating Procedure for Determination of Benzo[a]pyrene in Mainstream Cigarette Smoke Under ISO and Intense Smoking conditions Tobacco Free Initiative Tobacco Laboratory Network (TobLabNet)

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

No.: TobLabNet SOP 03 WHO Date: June 2014 Official Method

SOP 05

World Health Organization Standard operating procedure Tobacco Laboratory for determination ofNetwork benzo[a]pyrene in mainstream cigarette smoke under Standard operating procedure for method ISO and intense smoking conditions Determination of tobacco-speciﬁc nitrosamines in mainstream cigarette smoke under ISO and intense smoking conditions

Method:

Determination of tobacco-speciﬁc nitrosamines in mainstream cigarette smoke under ISO and intense smoking conditions 3-(1-Nitrosopyrrolidin-2-yl)pyridine(CAS# 16543-55-8) 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (CAS# 64091-91-4) N-Nitrosoanatabine (CAS# 71267-22-6) N-Nitrosoanabasine (CAS# 37620-20-5)

Analytes:

Matrix:

Tobacco cigarette mainstream smoke particulate matter

Last update: June 2014

World Health Organization Network SOP SOP World Health OrganizationTobacco Tobacco Laboratory Laboratory Network 0305 Determination of tobacco-speciﬁ c nitrosamines in mainstream tobacco smoke Standard operating procedure for determination of benzo[a]pyrene in mainstream cigarette smoke

3 1

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

WHO Library Cataloguing-in-Publication Data WHO Library Cataloguing-in-Publication Data Standard operating procedure for determination of tobacco-speciﬁc nitrosamines in mainstream cigarette smoke under ISO and intense smoking conditions. Standard operating procedure for determination of benzo[a]pyrene in mainstream cigarette smoke under ISO and intense smoking conditions. 1.Nitrosamine – analysis. 2.Tobacco – chemistry. 3.Smoke – analysis. 4.Consumer product safety. 5.Materials testing – methods.I.World Health Organization. 1.Benzo[a]pyrene – analysis. 2.Tobacco – chemistry. 3.Smoke – analysis. 4.Consumer Product Safety. 5.Materials Testing – methods. I.World Health ISBN 978 92 4 Organization. 150666 3 (NLM classiﬁcation:QV 137) ISBN 978 92 4 150832 2 (NLM classification: QV 137)

© World Health Organization 2014 © World Health Organization 2015 All rights reserved. Publications of the World Health Organization are available on the WHO website (www.who.int) or can be purchased from WHO Press, World Health All rights reserved. Publications ofOrganization, the World Health Organization are available on the website (tel.: (www. 20 Avenue Appia, 1211 Geneva 27,WHO Switzerland +41 22 791 3264; who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva fax: +41 22 791 4857; e-mail: bookorders@who.int). 27, Switzerland (tel.: +41 22 791 Requests 3264; fax: for +41 22 791 4857; permission to reproduce or translate WHO publications –whether for e-mail: bookorders@who.int). sale or for non-commercial distribution – should be addressed to WHO Press through the WHO website (www.who.int/about/licensing/copyright_form/en/index.html). Requests for permission to reproduce or translate WHO publications –whether for sale or for non-commercial distribution– should be addressed to WHO Press through the WHO website (www.who.int/about/licensing/ copyright_form/en/index.html). The designations employed and the presentation of the material in this publication do not imply the expression of any whatsoever on the part The designations employed and the presentation of the material in opinion this publication do not imply the of the World Health Organization concerning the legal Organization status of anyconcerning country, territory, city or area or of expression of any opinion whatsoever on the part of the World Health the legal its orauthorities, concerning the delimitation its frontiers Dotted status of any country, territory, city orauthorities, area or of its or concerning the of delimitation of or its boundaries. frontiers lines borderlines lines for which there may not yet be full or boundaries. Dotted and dashed lineson onmaps maps represent represent approximate approximate border for which there may not agreement. yet be full agreement.

The mention of specific companies or of certainof manufacturers’ products not imply that they are The mention specific companies or does of certain manufacturers’ products does not endorsed or recommended by theimply Worldthat Health Organization in preference to others ofthe a similar they are endorsed or recommended by Worldnature Healththat Organization in are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by and omissions preference to others of a similar nature that are not mentioned. Errors initial capital letters. excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information All reasonable precautions have been taken by the World Health Organization to contained in this publication. However, material is being distributed without warranty of published any verifythe thepublished information contained in this publication. However, the material kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the is being distributed without warranty of any kind, either expressed or implied. The reader. In no event shall the World Health Organization be liable for damages arising from its use. responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. Printed in Switzerland

4 ii

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

No.: SOP 05 Date: February 2015

No.: SOP 03 Date: June 2014

World Health World Health Organization Organization Tobacco Laboratory Laboratory Network Network Tobacco Standard operating operating procedure procedure for for method Standard method Determination of benzo[ﬁ ac ]pyrene Determination of tobacco-speci nitrosamines in mainstream cigarette smoke under ISO in mainstream cigarette smoke under and intense smoking conditions ISO and intense smoking conditions

Method: Method: Analytes: Analytes: Matrix:

Determination of tobacco-speciﬁc nitrosamines in mainstream cigarette smoke under ISO and intense smoking conditions Determination of benzo[a]pyrene in mainstream 3-(1-Nitrosopyrrolidin-2-yl)pyridine(CAS# 16543-55-8) cigarette smoke under ISO and intense smoking conditions 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Benzo[a]pyrene (CAS # 50-32-8) (CAS# 64091-91-4) N-Nitrosoanatabine (CAS# 71267-22-6) Tobacco cigarette mainstream smoke particulate matter

N-Nitrosoanabasine (CAS# 37620-20-5) Last update: February 2015 Matrix: Tobacco cigarette mainstream smoke particulate matter Last update: June 2014

5 1

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

WHO Library Cataloguing-in-Publication Data Standard operating procedure for determination of tobacco-speci ﬁc nitrosamines in mainstream No.: SOP 05 cigarette smoke under ISO and intense smoking conditions. Date: February 2015 1.Nitrosamine – analysis. 2.Tobacco – chemistry. 3.Smoke – analysis. 4.Consumer product safety. 5.Materials testing – methods.I.World Health Organization. ISBN 978 92 4 150666 3 (NLM classiﬁcation:QV 137)

The mention of specific companies or of certain manufacturers’ products does not imply ©preference World Health Organization 2014nature that they are endorsed or recommended by WHO in to others of a similar that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by capital letters.

All rights reserved. Publications of the World Health Organization are available on the WHO website (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications –whether for sale or for non-commercial distribution – should be addressed to WHO Press through the WHO website (www.who.int/about/licensing/copyright_form/en/index.html). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use.

No machine smoking regimen can represent all human smoking behaviour: machine smoking testing is useful for characterizing cigarette emissions for design and regulatory purposes, but communication of machine measurements to smokers can result in misunderstanding about differences between brands in exposure and risk. Data on smoke emissions 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 risk. Representing differences in machine measurements as differences in exposure or risk is a misuse of testing with WHO TobLabNet-recommended methods.

