Smoke Analytes Sub-Group - CORESTA | Home...Smoke Analytes Sub-Group Technical Report 2016 Joint Experiment on Aromatic Amines in Mainstream Cigarette Smoke by LC-MS/MS November 2019

Smoke Analytes Sub-Group

Technical Report

2016 Joint Experiment on

Aromatic Amines in Mainstream

Cigarette Smoke by LC-MS/MS

November 2019

Author and SMA Sub-Group Coordinator:

Jana Jeffery, Ph.D., British American Tobacco, U.K.

Study Project Leaders:

Michael Intorp, Ph.D., ITL-Reemtsma, Germany

Jana Jeffery, Ph.D., British American Tobacco, U.K.

Statistical Analysis (2019):

Oscar M. Camacho – Graphs, British American Tobacco, U.K.

Guidance on data interpretation:

Michael J. Morton, Ph.D., Altria Client Services, U.S.A.

Table of Contents

1. SUMMARY ...................................................................................................................... 3

2. INTRODUCTION ............................................................................................................ 4

3. ORGANISATION ............................................................................................................ 4

3.1 Participants .............................................................................................................................. 4

3.2 Protocol .................................................................................................................................... 5

3.2.1 Sample Shipment ......................................................................................... 5

3.2.2 Sample Preparation ...................................................................................... 5

3.2.3 Sample Analysis and Data Reporting .......................................................... 5

4. DATA – SUMMARY DESCRIPTIVE ANALYSIS ....................................................... 6

5. DATA – STATISTICAL ANALYSIS ............................................................................. 9

5.1 Outlier Detection.................................................................................................................... 9

5.2 Repeatability and Reproducibility ...................................................................................... 9

6. RECOMMENDATIONS .................................................................................................. 9

7. APPENDICES .................................................................................................................. 9

Appendix A: Study Protocol ........................................................................................... 10

Appendix B: Analytical Method ..................................................................................... 13

Appendix C: Data Summary ........................................................................................... 24

Appendix D: Full Data Sets ............................................................................................ 26

Appendix E: Raw Data Plots .......................................................................................... 29

SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 3/35

1. Summary

From 2014 to 2017, the CORESTA Special Analytes Sub Group[1] (SPA SG) evaluated

analytical methods for quantitative measurement of aromatic amines (o-toluidine,

2,6-dimethylaniline, o-anisidine, 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl

and 4-aminobiphenyl) in mainstream cigarette smoke.

At the beginning of the project, a survey was conducted to get insight into methodologies used

for measurement of aromatic amines by the SG. Due to their physico-chemical properties and

presence at trace levels (typically ng/cigarette), the analysis of these analytes is challenging.

Although most laboratories employed methods using Gas Chromatography Mass Spectrometry

(GC/MS), there was a group of laboratories who used methods based on Liquid

Chromatography Mass Spectrometry (LC-MS/MS). Both approaches were evaluated through a

series of joint experiments. Although the GC/MS based method was taken forward for a

Collaborative Study (the results were captured in the separate report SMA-048-2-CTR[2]), it

was agreed by the SG that the findings of the LC-MS/MS method evaluation will be captured

in this Technical Report.

The advantage of an LC-MS/MS based approach is in the omission of the derivatisation step -

compared to traditionally used GC/MS - which can simplify the method and increase sample

throughput. Due to very low yields of aromatic amines in the mainstream smoke, a clean-up

step (e.g. Solid Phase Extraction, SPE) is required to remove unwanted matrix components and

to concentrate target analytes. Even with this significant advantage, the SG chose to pursue a

GC/MS approach because GC/MS was most prevalent in the participating laboratories.

A Joint Experiment (JE) with LC-MS/MS was conducted in 2016 and was aimed at the

comparison of two SPE approaches; using (i) a single cartridge (MCX) or (ii) two cartridges

with different stationary phases (dual SPE). Kentucky reference cigarettes 3R4F smoked under

both ISO and intense[3] smoking regimes were selected as a testing matrix.

Four laboratories from one country participated in the study. Due to a very small data set, only

a limited comparison was possible. Statistical analysis (t-test) was carried out to compare mean

smoke yields of aromatic amines achieved by participating laboratories using both clean-up

strategies. No statistically significant differences were observed between the two limited data

sets.

For the ISO smoking regime, the relative differences between results obtained from both SPE

clean-ups were in the range from 1 % (o-toluidine) to 13 % (1-aminonaphthalene) for

5 cigarettes smoked and from 3 % (3-aminobiphenyl) to 40 % (1- and 2-aminonaphthalene) for

2 cigarettes smoked. The biggest variation of aromatic amines yields was observed when

2 cigarettes were smoked under ISO smoking regime, simulating low tar yield product, thus

representing the lowest yields of the mainstream smoke.

For the intense smoking regime, the difference of results between both SPE clean-up methods

varied between 2 % (3-aminobiphenyl) – 22 % (o-anisidine).

A larger study employing more laboratories would be required to further investigate both

methods and to confirm the findings.

[1] Since February 2017 the name SPA SG was changed to Smoke Analytes Sub Group (SMA SG, SMA). [2] 2016 Collaborative Study on Aromatic Amines in Mainstream Cigarette Smoke; available at

https://www.coresta.org/2016-collaborative-study-aromatic-amines-mainstream-cigarette-smoke-32272.html [3] At the time of the study Health Canada Intense (HCI) smoking regime was used and is therefore referred to in

the document. Since Q4 2018, an ISO intense smoking regime standard ISO 20778 was adopted.

https://www.coresta.org/2016-collaborative-study-aromatic-amines-mainstream-cigarette-smoke-32272.html


2. Introduction

Aromatic amines (o-toluidine, 2,6-dimethylaniline, o-anisidine, 1-aminonaphthalene,

2-aminonaphthalene, 3-aminobiphenyl and 4-aminobiphenyl) in mainstream cigarette smoke

are challenging analytes for analysis. Due to their physico-chemical properties and low yields

(ng/cigarette), analytical methods require a clean-up step(s) for removal of unwanted matrix

co-extracts. Traditionally and frequently used GC/MS-based methods require the use of a

derivatisation step to allow for measurement of the target analytes derivatives. The advantage

of an LC-MS/MS based approach is in the omission of the derivatisation step, thus decreasing

a laborious aspect of the analysis, potentially improving method performance and sample

throughput.

The LC-MS/MS method offered as a candidate method for the study comprised of a collection

of mainstream cigarette smoke particulate matter on a 44 mm Cambridge Filter Pad (CFP),

while the gas phase was trapped with one impinger containing hydrochloric acid (HCl) solution.