6 ii

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

No.: SOP 05 Date: February 2015

No.: SOP 03 Date: June 2014

FOREWORD This document was prepared by members of the World Health Organization (WHO) Tobacco Laboratory Network (TobLabNet) as an analytical method standard operating procedure (SOP) for measuring benzo[a]pyrene (B[a]P) in mainstream cigarette smoke under International Organization for Standardization (ISO) and intense smoking conditions.

INTRODUCTION In order to establish comparable measurements for testing tobacco products globally, consensus methods are required for measuring specific contents and emissions of cigarettes. The Conference of the parties to the WHO Framework Convention on Tobacco Control (WHO FCTC) at its third session in Durban, South Africa, in November 2008, “recalling its decisions FCTC/COP1(15) and FCTC/COP2(14) on the elaboration of guidelines for implementation of Articles 9 (Regulation of the contents of tobacco products) and 10 (Regulation of tobacco product disclosures) of the WHO FCTC, noting the information contained in the report of the working group to the third session of the Conference of the Parties on the progress of its work … requested the Convention Secretariat to invite WHO’s Tobacco Free Initiative to … validate, within five years, the analytical chemical methods for testing and measuring cigarette contents and emissions” (FCTC/COP/3/REC/1).

World Health Organization Tobacco Laboratory Network

Standard operating procedure for method

Using the criteria for prioritization set at its third meeting in Ottawa, Canada, in October 2006, the working group on Articles 9 and 10 identified the following contents for which methods for testing and measurement (analytical chemistry) should be validated as a priority: • nicotine • ammonia • propylene glycol propane-1,2-diol) • glycerol (propane-1,2,3-triol) • triethylene glycol (2,2-ethylenedioxybis(ethanol)). mainstream cigarette smoke under ISO and intense Measurement of these contents will require validation of three methods: one for Analytes: 3-(1-Nitrosopyrrolidin-2-yl)pyridine(CAS# 16543-55-8) nicotine, one for ammonia and one for humectants. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Using the criteria for prioritization set at the meeting in Ottawa mentioned above, (CAS# 64091-91-4) the working group identified the following emissions in mainstream smoke for which N-Nitrosoanatabine (CAS# 71267-22-6) methods for testing and measurement (analytical chemistry) should be validated as a priority: N-Nitrosoanabasine (CAS# 37620-20-5) Matrix: Tobacco cigarette mainstream smoke particulate matter Last update: June 2014 smoking conditions Method: Determination of tobacco-speciﬁc nitrosamines in

Determination of tobacco-speciﬁc nitrosamines in mainstream cigarette smoke under ISO and intense smoking conditions

7 1

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

WHO Library Cataloguing-in-Publication Data Standard operating procedure for determination of tobacco-speciﬁc nitrosamines in mainstream • 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) cigarette smoke under ISO and intense smoking conditions.

• N-nitrosonornicotine (NNN) • acetaldehyde

1.Nitrosamine – analysis. 2.Tobacco – chemistry. 3.Smoke – analysis. 4.Consumer product safety. 5.Materials testing – methods.I.World Health Organization. ISBN 978 92 4 150666 3 (NLM classiﬁcation:QV 137)

• acrylaldehyde (acrolein) • benzene • benzo[a]pyrene • 1,3-butadiene • carbon monoxide • formaldehyde

Measurement of these emissions with the two smoking regimens described below will The designations employed and the presentation of the material in this publication do require validation of five methods: one for tobacco-specific nitrosamines (NNK and not imply the expression of any opinion whatsoever on the part of the World Health NNN), one for B[a]P, one for Organization aldehydesconcerning (acetaldehyde, acrolein and formaldehyde), the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. one for volatile organic compounds (benzene, 1,3-butadiene) and one for carbon Dotted lines on maps represent approximate border lines for which there may not yet be full monoxide). agreement. The table below sets out the two smoking regimens for validation of the test methods The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in referred to above. Smoking regimen

Puff volume Puff frequency Filter (ml) All reasonable precautions have been taken by the ventilation World Health Organization to verify the information contained in this publication. However, holes the published material is being distributed without warranty of any kind, either expressed or implied. The 35 for the interpretation Once every No lies with the reader. In no ISO regimen: ISO 3308: responsibility and use of the material be liable modification for damages arising from its use. 60 s Routine analytical cigarette event shall the World Health Organization smoking machine—definitions and standard conditions 55 Once every All ventilation Intense regimen: Same as 30 s holes must be ISO 3308, but modified as blocked 100% indicated as described in WHO TobLabNet SOP 01. This SOP was prepared to describe the procedure for the determination of B[a]P in mainstream cigarette smoke under ISO and intense smoking conditions. 1 SCOPE This method is suitable for the quantitative determination of B[a]P in mainstream cigarette smoke by gas chromatography coupled with mass spectrometry (GC–MS).

preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters.

8 ii

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

B[a]P is a polycyclic aromatic hydrocarbon. It was classified as carcinogenic to humans (Group 1) by the International Agency for Research on Cancer (IARC) in 2012. It is formed during incomplete combustion of organic matter. Inhalation, oral ingestion and dermal absorption are important routes of entry.

No.: SOP 03 Date: June 2014

2 2.1 2.2 2.3 2.4 2.5 2.6 2.7

REFERENCES ISO 3402: Tobacco and tobacco products—Atmosphere for conditioning and testing. ISO 4387: Cigarettes—Determination of total and nicotine-free dry particulate matter using a routine analytical smoking machine. World Health Organization. Standard operating procedure for intense smoking of cigarettes. Geneva: Tobacco Laboratory Network (WHO TobLabNet SOP 01). ISO 3308: Routine analytical cigarette-smoking machine—Definitions and standard conditions. sampling. Vienna: Laboratory and Scientific Section; 2009 (http://www.unodc. org/documents/scientific/Drug_Sampling.pdf).

World Health Organization Tobacco Laboratory Network United Nations Office on Drugs and Crime. Guidelines on representative drug

Standard operating procedure for method ISO 8243: Cigarettes—Sampling.

Determination of tobacco-speciﬁc nitrosamines 2.8 ISO 5725-2: Accuracy (trueness and precision) of measurement methods and in mainstream cigarette smoke under results—Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method. ISO and intense smoking conditions World Health Organization. Method validation report on World Health Organization TobLabNet official method: Determination of benzo[a]pyrene in mainstream cigarette smoke under ISO and Intense smoking conditions. Geneva: Tobacco Laboratory Network, forthcoming

ISO 5725-1: Accuracy (trueness and precision) of measurement methods and results—Part 1: General principles and definitions.

2.9

Method: TERMS AND Determination DEFINITIONS of tobacco-speciﬁc nitrosamines in mainstream cigarette smoke under ISO and intense smoking conditions 3.1 TPM: total particulate matter

3-(1-Nitrosopyrrolidin-2-yl)pyridine(CAS# 16543-55-8) 3.2 Analytes: B[a]P: benzo[ a]pyrene

3.3 3.4

B[a]P-d12: deuterium-labelled B[a]P (CAS# 64091-91-4)

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

Matrix:

N-Nitrosoanatabine (CAS# 71267-22-6) Tobacco products : Products made entirely or partly of leaf tobacco as the raw material that manufactured for smoking, sucking, chewing or snuffing Nare -Nitrosoanabasine (CAS# 37620-20-5) (Article 1(f) of the WHO FCTC) Tobacco cigarette mainstream smoke particulate matter

Last update: June 2014

9 1

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

WHO Library Cataloguing-in-Publication Data

3.5 3.6 3.7

Standard operating procedure for determination of tobacco-speci ﬁc nitrosamines in mainstream Intense regimen : Parameters for smoking tobacco products that include a cigarette smoke under ISO and intense smoking conditions. 55-mL puff volume, a 30-s puff interval, a 2-s puff duration and 100% blocking of the filter ventilation holes– analysis. 2.Tobacco – chemistry. 3.Smoke – analysis. 4.Consumer product 1.Nitrosamine

ISO regimen: Parameters for smoking tobacco products that include a 35978 puff 92 4 150666 3 a 2-s puff duration (NLM classi cation:QV 137) of mL puff volume,ISBN a 60-s interval, andﬁno blocking the filter ventilation holes Laboratory sample: Sample intended for testing in a laboratory, consisting of a single type of product delivered to the laboratory at one time or within a All rights reserved. Publications of the World Health Organization are available on the specified period © World Health Organization 2014

safety. 5.Materials testing – methods.I.World Health Organization.

WHO website (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 from Geneva 27, Switzerland (tel.: +41 22 791 3264; 3.8 Test sample: Product to be tested, taken at random the laboratory sample fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications –whether for 3.9 Test portion: Random portion from the test sample to be used for a single sale or for non-commercial distribution – should be addressed to WHO Press through determination the WHO website (www.who.int/about/licensing/copyright_form/en/index.html). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health METHOD SUMMARY Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there mayis not yet be full Mainstream smoke total particulate matter from the cigarette test sample agreement.

4 4.1 4.2 4.3 4.4 4.5

trapped onto a glass-fibre filter pad (commonly referred to as a Cambridge filter pad). The mention of specific companies or of certain manufacturers’ products does not

preference others a similar nature are not breakthrough mentioned. Errorsof and omissions The number of cigarettes may to have toof be adjusted tothat prevent excepted, the names of proprietary products are distinguished by initial capital letters. the filter pad. If the TPM exceeds 600 mg for a 92-mm filter pad or 150 mg for a 44-mm filter pad, number of cigarettes smoked onto each pad Organization must Allthe reasonable precautions have been taken by the World Health to verify the information contained in this publication. However, the published material be decreased. for the interpretation and use of theis material lies with the reader. In no A solution containing responsibility an isotope-labelled internal standard spiked onto the event shall the World Health Organization be liable for damages arising from its use. pad, which is extracted with cyclohexane. is being distributed without warranty of any kind, either expressed or implied. The

imply that they are endorsed or recommended by the World Health Organization in

The cyclohexane extract is eluted through a silica solid-phase extraction cartridge. The eluent collected is analysed by gas chromatography–mass spectrometry (GC–MS) with electron ionization detection. A calibration curve is created by plotting the peak area ratio of B[a]P to B[a]P-d12 from the reconstructed ion chromatogram against known concentrations of B[a]P in standards. The B[a]P concentration in the sample is obtained from the calibration curve.

SAFETY AND ENVIRONMENTAL PRECAUTIONS CAUTION: Benzo[a]pyrene is a human carcinogen. Precautions shall be taken to avoid human exposure. B[a]P and its solutions should be handled in an adequately ventilated fume hood, glove box or equivalent. The laboratory shall establish procedures for disposal of solutions containing B[a]P.

10 ii

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

5.1 5.2

Take routine safety and environmental precautions, as in any chemical laboratory activity. The testing and evaluation of certain products with this test method may require the use of materials and or equipment that could be hazardous or harmful to the environment; this document does not address all the safety aspects associated with use of the method. All persons using this method have the responsibility to consult the appropriate authorities and to establish health and safety practices as well as environmental precautions in conjunction with any existing applicable regulatory requirements prior to its use.

No.: SOP 03 Date: June 2014

5.3 Special care should be taken to avoid inhalation or oral or dermal exposure to harmful chemicals. Use a chemical fume hood, and wear an appropriate laboratory coat, gloves and safety goggles when preparing or handling undiluted materials, standard solutions, extraction solutions or collected samples.

6 6.1 6.2 6.3

World Health Organization Tobacco Laboratory Usual laboratory apparatus, in particular: Network APPARATUS AND EQUIPMENT Equipment required to condition cigarettes as specified in ISO 3402 [2.1] Equipment required to mark buttprocedure length as specified in ISO 4387 [2.2] Standard operating for method Equipment required to cover filter ventilation holes for the intense regimen as specified in WHO TobLabNet SOP 01 [2.3]

6.4

Determination of tobacco-speciﬁc nitrosamines Equipment required to perform smoking of tobacco products as specified in ISO [2.4] in3308 mainstream cigarette smoke under 6.5 Analytical balance of measurement to conditions at least four decimal places ISO and capable intense smoking 6.6 6.7 Vortexer or wrist-action shaker or equivalent Pipettes and tips capable of accurately dispensing volumes of 10–1000 μL Volumetric pipette(s) or equivalent, 10 mL, 40 mL and 100 mL Volumetric flasks, 10 mL, 25 mL and 100 mL Method: Determination of tobacco-speciﬁc nitrosamines in

6.8 6.9 6.10

smoking conditions 6.11 Silica solid-phase extraction manifold and cartridges (500 mg): pure silica Analytes: 3-(1-Nitrosopyrrolidin-2-yl)pyridine(CAS# 16543-55-8) unbound phase 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Note: e.g. Sep-pak Vac silica cartridge (Waters), Bond Elut Jr SI cartridge (CAS# 64091-91-4) (Agilent), Supelclean LC-Si cartridge (Supelco), Strata SI-1 Silica (Phenomenex) N-Nitrosoanatabine (CAS# 71267-22-6) or equivalent

Erlenmeyer flasks, 100 mL, 200 mL or suitable flask mainstream cigarette smoke under ISO and intense

6.12

Matrix:

N-Nitrosoanabasine (CAS# 37620-20-5) Glass transfer pipettes or equivalent

Tobacco cigarette mainstream smoke particulate matter

Last update: June 2014

11 1

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

WHO Library Cataloguing-in-Publication Data

6.13 6.14 6.15 6.16

Standard operating procedure of tobacco-speci c nitrosamines in mainstream Rotary evaporator with suitable flasks,for ordetermination Turbovap with test tubesﬁor equivalent cigarette smoke under ISO and intense smoking conditions.