The CFP was extracted in an ultrasonic bath using the impinger solution. The crude extract was

purified using Solid Phase Extraction (SPE) and analysed by Liquid Chromatography-Mass

Spectrometry (LC-MS/MS).

This Joint Experiment (JE) was conducted in 2016 and was focused on evaluation of the SPE

clean-up procedure. The method was based on an on-line SPE system that was directly

connected to a triple quadrupole mass analyser, however many laboratories did not have such

equipment. Subsequently, an off-line SPE clean-up was developed to offer an alternative

approach.

The aim of this JE was to compare two off line SPE set-ups using either a single cartridge

(MCX) or two cartridges (dual SPE). The JE included 4 laboratories from one country and used

one cigarette product (Kentucky reference 3R4F) smoked under both ISO and intense smoking

regimes. Aromatic amine yields were reported in units of nanogram per cigarette (ng/cigarette).

3. Organisation

3.1 Participants

The laboratories that participated in the Joint Experiment are listed in alphabetical order in

Table 1. To ensure anonymity of the results, each laboratory was given a unique laboratory

code that was used for reporting of the data and was shared with each laboratory separately.

Laboratory codes were different to the order of participating laboratories in Table 1.

Table 1. Participating laboratories in 2016 Joint Experiment

Participants

CNTC Beijing Cigarette Factory, China

China NT QS&TC, China

China Tobacco Zhejiang Industrial Co., Ltd., China

China Tobacco Henan Industrial Co. Ltd. of CNTC, China


3.2 Protocol

The Study Protocol is described briefly below and in full in Appendix A.

3.2.1 Sample Shipment

Participating laboratories were asked to obtain 3R4F Kentucky Reference Cigarettes

themselves.

Table 2. Reference cigarette included in the Joint Experiment

Sample Characteristics ISO NFDPM

(mg/cigarette, pack data)

Cigarette Length (mm)

Tipping Length (mm)

Filter Length (mm)

Butt Length (mm)

3R4F Kentucky Reference 3R4F 8 84 32 27 35

3.2.2 Sample Preparation

Participating laboratories were asked to follow Study Protocol (Appendix A) and the

analytical method for determination of aromatic amines by LC-MS/MS (Appendix B).

The experiments defined a specific number of cigarettes to be smoked per each replicate

for each smoking regime (ISO and intense) to generate samples with different tar levels.

Five replicates of each product and each smoking regime were requested to be generated

following the randomised order specified in the Study Protocol (Appendix A).

For investigation of the SPE clean-up, two off-line SPE set-ups were assessed, a single

SPE cartridge clean-up with MCX mixed mode phase and dual SPE clean-up using PRS

and SP C-18 cartridges connected in series (Figure 1).

Laboratories were asked to document any deviations from the study protocol and to

submit those with the results. Templates were provided for recording of smoke data

(smoke machine, number of cigarettes, puff count and Total Particulate Matter, TPM) and

for aromatic amines mainstream smoke yields.

Figure 1. A design of experiment for the comparison of SPE approaches

3.2.3 Sample Analysis and Data Reporting

Five replicates for each sample and for each smoking regime following the randomised

protocol were requested (Appendix A).

Aromatic amines were quantified using internally standardised calibration curves. Six

internal standards were used for quantification of the target analytes (o-toluidine-d7,

1-aminonaphthalene-d7, 2-aminonaphthalene-d7, 3-aminobiphenyl-d9, 4-aminobiphenyl-

d9 and o-anisidine–d3).

The yields of aromatic amines were reported in units of nanogram per cigarette

(ng/cigarette) in the templates provided to participants.


4. Data – Summary Descriptive Analysis

The results were received altogether from 4 laboratories (Table 3). Due to a very small data set

available and a range of experiments carried out, only a very limited analysis was conducted.

Table 3. Submitted data sets

Participant

Dual SPE 2 cigs

Dual SPE 5 cigs

MCX 2 cigs

MCX 5 cigs

Dual SPE 3 cigs

MCX 3 cigs

ISO smoking regime Intense smoking regime

Laboratory 1 Yes Yes No No Yes No

Laboratory 2 Yes Yes Yes Yes Yes Yes

Laboratory 3 No No Yes Yes No Yes

Laboratory 4 Yes Yes No No Yes No

Total data sets 3 3 2 2 3 2

For the sample generation, all laboratories used linear smoking machines and smoke the same

quantity of 3R4F cigarettes; 2 and 5 for ISO smoking regime and 3 for ISO intense smoking

regime.

Mean yields for all participating laboratories and both smoking regimes and Standard Deviation

(SD) are summarised in Table 4/Figure 2 for ISO smoking regime and in Table 5/Figure 3 for

intense smoking regimes, respectively.

Table 4. Mean yields for ISO mainstream smoke across all participating laboratories

Analyte

Dual SPE (5 cigarettes)

MCX (5 cigarettes)


MCX (2 cigarettes)

ISO mainstream smoke yields (ng/cigarette)

mean SD mean SD mean SD mean SD

1-aminonaphthalene 11,4 0,3 13,1 5,1 7,7 4,7 12,7 7,2

2-aminonaphthalene 6,7 0,3 7,6 2,9 4,4 2,6 7,4 4,1

3-aminobiphenyl 1,5 0,3 1,7 0,9 1,8 0,2 1,8 0,8

4-aminobiphenyl 0,6 0,1 0,7 0,3 0,7 0,2 0,8 0,4

o-toluidine 51,0 1,5 50,5 24,5 41,0 8,4 52,6 29,6

o-anisidine 1,9 0,3 2,1 1,0 1,5 0,5 2,1 1,0

2,6-dimethylaniline 10,2 0,2 10,8 5,3 9,0 1,7 10,6 6,1


Table 5. Mean yields for intense mainstream smoke across all participating laboratories

Analyte


MCX (3 cigarettes)

Intense mainstream smoke yields (ng/cigarette)

mean SD mean SD

1-aminonaphthalene 24,6 0,5 29,6 1,1

2-aminonaphthalene 15,5 0,4 15,5 0,5

3-aminobiphenyl 3,8 0,04 3,9 0,1

4-aminobiphenyl 1,8 0,04 1,6 0,1

o-toluidine 143,2 3,1 130,2 1,0

o-anisidine 5,1 0,1 4,2 0,1

2,6-dimethylanilin 24,6 0,5 25,1 0,8

Figure 2. Mean yields for ISO mainstream smoke amongst participating laboratories. NB. Dual

SPE data originated from 3 data sets, MCX data from 2 data sets.