GC–MS system equipped with a computerized control, data acquisition and 1.Nitrosamine – analysis. 2.Tobacco – chemistry. 3.Smoke – analysis. 4.Consumer product processing system. system must be capable of operating the mass safety.The 5.Materials testing – methods.I.World Health Organization. spectrometer in order to obtain chromatographic data in single-ion monitoring ISBN 978 92 4 150666 3 classiﬁcation:QV 137) detection mode. The gas chromatograph must be(NLM configured to perform splitless injections on a capillary column. It is recommended that the gas chromatograph be equipped with an autosampler forOrganization sample injection. © World Health 2014 Column: Fused silica capillary column with a methylphenyl (5%) polysiloxane All rights reserved. Publications of the World Health Organization are available on the stationary phase, a 30-m column with 0.25-mm internal diameter and 0.25WHO website or can be purchased from WHO Press, World Health µm film thickness is suitable for (www.who.int) this analysis. Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Note: e.g. DB-5ms (Agilent) or equivalent Requests for permission to reproduce or translate WHO publications –whether for sale or for non-commercial distribution – should be addressed to WHO Press through Autosampler vials, 2 the mLWHO or equivalent website (www.who.int/about/licensing/copyright_form/en/index.html). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of REAGENTS AND SUPPLIES its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted on least maps represent approximate border lines for which there may not yet be full All reagents shall belines of at analytical reagent grade unless otherwise agreement.

7 7.1 7.2 7.3

noted. Reagents are identified by their Chemical Abstract Service (CAS) registry numbers when Theavailable mention of.specific companies or of certain manufacturers’ products does not

preference to others of a similar nature that are not mentioned. Errors and omissions Cyclohexane (110-82-7)

imply that they are endorsed or recommended by the World Health Organization in

B[a]P (50-32-8)

excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to

verify the information contained in this publication. However, the published material 7.4 B[a]P-d12 (63466-71-7)

Note: B[a]P and precautions shall be taken when manipulating these compounds or any solution containing these compounds.

is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no B[a]P-d12 are carcinogenic to humans. Appropriate safety event shall the World Health Organization be liable for damages arising from its use.

PREPARATION OF GLASSWARE Clean and dry glassware in a manner to avoid contamination.

PREPARATION OF SOLUTIONS Not applicable

PREPARATION OF STANDARDS The method for preparing standard solutions described below is for reference purposes and can be adjusted if necessary.

12 ii

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

10.1

Isotope-labelled internal standard Prepare in accordance with either option A or B. Option A 10.1.1 Primary B[a]P-d12 stock solution (200 μg/mL)

No.: SOP 03 Date: June 2014

10.1.1.1 Weigh approximately 0.005 g of B[a]P-d12 into a 25 mL volumetric flask on a four-decimal place balance, and record the weight to the nearest 0.0001 g. 10.1.1.2 Dissolve B[a]P-d12 internal standard in 25 mL of cyclohexane, and mix well. 10.1.2 10.1.1.3 Label, and store at –20 ± 5 °C in amber vials.

B[a]P-d12 spiking solution (2 μg/mL)

10.1.2.1 Pipette 1 mL of the primary B[a]P-d12 stock solution (10.1.1) into a 100-mL volumetric flask.

World Health Organization 10.1.2.2 Dilute to the mark with cyclohexane, and shake well. Tobacco Laboratory Network 10.1.2.3 Label and store at –20 ± 5 °C.

Determination of tobacco-speci ﬁc nitrosamines 10.1.4 Prepare B[a]P-d12 spiking solution (2 μg/mL). in mainstream cigarette under 10.1.4.1 Pipette 200 µL of smoke 1000 µg/mL B[a]P-d12 stock solution (10.1.3) into a 100-mL volumetric flask. ISO and intense smoking conditions 10.1.4.2 Dilute to the mark with cyclohexane, and shake well. 10.1.4.3 Label and store at –20 ± 5 °C. 10.2 Native standards 10.2.1 Primary B[a]P stock solution (200 µg/mL)

10.1.3

Option B Standard operating procedure for method

Obtain primary B[a]P-d12 stock solution of 1000 µg/mL in methylene chloride from a commercial supplier, label, and store at –20 ± 5 °C.

Method: Determination of approximately tobacco-speciﬁ0.005 c nitrosamines in a 25-mL 10.2.1.1 Weigh g B[a]P into mainstream cigarette smoke under ISO and intense volumetric flask on a four-decimal place balance, and smoking conditions record the weight to the nearest 0.0001 g.

10.2.1.2 Dissolve the B[a]P standard in 25 mL of cyclohexane, 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and mix well. 10.2.2 10.2.1.3 Label and store at –20 ± 5 °C. N-Nitrosoanatabine (CAS# 71267-22-6) Secondary B[a]P stock (CAS# solution (1 µg/mL) N-Nitrosoanabasine 37620-20-5) (CAS# 64091-91-4)

Analytes:

3-(1-Nitrosopyrrolidin-2-yl)pyridine(CAS# 16543-55-8)

Matrix: Tobacco mainstream smoke particulate matter 10.2.2.1 cigarette Pipette 0.5 mL of the primary B[a]P stock solution ( Last update: June 2014 10.2.1) into a 100-mL volumetric flask.

13 1

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

WHO Library Cataloguing-in-Publication Data

Standard operating for determination of tobacco-speci ﬁcmix nitrosamines 10.2.2.2 Diluteprocedure to the mark with cyclohexane, and well. in mainstream cigarette smoke under ISO and intense smoking conditions.

10.2.2.3

The standard solutions are stable for up to 6 months if stored at 3 –20 ± 5 °C. ISBN 978 92 4 150666 (NLM classiﬁcation:QV 137) 10.3

1.Nitrosamine – analysis. 2.Tobacco – chemistry. 3.Smoke – analysis. 4.Consumer product safety. 5.Materials testing – methods.I.World Health Organization.

Label and store at –20 ± 5 °C.

Note: Sonication may be used in the preparation of primary stock © World Health Organization solutions to help dissolve B[a]P and B[a]P-d12 standards 2014 completely. In this case, first dissolve the standards in about 15 mL of solvent. After sonication, let the solution stand until it cools to working All rights reserved. Publications of the World Health Organization are available on the temperature (e.g. room temperature), dilute from to the mark with WHO website (www.who.int) or can then be purchased WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; cyclohexane. fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications –whether for standard solutions sale or for non-commercial distribution – should be addressed to WHO Press through the WHO website (www.who.int/about/licensing/copyright_form/en/index.html).