Figure 3. Mean yields for intense mainstream smoke amongst participating laboratories. NB.

Dual SPE data originated from 3 data sets, MCX data from 2 data sets.

Statistical analysis (t-test) was carried out to compare mean smoke yields of aromatic amines

achieved by participating laboratories and both clean-up approaches. No statistically significant

differences were observed between the two limited data sets.

For the ISO smoking regime, the relative differences between results obtained from both SPE

clean-ups were in the range from 1 % (o-toluidine) to 13 % (1-aminonaphthalene) for

5 cigarettes smoked and from 3 % (3-aminobiphenyl) to 40 % (1- and 2-aminonaphthalene) for

2 cigarettes smoked. The biggest variation was observed when 2 cigarettes were smoked under

ISO smoking regime, simulating a low tar yield product, thus representing the lowest yields of

the mainstream smoke.

For the intense smoking regime, the difference of results between both SPE clean up varied

between 2 % (3-aminobiphenyl) – 22 % (o-anisidine).

The summary of the mainstream smoke yields results for each participating laboratory is in

Appendix C. The smoke data and target analytes individual yields collected from all

participating laboratories are summarised in Appendix D. Raw data plots demonstrating the

spread of the results (individual replicates and mean for each analyte and smoking regime) for

each participating laboratory are summarised in Appendix E.


5. Data – Statistical Analysis

5.1 Outlier Detection

Due to a very limited amount of data, outlier evaluation and detection was not conducted.

5.2 Repeatability and Reproducibility

Repeatability (r) and reproducibility (R) were out of the scope of this Joint Experiment.

6. Recommendations

Through this JE conducted in 2016, an investigation was conducted on a specific aspect of LC-

MS/MS method for measurement of 7 aromatic amines – a comparison of two off line SPE

approaches for a purification of crude mainstream smoke extracts.

The study was conducted by a small number of laboratories (4) and data analysis of the

submitted results was limited.

A larger study employing more laboratories would be required to further investigate both clean-

up approaches to confirm the findings and to allow for comparison of both analytical methods

(GC, LC).

7. Appendices

Appendix A: Study Protocol

Appendix B. Analytical Method

Appendix C: Data Summary

Appendix D: Full Data Sets

Appendix E: Raw Data Plots


Appendix A: Study Protocol

1. CORESTA Special Analytes Sub-Group

Pre-study on Aromatic Amines by LC-MS/MS -Study Protocol

1) Objective

The aim of this pre-study is to give laboratories an opportunity to apply and to assess LC-

MS/MS method for a quantitative measurement of seven aromatic amines (o-toluidine; 2,6-

dimethylanilin, o-anisidine, 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl

and 4-aminobiphenyl) in the KR 3R4F mainstream cigarette smoke generated under ISO and

Health Canada Intense smoking regimes.

The pre-study will enable the SG to select a suitable method for further joint experiments in

order to choose an appropriate candidate for a robust and less time consuming CORESTA

Recommended Method (CRM).

2) Study coordinators

Michael Intorp

E-mail: [email protected]

Tel: 00 49 408220 2352

Fax: 00 49 408220 2241

Jana Ticha

E-mail: xxxxxxxxxxxxxxxx

Tel: 00 44 238 0793 051

Fax: 00 44 238 0793 962

3) Analytes and Methods

The analytes included in the study are o-toluidine, 2,6-dimethylanilin, o-anisidine,

1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl and 4-aminobiphenyl.

Laboratories are asked to follow the Study Protocol and provided LC-MS/MS method as

much as practicable. Particularly the clean-up procedures suggested in the method will be

in the focus of this pre-study. Laboratories are asked to perform both offline clean up

procedures in order to study possible matrix effects.

Any deviations to the methods must be recorded and shared with SG coordinators.

TPM, puff count data, number of cigarettes per replicate and the type of smoke machine

should be recorded in the provided template.


4) Samples

Kentucky Reference KR 3R4F

Laboratories should order KR 3R4F Reference Cigarettes directly from University of

Kentucky. Please include shipping instructions and other requirements for shipping the

cigarettes to the individual laboratory.

5) Smoking plans

ISO and Health Canada Intense (HCI) smoking regimes are requested.

Five replicates for the reference cigarette should be generated per method (per SPE clean-

up strategy) and per smoking regime. One sample extract can be used for both SPE clean up

steps. See flowchart below as a guidance.

For ISO smoking regime please smoke 2 and 5 cigarettes per replicate in case a linear

smoking machine is used and 2 and 10 cigarettes per replicate in case a rotary machine is

used.

For HCI smoking regime the number of cigarettes per replicate could be adjusted

individually to avoid overloading of CF pads.

Note: Reason for using two different amounts of cigarettes is to simulate different tar

deliveries in the absence of KR 1R5F.

6) Data Submission

Laboratories are asked to provide data on as many of the requested aromatic amines (o-

toluidine, 2,6-dimethylanilin, o-anisidine, 1-aminonaphthalene, 2-aminonaphthalene, 3-

aminobiphenyl and 4-aminobiphenyl) as possible.

The template for results will be provided. Please supply data in the requested format without

creating new cells or rows in the spreadsheet.

Results should be reported back to Michael Intorp and Jana Ticha.

ISO 2 cig (LM, RM) ISO – 5 cig (LM) or 10 cig (RM ) HCI eg 5 cig

CFP CFP CFP

Clean up (offline) Clean up (offline) Clean up (offline)

MCX 1.PRS/ MCX 1. PRS MCX 1. PRS 2. SP C-18 2. SP C-18 2. SP C-18


7) Data Analysis

The data will be analysed statistically (organized by SG coordinators).

8) Timescale

Mid July 2016

All documents are distributed to participating laboratories:

• Protocol of the study,

• Method drafts,

• Report form.

End July – mid September 2016

Laboratories perform the study.

Mid of September 2016

The results must be sent to the data coordinators (Michael Intorp and Jana Ticha) by 15th

September.

End of September 2016

The statisticians perform the data analysis and SG coordinators format the data for

presentation.

8th October 2016

The SPA SG will review the JE results during the meeting in Berlin.


Appendix B: Analytical Method

Determination of 7 aromatic amines in mainstream cigarette smoke by LC-MS/MS

1. Field of application

This method is applicable to the determination of 7 aromatic amines (AAs) (i.e. o-toluidine, 1-

naphthylamine, 2-naphthylamine, 2,6-dimethylanilin, 3-aminobiphenyl, 4-aminobiphenyl and

o-anisidine ) in mainstream cigarette smoke[4].

2. Normative references

2.1 ISO 3308:2000/Amd 1:2009. Routine analytical cigarette-smoking machine – Definitions

and standard conditions

2.2 ISO 8243:2006. Cigarettes – Sampling

2.3 ISO 3402:1999. Tobacco and tobacco products – Atmosphere for conditioning and testing

2.4 Health Canada Official Method T–115: December 1999. Determination of “Tar”, Nicotine

and Carbon Monoxide in Mainstream Tobacco Smoke

2.5 ISO 4387:2000 Cigarettes – Determination of Total and Nicotine-free Dry Particulate

Matter Using a Routine Analytical Smoking Machine

3. Method summary

Cigarettes are smoked on a routine analytical smoking machine under ISO 3308 or

Health Canada Intense (HCI) T-115. The particulate phase (PP) of mainstream cigarette

smoke is collected on a 44 mm Cambridge Filter Pad (CFP), while the gas phase (GP)

is trapped with one impinger containing 25 mL 0.6N hydrochloric acid (HCl) solution.

The CFP is extracted in an ultrasonic bath with the impinger solution. The extract is

purified using Solid Phase Extraction (SPE) and then analyzed by Liquid

Chromatography-Mass Spectrometry (LC-MS/MS).

4. Apparatus

4.1 A 20-port linear smoking machine (Cerulean, UK) is used as the smoking engine for

puffing the cigarette.

4.2 SPE clean up system – either automated on line SPE system or off line SPE system

4.2.1 Fully-automated on-line SPE Symbiosis liquid chromatography coupled with a triple

quadruple mass spectrometer and equipped with:

• A cation-exchange cartridge such as Bond Elute PRS with average sorbent

content of 15.63 mg and mean particle size of 54 µm (e.g. Spark Holland), or

equivalent and

• A reversed phase (RP) cartridge such as HySphere-C18 HD with average sorbent

content of 18.22 mg (e.g. Spark Holland), or equivalent,

OR

[4] The method is validated for additional 3 aromatic amines (m-toluidine, p-toluidine and m-anisidine). However

as they are not required by FDA, these substances are not included in this method.


4.2.2 Off-line SPE

4.2.2.1 One stage clean-up – Oasis MCX or

4.2.2.2 Two stage clean-up – a cation exchange SPE cartridge (Bond Elute PRS)

followed by a reversed phase (RP) C18 high density SPE cartridge such as Waters

Oasis HLB cartridge or equivalent. Note. Recommended sorbent amount is

200 mg and 6 cc cartridge volume.

4.3 HPLC column: Agilent ZORBAX Eclipse Plus Phenyl-Hexyl column (2.1 mm ×150 mm

i.d., 3.5 μm) or equivalent.

4.4 Ultrasonic instrument.

4.5 General laboratory equipment.

5. Reagents

5.1 o-toluidine (o-TOL), 1-naphthylamine (1-NA), 2-naphthylamine (2-NA),

3-aminobiphenyl (3-ABP), 4-aminobiphenyl (4-ABP), o-anisidine (o-ASD) and

2,6-dimethylanilin (2,6-DMA) (minimum 98 % purity).

5.2 o-toluidine-d7 as the internal standard for o-TOL and 2,6-DMA. 1-NA-d7 is used for

1-NA. And 2-NA-d7 is used for 2-NA. 3-ABP-d9 is used for 3-ABP. 4-ABP-d9 is used for

4-ABP. And o-ASD–d3 is used for o-ASD (minimum 98 % purity).

5.3 Methanol, acetonitrile (LC/MS grade).

5.4 Deionised water purified with a Milli-Q system or equivalent.

5.5 Hydrochloric acid (HCl, 36-38 %), analytical grade

5.6 Formic acid (FA, 98 %), analytical grade

5.7 Aqueous ammonia (NH4OH, 28 %, analytical grade).

6. Preparation of solutions

6.1 Extraction Solution (0.6M HCl solution)

Add 50 mL of HCl (36-38 %) into a 1000 mL volumetric flask containing at least 300 mL

of deionized water and dilute to the mark with deionized water.

6.2 2 % Ammonia solution

Add 80 mL ammonia (28 %) into a 1000 mL volumetric flask and dilute to the mark with

deionized water.

6.3 2 % (v/v) Formic acid solution

Add 20 mL of FA (98 %) into 1000 mL volumetric flaks containing at least 300 mL of

deionized water. Dilute to the mark with deionized water.

6.4 10 % (v/v) Methanol

Add 100 mL of methanol into 1000 mL volumetric flaks and dilute to the mark with

deionized water.

6.5 HPLC Mobile Phase A (water)

100 % deionized water.

6.6 HPLC Mobile Phase B (0.25 % formic acid solution in acetonitrile)

Add 2.5 mL of FA (98 %) to 300 mL methanol in 1000 mL volumetric flask and dilute to

the mark with acetonitrile.

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7. Preparation of standards

7.1 Preparation of Internal Standard Solutions

7.1.1 Primary Solution

Weigh approximately 5 mg each of o-TOL-d7, 1-NA-d7, 2-NA-d7, 3ABP-d9,

4ABP-d9 and o-ASD-d3 into individual 25 mL volumetric flasks, dilute in

methanol and mix well. The concentration of each solution is approximately 200

µg/mL.

7.1.2 Combined Secondary Solution

Transfer 2.5 mL of primary solution of o-TOL-d7, 0.5 mL of 1-NA-d7 and 2-NA-

d7, 0.25 mL of 3ABP-d9, 4ABP-d9 and o-ASD-d3 into a 100 mL volumetric flask,

respectively. Add methanol to each flask to the mark and mix well. The

concentration of this solution is approximately 5 µg/mL of o-TOL-d7, 1 µg/mL of

1-NA-d7 and 2-NA-d7 and 0.5 µg/mL of 3ABP-d9, 4ABP-d9 and o-ASD-d3.

7.2 Preparation of Calibration Standard Solutions

7.2.1 Primary Single AAs Solutions

Weigh approximately 10 mg each of o-TOL, 1-NA, 2-NA, 3-ABP, 4-ABP, 2,6-

DMA, o-ASD and put into individual 100 mL volumetric flasks, respectively. Add

methanol to each flask to the mark and mix well. The concentration of each solution

is approximately 100 µg/mL.