B[a]P working

Prepare working standard solutions as stated below and summarized in Table 1. The designations employed and the presentation of the material in this publication do Organization concerning the legal status of any country, territory, city or area or of Table 1. Working standard solutions of B[a]P Standard not imply the expression of any opinion whatsoever on the part of the World Health

1 2 3 4 5 6

its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines of on maps represent lines there may not yet be full Volume Volume approximate of Totalborder volume (mL)for which Approximate agreement. secondary B[a]P-d12 spiking concentration B[a]P stock solution (2 μg/ of B[a]P working The mention of specific companies or of certain manufacturers’ products does not standard solution mL from 10.1.2 or standard solution imply (μL) that they are endorsed Health Organization in (1 μg/mL) 10.1.4) (μL) or recommended by the World (ng/mL) preference to others of a similar nature that are not mentioned. Errors and omissions 20 100 10 are distinguished 2by initial capital letters. excepted, the names of proprietary products 40 100 10 4 All reasonable precautions 80 100 have been taken 10 by the World Health 8 Organization to verify the information contained in this publication. However, the published material 200 100 warranty of any 10kind, either expressed 20 or implied. The is being distributed without 400 10of the material lies 40 responsibility for the 100 interpretation and use with the reader. In no event Health Organization be liable for damages 600 shall the World 100 10 60 arising from its use.

Note: All solvents and solutions must be equilibrated to room temperature before use. Spike variable volumes of the secondary B[a]P stock solution from 10.2.2 into a 10-mL volumetric flask. Add 100 μL of the B[a]P-d12 spiking solution prepared in 10.1.2 or 10.1.4.

10.3.1 10.3.2

10.3.3 If option A is chosen, the concentrations of B[a]P-d12 spiking solution (10.1.2) can be determined from the equation: 10.3.4 Final concentration (µg/mL) = x × 400, where x is the original weight (in g) of standard as determined in 10.1.1.1. When option B is chosen, the concentration of B[a]P-d12 spiking solution (10.1.4) is computed according to the value provided by the supplier.

14 ii

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

10.3.5

Fill to the mark with cyclohexane, and mix well.

No.: SOP 03 Date: June 2014

10.3.6

The final concentrations of standard are determined from the equation: Final concentration (ng/mL) = x × y × 20. Note: The equation in 10.3.6 is derived as follows: The concentrations of B[a]P working standard solutions (in ng/mL) can be calculated from: x (g) 0.5 mL y (μL) 1 mL 1 000 000 000 ng × × × × 25 mL 100 mL 10 mL 1000 μL 1g = x × 0.5 × 1000 × y (ng) 25 mL = x × y × 20

10.3.7

World Health Organization Tobacco Laboratory Network

where x is the original weight (in g) of standard as determined in 10.2.1.1 , and y is the volume (in μL) of secondary B[a]P stock solution as described in 10.2.2 and Table 1.

Standard operating procedure for method

The range of the standard solutions may be adjusted in accordance with the equipment used and the samples to be tested, keeping in mind a possible effect on the sensitivity limits of the method.

Determination of tobacco-speciﬁc nitrosamines in mainstream cigarette smoke under 11.1 Sample cigarettes according to ISO 8243 [2.6]. Alternative approaches may be used to obtain a representative laboratory sample in accordance with ISO and intense smoking conditions individual laboratory practice or when required by specific regulation or the 11 Sampling availability of samples. 11.2 Constitution of test sample 11.2.1 Divide the laboratory sample into separate units (e.g. packet, container), if applicable. Method: TakeDetermination of tobacco-speci ﬁc nitrosamines in 11.2.2 an equal amount of product for each test sample from mainstream cigarette smoke under ISO and intense at least √n [2.5] of the individual units (e.g. packet, container). smoking conditions Analytes: 3-(1-Nitrosopyrrolidin-2-yl)pyridine(CAS# 16543-55-8) (CAS# 64091-91-4)

CIGARETTE PREPARATION 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone N-Nitrosoanatabine (CAS# 71267-22-6)

12.1 Condition all cigarettes to be smoked in accordance with ISO 3402 [2.1]. 12.2 Mark cigarettes at a butt length(CAS# in accordance with ISO 4387 [2.2] and N-Nitrosoanabasine 37620-20-5) WHO TobLabNet SOP 01 [2.3]. Matrix: Tobacco cigarette mainstream smoke particulate matter Last update: June 2014

15 1

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

WHO Library Cataloguing-in-Publication Data operating procedure for determination of tobacco-speci ﬁc nitrosamines in mainstream 12.3 Prepare testStandard samples to be smoked in accordance with either ISO or cigarette smoke under ISO and intense smoking conditions. intense smoking conditions as specified in WHO TobLabNet SOP 01 [2.3]. 1.Nitrosamine – analysis. 2.Tobacco – chemistry. 3.Smoke – analysis. 4.Consumer product safety. 5.Materials testing – methods.I.World Health Organization.

PREPARATION OF THE SMOKING ISBN 978 92 4 150666MACHINE 3

(NLM classiﬁcation:QV 137)

13.1 Ambient conditions 13.2

The ambient conditions for smoking are specified in ISO 3308 [2.4].

Smoking machine specifications All rights reserved. Publications of the World Health Organization are available on the Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 Follow ISO 3308 [ 2.4 ] smoking machine specifications, except for22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). intense smoking, for which the smoking machine should be prepared as Requests for permission to reproduce or translate WHO publications –whether for described in WHO TobLabNet SOP 01 [2.3distribution ]. sale or for non-commercial – should be addressed to WHO Press through the WHO website (www.who.int/about/licensing/copyright_form/en/index.html). WHO website (www.who.int) or can be purchased from WHO Press, World Health

14 14.1 14.2 14.3 14.4

not imply the expression of any opinion whatsoever on the part of the World Health SAMPLE GENERATION Organization concerning the legal status of any country, territory, city or area or of

The designations employed and the presentation of the material in this publication do

its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted Smoke a sufficient number of cigarettes on the specified smoking machine lines on maps represent approximate border lines for which there may not yet be full such that breakthrough does not occur and the concentrations of the B[a]P agreement. fall within the calibration range prepared for the analysis. they are endorsed or recommended World Health Organization in Smoke the cigarette imply test that samples, and collect the TPM by asthe specified in ISO preference to others of a similar nature that are not mentioned. Errors and omissions 4387 [2.2] or WHO TobLabNet SOP 01 [2.3]. The mention of specific companies or of certain manufacturers’ products does not

excepted, the names of proprietary products are distinguished by initial capital letters.

Include at least one reference test sample for quality control, if applicable. All reasonable precautions have been taken by the World Health Organization to

When testing sample for the first time, evaluate breakthrough oforthe is types being distributed without warranty of any kind, either expressed implied. The responsibility for the interpretation and the material If lies withTPM the reader. In no filter pad. The number of cigarettes might have touse beofadjusted. the shall the World Health Organization be liable for damages arising from its use. exceeds 600 mg for event a 92-mm filter pad or 150 mg for a 44-mm filter pad, decrease the number of cigarettes smoked onto each pad. The numbers of cigarettes to be smoked per measurement for linear and rotary smoking machines in ISO and intense smoking regimes are shown in Table 2. Table 2. Suggested numbers of cigarettes to be smoked for one measurement on linear and rotary smoking machines ISO smoking regimen Linear No. of cigarettes per pad No. of pads per result 5 4 Rotary 20 1 Intense smoking regimen Linear 3 3 Rotary 10 1

verify the information contained in this publication. However, the published material

Note: The number of cigarettes to be smoked should be adjusted such that breakthrough does not occur and the concentrations of B[a]P fall within the calibration range prepared for the analysis.