7.2.2 Mixed AAs Stock Solution (I)

Transfer 5 mL of the primary single AAs solutions of o-TOL, 2 mL of the primary

single AAs solution of 1-NA, 1 mL of the primary single AAs solution of 2-NA,

0.5 mL of the primary single AAs solutions of 3-ABP and o-ASD, 0.25 mL of the

primary single AAs solution of 4-ABP and 2.5 mL of the primary single AAs

solution of 2,6-DMA into a 25 mL volumetric flask. Add methanol to each flask to

the mark and mix well. The concentration of this solution is approximately 20

µg/mL of o-TOL, 8 µg/mL of 1-NA, 4 µg/mL of 2-NA, 2 µg/mL of 3-ABP and o-

ASD, 1 µg/mL of 4-ABP and 10 µg/mL of 2,6-DMA.

7.2.3 Mixed AAs Stock Solution (II)

Transfer 0.625 mL of the mixed methanol stock solution (I) into a 25 mL volumetric

flask. Add 0.6N HCl solution to each flask to the mark and mix well. The

concentration of this solution is approximately 500 ng/mL of o-TOL, 200 ng/mL of

1-NA, 100 ng/mL of 2-NA, 50 ng/mL of 3-ABP and o-ASD, 25 ng/mL of 4-ABP

and 250 ng/mL of 2,6-DMA.

7.2.4 Working Standard Solutions

Prepare 6 working standard solutions that cover the concentration range of interest.

Add selected volumes of solutions listed in Table 1 in a 25 mL volumetric flask and

dilute to the mark with 0.6N HCl solution. Calculate the exact concentrations for

each standard and record (Table 2).

Note: Each laboratory should establish the most suitable calibration range depending on

the equipment used and the type of samples to be analysed. The standard preparation

procedure is given as an example and is applicable for the range of the products in a

collaborative study.


Table 1. Preparation of working standard solutions for calibration

S1(mL) S2 (mL) S3 (mL) S4 (mL) S5 (mL) S6 (mL)

Internal standard solution

0.05 0.05 0.05 0.05 0.05 0.05

Mixed AAs stock solution (II)

0.05 0.15 0.45 1.0 2.0 5.0

Table 2. Concentration of each calibration standard

S1(ng/mL) S2 (ng/mL) S3 (ng/mL) S4 (ng/mL) S5 (ng/mL) S6 (ng/mL)

o-TOL 1.0 3.0 9.0 20.0 40.0 100.0

1-NA 0.4 1.2 3.6 8.0 16.0 40.0

2-NA 0.2 0.6 1.8 4.0 8.0 20.0

3-ABP 0.1 0.3 0.9 2.0 4.0 10.0

4-ABP 0.05 0.15 0.45 1.0 2.0 5.0

o-ASD 0.1 0.3 0.9 2.0 4.0 10.0

2,6-DMA 0.5 1.5 4.5 10.0 20.0 50.0

8. Procedures

8.1 Smoking and sample preparation

Cigarettes are sampled according to ISO 8243:2006, and all the cigarettes to be smoked and

conditioned following the ISO 3402:1999. Cigarettes are smoked according to ISO 3308:2000

and/or Health Canada Official Method T–115. Typically five cigarettes per port are smoked

under ISO regime and three cigarettes per port are smoked under Health Canada Intense

smoking regime. All samples are smoked to a butt mark of 3 mm past the tipping paper

overwrap.

The particulate phase (PP) of cigarette smoke is collected on a CFP, while the gas phase (GP)

is trapped with one impinger containing 25 mL of 0.6N HCl solution. After smoking, the filter

pad is extracted with 0.6N HCl solution in the impinger and 50 µL of internal standard solution

is added in an ultrasonic bath for 30 min with temperature maintained at below 40 °C. The

extract is purified with two on-line or off-line SPE cartridges then analysed with LC-MS/MS.

8.2 Examples of SPE conditions

8.2.1 Online SPE conditions[5]

The extract is purified on the system of two SPE cartridges: cation-exchange cartridge (Bond

Elute PRS with average sorbent content of 15.63 mg and mean particle size of 54 µm or

equivalent) and reversed phase cartridge (HySphere-C18 HD with average sorbent content of

18.22 mg or equivalent). The conditions for on-line SPE operation are listed on Table 3.

[5] This procedure is called mXLC (multidimensional online SPE).


Table 3. Conditions for online SPE

First cartridge PRS cation exchange cartridge

Second cartridge HySphere C18 HD.

Activation PRS + C18 HD Methanol 1.0 mL (5 mL /min)

Equilibration PRS 2 % FA 1.0 mL (5 mL /min)

Sample loading PRS 2 % FA 0.8 mL (0.8 mL /min)

Wash PRS 1 2 % FA 1.0 mL (2 mL /min)

Wash PRS 2 10 % MeOH + 2 % FA 0.5 mL (2 mL /min)

Equilibration C18 HD 2 % NH4OH 1.0 mL (5 mL /min)

Transfer from PRS to C18 HD 2 % NH4OH 0.4 mL (0.8 mL /min)

Wash C18 HD 2 % NH4OH 1.0 mL (2 mL /min)

Elution C18 HD 13.0 minutes normal elution with pump gradient

8.2.2 Optimized off-line SPE clean-up procedure

For off-line SPE, there are two methods that can be applied. Both have a comparable cleanup

effect. The one cartridge procedure uses a mixed mode cation exchange cartridge (Oasis MCX

or equivalent); the two cartridge method uses a cation exchange cartridge (Bond Elute PRS or

equivalent) and a C18 cartridge (Sep-Pak C18 or equivalent). The sample went through the PRS

cartridge first and as a second clean-up step through C18 cartridge. The conditions for both off-

line SPE clean-up strategies are listed in the Tables 4 and 5.

Note: Although the recommended offline SPE method brings sufficient results, the online SPE

method reaches higher response for all AAs leading to lower limits of detection.