16 ii

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

14.5

Record the number of cigarettes and total puffs for each filter pad.

No.: SOP 03 Date: June 2014

14.6 After smoking the required number of test samples, perform five clearing puffs, and remove the pad holder from the smoking machine.

15 15.1

SAMPLE PREPARATION Extraction of filter pads

15.1.1 Remove the pads from the holders. Fold each pad loosely in half and then in half again, with the TPM on the inside. Use the side opposite to that on which TPM is collected to wipe the inner surface of the pad holder, thus including any particulate matter that may have been left in the holder. Transfer the filter pad(s) into a 100-mL Erlenmeyer or suitable flask for a 44-mm pad or a 200-mL Erlenmeyer or suitable flask for a 92-mm pad. Note: In the ISO regimen, four 44-mm pads for a linear smoking machine or one 92-mm pad for a rotary smoking machine are extracted into one flask. In the intense regimen, three 44-mm pads for a linear smoking machine or one 92-mm pad for a rotary smoking machine are extracted into one flask. 15.1.2 Add cyclohexane (40 mL for 44-mm pads, 100 mL for 92-mm pads) to the flask. 15.1.3 Spike each flask with B[a]P-d12 spiking solution (10.1.2 or 10.1.4) (40 μL for a 44-mm pad, 100 μL for a 92-mm pad)

World Health Organization Tobacco Laboratory Network

Standard operating procedure for method

Determination of tobacco-speciﬁc nitrosamines 15.1.4 a vortexer, wrist-action or equivalent device, extract the B[a]P in Using mainstream cigarette smoke under from the pad by shaking at 200 rpm for 60–80 min. ISO and intense smoking conditions 15.2 Sample clean-up

15.2.1 Condition a solid-phase extraction cartridge with 10 mL of cyclohexane, and discard the eluate. Do not let the cartridge run dry. 15.2.2 Pipette 10 mL of sample extract onto the cartridge, and allow it to pass through the cartridge. Collect the eluate. Method: of tobacco-speci ﬁc nitrosamines 15.2.3 EluteDetermination the cartridge with two further 15-mL aliquots ofin cyclohexane, mainstream cigarette smoke under ISO and intense allowing the cartridge to run dry after the last aliquot is passed through. smoking conditions

15.2.4 Evaporate the cyclohexane solution almost to dryness. Analytes: 3-(1-Nitrosopyrrolidin-2-yl)pyridine(CAS# 16543-55-8)

Note4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone : The conditions of the rotary evaporator are to be set at 55 °C 64091-91-4) with (CAS# a vacuum pressure of 30 kPa (300 mbar) (approximately 10 min).N The vacuum pressure can 71267-22-6) be adjusted for the equipment. The -Nitrosoanatabine (CAS# conditions of the Turbovap are to be set at 30 °C in a steady stream N-Nitrosoanabasine (CAS# 37620-20-5) of nitrogen (approximately 15 min). Matrix: Tobacco cigarette mainstream smoke particulate matter Last update: June 2014

17 1

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

WHO Library Cataloguing-in-Publication Data Standard procedureinto for determination of dissolve tobacco-speci ﬁcextract. nitrosamines in mainstream 15.2.5 Pipette 1 mL operating of cyclohexane the flask to the Cap cigarette smoke under ISO and intense smoking conditions. the flask, and rotate it carefully to rinse the inner surface. Take an aliquot for GC–MS testing. 1.Nitrosamine – analysis. 2.Tobacco – chemistry. 3.Smoke – analysis. 4.Consumer product

Important note : The final concentration of B[a]P-d12 internal 978sample 92 4 150666 3 (NLM classiﬁcation:QV 137) standardISBN in the is approximately 20 ng/mL. © World Health Organization 2014

safety. 5.Materials testing – methods.I.World Health Organization.

16 16.1

SAMPLE ANALYSIS All rights reserved. Publications of the World Healthsmoke Organization are available on the This method for quantifying B[a]P in mainstream cigarette involves WHO website (www.who.int) or can be purchased from WHO Press, World Health GC–MS. The analytes are resolved potentially interfering Organization, 20 Avenuefrom Appia, other 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). substances on a GC column. Comparison of the area ratio (native analyte to Requests for permission to reproduce or translate WHO publications –whether for isotope-labelled analyte) of the unknowns with the area ratio (native analyte sale or for non-commercial distribution – should be addressed to WHO Press through to isotope- labelled analyte) the known standard concentrations yields the the WHOof website (www.who.int/about/licensing/copyright_form/en/index.html). concentrations of the analyte. not imply the expression of any opinion whatsoever on the part of the World Health GC–MS operating conditions: example The designations employed and the presentation of the material in this publication do Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted Fused silica capillary column with a methylphenyl (5%) lines on maps represent approximate border lines for which there may not yet be full polysiloxane stationary phase, a 30-m column with a agreement.

GC column Injector

The mention of specific companies or of certain manufacturers’ products does not is suitable (DB-5ms Agilent or equivalent). imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions temperature excepted, 280 °C the names of proprietary products are distinguished by initial capital letters.

0.25-mm internal diameter and 0.25-µm film thickness

Mode Flow rate Injection

Constantprecautions flow All reasonable have been taken by the World Health Organization to verify the information contained in this publication. However, the published material 1.2 distributed mL/min without warranty of any kind, either expressed or implied. The is being responsibility for the interpretation and use of the material lies with the reader. In no event shall the Health Organization be liable for damages arising from its use. 1 µL or 2 World µL splitless

Column temperature 150 °C for 0 min; 6 °C/min to 260 °C, hold at 260 °C for 7 min; 50 °C/min to 290 °C, hold at 290 °C for 20 min Transfer line temperature MS source Ion traces Dwell time Ionization mode 280 °C 230 °C B[a]P: m/z 252 B[a]P-d12: m/z 264 50 ms Electron ionization

Note: The operating parameters may have to be adjusted to the instrument and column conditions and the resolution of the chromatographic peaks.

18 ii

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

16.2

General analytical information

No.: SOP 03 Date: June 2014

16.2.1 For the conditions described here, the expected sequence of elution will be B[a]P-d12, B[a]P. 16.2.2 Differences in e.g. temperature, gas flow rate and the age of the column can be expected to alter retention times. 16.2.3 The sequence of determination of B[a]P will be in accordance with individual laboratory practice. This section gives an example. 16.2.4 Inject a blank solution (extraction solution minus labelled internal standard) to check for any contamination in the system or reagents. 16.2.5 Inject a standard blank (blank with labelled internal standard) to verify the performance of the GC–MS system. 16.2.6 Inject the calibration standards, the quality control and the samples. 16.2.7 Record the peak areas of B[a]P and B[a]P-d12.

16.2.8 standard blanks.

World Health Organization Calculate the relative response ratio of the B[a]P peak to the Tobacco Network B[a]P-d12 peakLaboratory (A /A ) for each standard solution, including B[a]P B[a]P-d12

16.2.9 Plot a graph of the concentration of B[a]P (X axis) against the area ratios (Y axis).

Standard operating procedure for method

16.2.10 The intercept should not be statistically significantly different from zero.

Determination of tobacco-speciﬁc nitrosamines 16.2.11 The standard curve should be linear over the entire standard range. in mainstream cigarette smoke under 16.2.12 Calculate the linear regression (Y = a + bx) from these data, and ISO intense smoking conditions useand both the slope (b) and the intercept (a) of the linear regression. If the linear regression, R2, is less than 0.99, the calibration should be repeated. If an individual calibration point differs by more than 10% from the expected value (estimated by linear regression), the point should be omitted.

16.2.13 Inject the quality controls and samples, and determine the peak with the appropriate software.ﬁc nitrosamines in Method: areas Determination of tobacco-speci 16.2.14 Analytes: Thesmoking peak ratio obtained for all test portions must fall within the conditions working range of the calibration curve; otherwise, the concentrations 3-(1-Nitrosopyrrolidin-2-yl)pyridine(CAS# 16543-55-8) of the standard and standard solutions or test portion solutions 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone should be adjusted. (CAS# 64091-91-4) N-Nitrosoanatabine (CAS# 71267-22-6) mainstream cigarette smoke under ISO and intense

See Annex 1 for representative chromatograms. N-Nitrosoanabasine (CAS# 37620-20-5) Matrix: Tobacco cigarette mainstream smoke particulate matter Last update: June 2014

19 1

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

WHO Library Cataloguing-in-Publication Data

17 17.1 17.2

Standard procedure for determination of tobacco-speciﬁc nitrosamines in mainstream DATA ANALYSIS ANDoperating CALCULATIONS cigarette smoke under ISO and intense smoking conditions.

The slope and intercept are determined from the relative responses of 1.Nitrosamine – analysis. 2.Tobacco – chemistry. 3.Smoke – analysis. 4.Consumer product B[a]P and B[a]P-d12 the relative concentrations B[a]P. safety.versus 5.Materials testing – methods.I.World Healthof Organization. Calculate the relative response ratio from the peak areas for each of the ISBN 978 92 4 150666 3 (NLM classi ﬁcation:QV 137) calibration standards (10.3): © World RF = AB[a]P / AB[a]P-d12 , Health Organization 2014

17.3 17.4 17.5

where RF is the relative response ratio, AB[a]P is the peak area of B[a]P All rights reserved. m/z Publications the World Health available on the (reconstructed ion chromatogram 252)of and AB[a]P-d12 is Organization the peak are area WHO website (www.who.int) or can be purchased from WHO Press, World Health of B[a]P-d12 (reconstructed ion 20 chromatogram m/z 264). Organization, Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; Plot a graph of the relative factor, AB[a]Por / translate AB[a]P-d12 (Y axis), versus Requests response for permission to reproduce WHO publications –whether for sale for or for non-commercial distribution – should be addressed WHO Press through concentration (X axis) each standard solution. Calculate thetolinear the WHO website (www.who.int/about/licensing/copyright_form/en/index.html). regression (Y = a + bx) from these data, and use both the slope (b) and the intercept (a) of the linear regression. The designations employed and the presentation of the material in this publication do Organization concerning the legal statusstandard of any country, territory, city or area or of The standard curve should be linear over the entire range. its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted not imply the expression of any opinion whatsoever on the part of the World Health fax: +41 22 791 4857; e-mail: bookorders@who.int).

lines on maps represent approximate borderfrom lines for which there may not yet be full The content of B[a]P (ng/cigarette) is determined the calculated agreement. relative response ratio for the test portion, the slope and the intercept obtained from the appropriate curve and the equation: The mention calibration of specific companies or of certain manufacturers’ products does not imply that they or recommended by the World Health Organization in Y are - a endorsed V×V e preference of a similar nature that are not mentioned. Errors and omissions M = to others× b c × n excepted, the names of V proprietary products are distinguished by initial capital letters. where M is the calculated content of B[a]P in nanograms per cigarette, Y is All reasonable precautions have been taken by the World Health Organization to the relative response ratio AB[a]P / AB[a]P-d12 , a is the intercept of the linear verify the information contained in this publication. However, the published material regression obtained from the standard calibration curve, b either is the slope of the is being distributed without warranty of any kind, expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no linear regression obtained from the standard calibration curve, V is the volume event shall the World Health Organization be liable for damages arising from its use. of the sample solution (1 mL), Ve is the volume of the extraction solution (40 mL for a 44-mm Cambridge filter pad, 100 mL for a 92-mm Cambridge filter pad), Vc is the volume of the aliquot of the extraction solution used in clean-up (10 mL), and n’ is the number of cigarettes smoked (see Table 2).

Alternative calculation procedures may be used if applicable.

SPECIAL PRECAUTIONS After installing a new column, condition it as specified by the manufacturer by injecting a tobacco sample extract under the specified instrument conditions. Injections should be repeated until the peak areas (or heights) of both B[a]P and B[a]P-d12 are reproducible.

19 19.1

DATA REPORTING Report individual measurements for each sample evaluated.

20 ii

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

19.2 20 20.1

Report results as nanograms per cigarette or as required. QUALITY CONTROL Control parameters

No.: SOP 03 Date: June 2014

Note: If the control measurements are outside the tolerance limits of the expected values, appropriate investigation and action must be undertaken. Note: Additional quality assurance procedures should be used if necessary in order to comply with the policies of individual laboratories. 20.2 20.3 Laboratory reagent blank To detect any contamination during sample preparation and analysis, include a laboratory reagent blank, as described in 16.2.5 The blank consists of all the reagents and materials used in analysing test samples and is analysed like a test sample. The blank should be assessed in accordance with the practices of individual laboratories.

World Health Organization Quality control sample Tobacco Laboratory Network

Standard operating procedure for method

To verify the consistency of the entire analytical process, analyse a reference cigarette (or an appropriate quality control sample) in accordance with the practices of the individual laboratory.