8.2.2.1 One cartridge clean-up (Oasis MCX)

Table 4. Conditions for offline SPE (Oasis MCX)

Cartridge Oasis MCX

Activation Methanol 10.0 mL (3 mL /min)

Equilibration 2 % FA 10.0 mL (3 mL /min)

Sample loading extracted sample 5-10 mL (1 mL /min)

Wash 2 % FA 10.0 mL (2 mL /min)

Elution 6 mL methanol containing 5 % NH4OH (0.8 mL /min)


8.2.2.2 Two cartridges clean-up

Table 5. Conditions for offline SPE (Two dimensional SPE)

First cartridge Bond Elute PRS

Activation (PRS) Methanol 10.0 mL (3 mL /min)

Equilibration ( PRS) 2 % FA 10.0 mL (3 mL /min)

Sample loading (PRS) extracted sample 5-10 mL (1 mL /min)

Wash (PRS) 2 % FA 10.0 mL (2 mL /min)

Second cartridge Sep-Pak C18

Equilibration (C18) 5 % NH4OH 10.0 mL (3 mL /min)

Transfer from PRS to C18 5 % NH4OH 5 mL (0.8 mL /min)

Wash (C18) 5 % NH4OH 10.0 mL (2 mL /min)

Elution (C18) 5 mL methanol/water (6:4) (0.8 mL /min)

8.3 Suggested LC-MS/MS separation and detection conditions

8.3.1 Mobile Phase A: water；Mobile Phase B: 30:69.75:0.25 (v/v/v) mixture of

methanol, acetonitrile and formic acid；HPLC column: Agilent ZORBAX Eclipse

Plus Phenyl-Hexyl column (2.1mm ×150 mm i.d., 3.5 μm) or equivalent. Gradient

elution conditions are shown in Table 6. The flow rate is 300 μL/min and the

column temperature is set at 30 ℃. Sample injection volume is 40 μL after online

SPE purification and 10μL after offline SPE purification.

The examples of separation of target analytes in solvent (calibration standards) and in the

matrix (1R5F, 3R4F) are shown in Appendix 1.

Note: Satisfactory separation of o, m and p- toluidine isomers should be achieved to prevent

overestimating of the analytical data.

Table 6. Gradient conditions for HPLC

Time (min) Solvent A (%) Solvent B (%)

0.0 95 5

20.0 75 25

22.0 75 25

24.0 60 40

24.1 5 95

26.0 5 95

26.1 95 5

30.0 95 5


8.3.2 Suggested mass spectrometry detection conditions

Ionization mode: positive ESI

Scan mode: multiple reaction monitoring (MRM)

Ion spray voltage: 5500 V

Ion source temperature: 500℃

Curtain gas: nitrogen; setting: 30 psi

Ion source gas 1 (GS1), setting: 70 psi; ion source gas 2 (GS2), setting: 70 psi

The parameters of precursor ion, qualitative and quantitative ion pairs, dwell time,

collision energy (CE), declustering potential (DP) are summarized in Table 6.

Table 7. The MS specific parameters for the detection of aromatic amines.

Analytes Precursor Ion (m/z)

Production Ion (m/z)

Dwell Time

（ms）

CE

（eV） DP (V)

CXP (eV)

o-TOL 108.1 91.0a

65.0b 50 24 92 11

1-NA 144.0 127.0a

77.0b 50 30 124 15

2- NA 144.0 127.0a

77.0b 50 30 124 15

3-ABP 169.9 152.1a

127.1b 50 34 85 18

4-ABP 169.9 152.1a

127.1b 50 36 50 12

o-ASD 124.0 92.0 a

109.0 b 50 30 106 13

m-ASD 124.0 92.0 a

109.0 b 50 30 106 13

2,6-DMA 122.1 77.0 a 50 35 98 14

105.0 b

o-TOL-d7 115.1 91.0a

65.0b 50 24 83 15

1- NA -d7 151.1 132.1a

81.0b 50 30 124 15

2- NA -d7 150.9 132.1a

81.0b 50 30 124 15

3-ABP-d9 179.0 160.0a

134.0b 50 35 104 14

4-ABP-d9 179.2 160.1a

134.1b 50 35 98 14

o-ASD-d3 127.1 92.0a

109.0b 50 30 96 13

a: the qualitative ion b: quantitative ion


9. Calibration

Inject successively each working standard solution (7.2.4) in LC-MS/MS system. Record

the area of aromatic amines and internal standard peaks. The internal standard method is

used in the quantitative analysis.

10. Determination and calculation

Calculate the concentration of aromatic amines according to the calibration curve. The

amount of aromatic amines per cigarette is calculated as follows (Equation 1):

Equation 1: Aromatic amines yields calculation

m =C × V

n

Where:

m is the mass of aromatic amines per cigarette expressed as ng/cig;

C is the concentration of aromatic amines in the elution solution expressed as ng/mL;

V is the volume of extraction solution expressed as mL;

n is the amount of the smoked cigarettes.


Figure 1: Chromatogram of Typical Aromatic Amines Calibration Standards. The concentrations

of AAs are 40 ng/mL for o-TOL, 16 ng/mL of 1-NA, 8 ng/mL for 2-NA, 4 ng/mL of 3-ABP and

o-ASD, 2 ng/mL for 4-ABP and 20 ng/mL for 2,6-DMA.


Figure 2. Examples of chromatograms of aromatic amines in 3R4F mainstream cigarette smoke

generated under ISO regime.


Figure 3. Chromatograms for aromatic amines in 1R5F mainstream cigarette smoke generated

under ISO regime.


Appendix C: Data Summary

Table C1. Mean 3R4F ISO mainstream smoke yields

Analyte

(ISO smoking regime)

Laboratory 1 Laboratory 2 Laboratory 3 Laboratory 4

Dual SPE

2 cigs

Dual SPE

5 cigs

Dual SPE

2 cigs

Dual SPE

5 cigs

MCX 2 cigs

MCX 5 cigs

MCX 2 cigs

MCX 5 cigs

Dual SPE

2 cigs

Dual SPE

5 cigs

1-amino naphthalene

Mean (ng/cigarette)

7,8 11,6 12,3 11,0 11,9 11,4 13,5 14,7 2,9 11,6

SD (ng/cigarette)

1,9 0,5 1,0 0,7 0,9 0,9 0,7 0,3 0,6 0,6

RSD (%) 25 4 8 7 8 8 5 2 19 5

2-amino naphthalene

Mean (ng/cigarette)

5,2 6,5 6,5 7,0 6,9 7,3 7,8 7,8 1,5 6,5

SD (ng/cigarette)

1,5 0,2 0,6 0,3 0,7 0,4 0,2 0,2 0,3 0,3

RSD (%) 29 3 9 4 10 5 2 3 17 4

3-amino biphenyl

Mean (ng/cigarette)

1,6 1,4 2,0 1,9 1,9 1,8 1,6 1,6 1,8 1,3

SD (ng/cigarette)

0,2 0,1 0,2 0,1 0,2 0,1 0,04 0,06 0,1 0,1

RSD (%) 12 6 8 6 9 7 3 3 9 10

4-amino biphenyl

Mean (ng/cigarette)