20.4 Make a new calibration curve after every 20 injections of test sample (20 sample extracts).

Determination of tobacco-speciﬁc nitrosamines in mainstream cigarette smoke under 21 METHOD PERFORMANCE SPECIFICATIONS and intense smoking conditions 21.1 LimitISO of reporting 21.2

The limit of reporting is set to the lowest concentration of the calibration standards used, recalculated to nanograms per cigarette Laboratory-fortified matrix recovery Recovery of analyte spiked onto the matrix is used as a surrogate measure of accuracy. Recovery is determined by spiking a known amount of Method: Determination of tobacco-speciﬁc nitrosamines in standard (after smoking) onto a filter pad with cigarette smoke and mainstream cigarette smoke under ISO and intense extracting the pad by the same method used for extracting samples. smoking conditions Unspiked pads are also analysed. The recovery is calculated from the Analytes: 3-(1-Nitrosopyrrolidin-2-yl)pyridine(CAS# 16543-55-8) following equation and is shown in Table 3: Recovery (%) =64091-91-4) 100 × (analytical result – unspiked result / spiked amount) (CAS#

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone N-Nitrosoanatabine (CAS# 71267-22-6) N-Nitrosoanabasine (CAS# 37620-20-5)

Matrix:

Tobacco cigarette mainstream smoke particulate matter

Last update: June 2014

21 1

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

WHO Library Cataloguing-in-Publication Data

Table 3. Mean and recovery of B[a]P spiked onto the matrix of tobacco-speciﬁc nitrosamines in mainstream Standard operating procedure for determination cigarette smoke under ISO and intense smoking conditions. Spiked amount (ng/cig) Mean (ng/cig) Recovery (%) 1.Nitrosamine – analysis. 2.Tobacco – chemistry. 3.Smoke – analysis. 4.Consumer product 2.91 safety. 5.Materials testing 2.93 100.58 – methods.I.World Health Organization. 6.79 6.82 100.39 (NLM classi ﬁcation:QV 137) 9.70 ISBN 978 92 4 150666 3 10.08 103.92

21.3

Analytical specificity

GC–MS provides analytical specificity. The retention time and molecular mass to charge ratio (m/z) are used to verify the specificity of the results All rights reserved. Publications of the World Health Organization are available on the for an unknown sample. WHO website (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications –whether for salecurves or for non-commercial distribution – should be addressed to WHO Press through calibration established are linear over the standard the WHO website (www.who.int/about/licensing/copyright_form/en/index.html). The designations employed and the presentation of the material in this publication do Organization concerning the legal status of any country, territory, city or area or of agreement.

21.4 Linearity 21.5

The B[a]P concentration range of 2–60 ng/mL.

Possible interference not imply the expression of any opinion whatsoever on the part of the World Health

No known components have both similar retention times and m/z B[a]P Dotted its authorities, or concerning the delimitation of its frontiers oras boundaries. lines on maps represent approximate border lines for which there may not yet be full or B[a]P-d12. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in andpreference reproducibility limits to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters.

Repeatability

An international collaborative study [2.9] conducted in 2012, involving testing All reasonable precautions have been taken and by thetwo World Health Organization to of three reference cigarettes (1R5F, 3R4F and CM6) commercial verify the gave information contained in this publication. However,indicated the published material brands by eight laboratories, the precision limits for the method is being distributed without warranty of any kind, either expressed or implied. The in Table 4. responsibility for the interpretation and use of the material lies with the reader. In no The difference between two single results found for matched cigarette samples by the same operator using the same apparatus within the shortest feasible time will exceed the repeatability, r, on average not more than once in 20 cases in the normal, correct application of the method. Single results for matched cigarette samples reported by two laboratories will differ by more than the reproducibility, R, on average no more than once in 20 cases with normal, correct application of the method.

event shall the World Health Organization be liable for damages arising from its use.

The test results were analysed statistically in accordance with ISO 5725-1 [2.7] and ISO 5725-2 [2.8] to give the precision data shown in Table 4.

22 ii

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

Table 4. Precision limits for the determination of B[a]P (ng/cigarette) in mainstream cigarette smoke from reference cigarettes Reference cigarette 1R5F 3R4F CM6 ISO smoking regimen n 8 8 7 mcig 1.46 5.99 13.70 rlimit 0.47 1.03 1.43 Rlimit 0.99 2.09 5.83 n 8 8 7 Intense smoking regimen mcig 6.47 14.51 25.86 rlimit 1.52 2.52 2.81 Rlimit 2.91 3.15 8.45

No.: SOP 03 Date: June 2014

Where 1R5F, 3R4F and CM6 are three reference cigarettes analysed in this study, n is the number of laboratories that participated, mcig is the mean value of B[a]P per cigarette, rlimit is the repeatability limit of B[a]P, and Rlimit is the reproducibility limit of B[a]P.

For the purpose of calculating r and R, one test result under ISO smoking conditions was defined as the average of seven individual replicates of the mean yield of four Cambridge filter pads (five cigarettes smoked per pad) from linear smoking machines and one Cambridge filter pad (20 cigarettes smoked per pad) from rotary smoking machines. Under intense smoking conditions, one test result was defined as the average of seven individual replicates of the mean yield of two sets of three Cambridge filter pads (three cigarettes smoked per pad) from linear smoking machines and two sets of one Cambridge filter pad (10 cigarettes smoked per pad) from rotary smoking machines. For more information, see reference [2.9].

World Health Organization Tobacco Laboratory Network

Standard operating procedure for method TEST REPORT

Determination of tobacco-speciﬁc nitrosamines The following information shall be included in the test report: in mainstream cigarette smoke under A reference to this method, i.e. WHO TobLabNet SOP 05 ISO and intense smoking conditions Date of receipt of the sample ● ●

● The results and their units

Method:

Analytes:

Matrix:

Tobacco cigarette mainstream smoke particulate matter

Last update: June 2014

23 1

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

WHO Library Cataloguing-in-Publication Data Standard operating procedure in for the determination of tobacco-speci ﬁc nitrosamines in mainstream Annex 1. Typical chromatograms obtained determination of benzo[ a]pyrene cigarette smoke under ISO and intense smoking conditions. in mainstream cigarette smoke 1.Nitrosamine – analysis. 2.Tobacco – chemistry. 3.Smoke – analysis. 4.Consumer product safety. 5.Materials testing – methods.I.World Health Organization. ISBN 978 92 4 150666 3 (NLM classiﬁcation:QV 137)

© World Health Organization 2014 All rights reserved. Publications of the World Health Organization are available on the WHO website (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications –whether for sale or for non-commercial distribution – should be addressed to WHO Press through the WHO website (www.who.int/about/licensing/copyright_form/en/index.html). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on represent approximate border lines for which there may not yet be full Figure 1. Representative chromatogram ofmaps a standard solution agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use.

Figure 2. Representative chromatogram of a sample solution

24 ii

Tobacco Free Initiative Tobacco Laborator y Network (TobLabNet)

No.: SOP 03 Date: June 2014

World Health Organization Tobacco Laboratory Network Standard operating procedure for method Determination of tobacco-speciﬁc nitrosamines in mainstream cigarette smoke under ISO and intense smoking conditions

Method:

Analytes:

Matrix:

Tobacco cigarette mainstream smoke particulate matter

Last update: June 2014

25 1

This document was prepared by members of the World Health Organization (WHO) Tobacco Laboratory Network (TobLabNet) as an analytical method standard operating procedure (SOP) for determination of benzo[a]pyrene in mainstream cigarette smoke under International Organization for Standardization (ISO) and intense smoking conditions.

9 789241 508322

Informations clés

Organisation Organisation mondiale de la santé (OMS)

Type de document Publications

Date d'adoption 3 juin 2015

Source Organisation mondiale de la santé