0,6 0,6 0,9 0,7 0,9 0,7 0,7 0,7 0,7 0,6

SD (ng/cigarette)

0,2 0,1 0,1 0,1 0,1 0,07 0,05 0,06 0,1 0,1

RSD (%) 31 13 8 7 9 10 6 5 12 12

o-toluidine

Mean (ng/cigarette)

38,4 52,7 50,4 49,7 49,6 46,8 55,6 54,1 34,2 50,7

SD (ng/cigarette)

5,2 3,5 3,0 2,7 2,9 1,6 1,6 2,4 3,3 4,6

RSD (%) 14 7 6 5 6 4 3 4 10 9

o-anisidine

Mean (ng/cigarette)

1,5 1,6 2,0 2,2 2,0 1,9 2,1 2,3 1,1 1,8

SD (ng/cigarette)

0,2 0,2 0,2 0,1 0,2 0,2 0,1 0,05 0,1 0,1

RSD (%) 17 15 8 6 10 8 8 2 9 6

2,6-dimethyl aniline

Mean (ng/cigarette)

10,1 10,3 9,9 10,0 9,6 10,2 11,6 11,4 7,1 10,4

SD (ng/cigarette)

0,9 0,3 0,9 0,8 0,7 0,8 0,5 0,4 0,4 0,3

RSD (%) 9 3 9 9 8 8 5 4 6 3


Table C2. Mean 3R4F intense mainstream smoke yields

Analyte

(ISO intense smoking regime)


Dual SPE Dual SPE MCX MCX Dual SPE

1-amino naphthalene

Mean (ng/cigarette)

23,4 26,8 27,5 31,7 23,6

SD (ng/cigarette) 0,5 1,7 2,7 0,9 0,9

RSD (%) 2 6 10 3 4

2-amino naphthalene

Mean (ng/cigarette)

15,8 15,4 14,1 16,9 15,2

SD (ng/cigarette) 0,5 1,3 1,2 0,3 0,5

RSD (%) 3 9 8 2 3

3-amino biphenyl

Mean (ng/cigarette)

3,5 4,4 4,1 3,6 3,4

SD (ng/cigarette) 0,3 0,4 0,3 0,1 0,3

RSD (%) 8 9 7 3 8

4-amino biphenyl

Mean (ng/cigarette)

1,8 1,7 1,6 1,5 1,9

SD (ng/cigarette) 0,1 0,1 0,1 0,1 0,04

RSD (%) 7 7 8 4 2

o-toluidine

Mean (ng/cigarette)

150,0 133,8 127,4 132,8 145,6

SD (ng/cigarette) 9,1 4,5 5,3 3,6 11,2

RSD (%) 6 3 4 3 8

o-anisidine

Mean (ng/cigarette)

5,6 4,5 4,3 4,1 5,3

SD (ng/cigarette) 0,4 0,4 0,4 0,2 0,3

RSD (%) 8 9 9 5 5


Mean (ng/cigarette)

25,1 25,0 23,9 26,4 23,7

SD (ng/cigarette) 1,2 2,0 2,1 0,8 1,0

RSD (%) 5 8 9 3 4


Appendix D: Full Data Sets

Table D1: 3R4F smoke data

Experiment:

Smoking regime/product and

a number of cigarettes

smoked/replicate


Puff Count

(per cig)

TPM (mg/cig)

Puff Count

(per cig)

Wet TPM

(mg/cig)

Puff Count

(per cig)

Wet TPM

(mg/cig)

Puff Count

(per cig)

Wet TPM (mg/cig)

ISO 3R4F

2 cigs

1 8,3 9,5 8,5 10,0 8,0 10,1 8,8 9,9

2 8,2 9,6 8,2 9,9 8,4 9,9 8,2 9,7

3 8,1 9,3 9,0 11,1 8,0 9,8 8,1 9,4

4 8,2 9,4 9,0 11,1 8,0 10,0 8,2 9,9

5 8,4 9,1 8,8 11,2 7,9 10,1 8,2 9,1

ISO 3R4F

5 cigs

1 8,4 9,3 8,9 10,7 8,0 9,9 8,5 9,3

2 8,4 9,3 8,3 10,2 7,9 10,0 8,1 9,6

3 8,2 9,2 8,8 11,0 8,0 9,4 8,3 8,9

4 8,3 9,4 8,7 10,4 7,9 9,6 8,4 9,1

5 8,1 9,4 8,7 10,7 8,0 10,0 8,4 9,5

HCI 3R4F

3 cigs

1 10,4 43,9 11,0 47,8 9,7 41,8 10,6 43,2

2 10,5 41,2 10,9 45,5 9,6 44,9 10,8 43,5

3 10,6 45,3 10,8 44,0 10,1 43,5 10,7 40,6

4 10,6 39,9 11,0 47,9 10,5 44,5 10,0 43,6

5 10,2 45,2 11,7 44,9 10,5 41,9 10,3 46,2


Table D2: 3R4F ISO mainstream smoke yields

Replicate Analyte


Dual SPE

2 cigs

Dual SPE

5 cigs

Dual SPE

2 cigs

Dual SPE

5 cigs

MCX 2 cigs

MCX 5 cigs

MCX 2 cigs

MCX 5 cigs

Dual SPE

2 cigs

Dual SPE

5 cigs

3R4F ISO mainstream smoke yields (ng/cigarette)

1

1-amino naphthalene

8,2 11,9 10,8 10,8 10,7 11,2 14,1 14,6 2,1 11,2

2 6,5 11,2 12,3 10,3 11,2 10,4 13,5 14,4 2,6 11,0

3 5,2 11,7 12,2 11,9 12,9 12,0 13,0 15,1 3,2 11,6

4 9,2 11,0 13,6 10,3 12,5 10,8 12,6 14,5 3,5 12,1

5 9,9 12,2 12,4 11,5 12,1 12,6 14,4 15,0 3,2 12,3

1

2-amino naphthalene

4,9 6,3 6,1 7,1 6,2 7,2 8,1 7,6 1,5 6,3

2 2,7 6,4 6,0 6,5 6,6 6,8 7,9 8,0 1,3 6,7

3 6,7 6,4 6,5 6,9 7,9 7,2 7,6 7,5 1,9 6,3

4 6,1 6,6 7,5 7,3 7,1 7,3 7,8 8,0 1,7 6,8

5 5,5 6,8 6,3 6,9 6,6 7,9 7,8 7,9 1,3 6,3

1

3-amino biphenyl

1,6 1,3 1,9 1,7 1,8 2,0 1,6 1,6 2,0 1,4

2 1,6 1,3 2,2 1,9 2,1 1,7 1,6 1,7 1,7 1,2

3 1,8 1,4 1,9 2,0 2,2 1,9 1,6 1,6 1,6 1,4

4 1,9 1,4 1,9 1,8 1,9 1,9 1,6 1,6 1,7 1,3

5 1,4 1,5 2,2 1,9 1,8 1,7 1,7 1,7 1,8 1,1

1

4-amino biphenyl

0,8 0,5 0,9 0,8 0,8 0,8 0,7 0,8 0,8 0,6

2 0,7 0,6 0,9 0,8 0,8 0,6 0,8 0,7 0,7 0,6

3 0,3 0,6 1,0 0,7 1,0 0,7 0,7 0,7 0,7 0,7

4 0,6 0,7 0,9 0,8 0,9 0,7 0,7 0,7 0,8 0,5

5 0,7 0,7 0,8 0,7 0,9 0,7 0,7 0,8 0,6 0,6

1

o-toluidine

35,1 49,5 47,5 49,1 47,3 45,5 53,4 53,5 31,4 49,5

2 39,6 52,5 49,1 49,3 48,2 45,0 57,4 52,3 32,2 44,8

3 41,3 55,6 55,3 54,0 54,0 49,1 56,3 51,5 35,1 48,9

4 44,4 49,0 49,7 46,7 51,2 47,5 56,1 56,0 39,6 54,0

5 31,3 56,9 50,7 49,2 47,5 46,8 54,6 57,0 32,6 56,5

1

o-anisidine

1,2 1,7 2,0 2,3 2,3 1,9 2,1 2,3 1,0 1,7

2 1,9 1,9 2,1 2,0 1,8 1,9 2,4 2,2 1,1 2,0

3 1,6 1,4 2,2 2,2 2,0 1,8 2,0 2,3 1,2 1,8

4 1,5 1,3 1,9 2,3 2,2 2,2 2,0 2,2 1,0 1,9

5 1,4 1,6 1,8 2,1 1,9 1,9 2,1 2,3 1,2 1,8

1


10,5 10,3 10,1 10,2 9,6 9,9 11,6 11,5 7,0 10,3

2 9,8 9,8 8,9 10,3 8,5 8,9 10,6 11,9 7,1 10,5

3 8,7 10,2 10,0 9,4 10,6 10,8 11,9 10,9 7,5 10,0

4 10,6 10,7 11,2 8,9 9,8 10,3 11,9 11,7 6,4 10,9

5 11,0 10,4 9,1 11,1 9,6 11,0 11,9 11,2 7,5 10,3


Table D3: 3R4F intense smoking regime mainstream smoke yields

Replicate Analyte


Dual SPE Dual SPE MCX MCX Dual SPE

3R4F intense mainstream smoke yields (ng/cigarette)

1

1-amino

naphthalene

23,6 24,4 26,5 31,8 23,2

2 22,9 26,4 24,6 30,8 24,1

3 23,6 28,8 29,1 30,6 22,4

4 24,0 27,9 31,3 32,3 23,8

5 22,9 26,3 26,0 32,8 24,7

1

2-amino

naphthalene

15,0 15,2 14,1 17,0 14,8

2 15,9 13,6 13,0 16,5 15,3

3 15,9 16,1 15,9 17,2 16,0

4 16,3 17,1 14,4 16,8 15,0

5 16,1 14,9 13,2 17,2 14,7

1

3-amino

biphenyl

3,3 4,6 4,1 3,6 3,2

2 3,7 4,1 3,9 3,6 3,8

3 3,7 4,8 4,3 3,6 3,3

4 3,9 4,5 4,5 3,4 3,1

5 3,2 3,9 3,8 3,7 3,5

1

4-amino

biphenyl

1,6 1,6 1,7 1,5 1,8

2 1,8 1,9 1,5 1,5 1,9

3 1,9 1,7 1,7 1,5 1,9

4 1,7 1,8 1,8 1,4 1,9

5 1,9 1,7 1,5 1,4 1,9

1

o-toluidine

160,2 135,0 129,7 130,0 161,8

2 157,9 127,4 120,5 136,7 152,2

3 148,3 136,0 130,3 129,2 134,2

4 138,3 131,7 133,2 136,7 139,2

5 145,1 139,2 123,5 131,7 140,8

1

o-anisidine

5,3 4,4 4,1 4,1 5,7

2 5,5 4,8 3,8 4,4 5,5

3 5,6 5,0 4,8 4,2 5,1

4 6,3 4,0 4,2 3,8 5,2

5 5,2 4,3 4,6 4,0 5,1

1

2,6-dimethyl

aniline

26,8 26,9 21,6 25,0 23,4

2 25,5 23,2 22,7 26,5 23,8

3 25,6 27,0 27,3 26,3 23,3

4 24,2 22,6 23,8 27,2 22,6

5 23,7 25,2 23,9 26,8 25,4


Appendix E: Raw Data Plots

Figure E1. 3R4F ISO mainstream cigarette smoke yields of 1-aminonaphthalene collected from

participating laboratories. Raw data plots left to right: Dual SPE 2 cigarettes (laboratory 1,

laboratory 2 and laboratory 4), dual SPE 5 cigarettes (laboratory 1, laboratory 2 and laboratory

4), MCX SPE 2 cigarettes (laboratory 2, laboratory 3 and laboratory 4) and MCX SPE 5 cigarettes

(laboratory 2, laboratory 3 and laboratory 4).

Figure E2. 3R4F ISO mainstream cigarette smoke yields of 2-aminonaphthalene collected from






Figure E3. 3R4F ISO mainstream cigarette smoke yields of 3-aminobiphenyl collected from





Figure E4. 3R4F ISO mainstream cigarette smoke yields of 4-aminobiphenyl collected from






Figure E5. 3R4F ISO mainstream cigarette smoke yields of o-anisidine collected from





Figure E6. 3R4F ISO mainstream cigarette smoke yields of o-toluidine collected from






Figure E7. 3R4F ISO mainstream cigarette smoke yields of 2,6-dimethylanilin collected from





Figure E8. 3R4F intense mainstream cigarette smoke yields of 1-aminonaphthalene collected from

participating laboratories (see the legend for colour coding). Raw data plots left to right: Dual

SPE (laboratory 1, laboratory 2, laboratory 4) and MCX SPE (laboratory 2 and laboratory 3).


Figure E9. 3R4F intense mainstream cigarette smoke yields of 2-aminonaphthalene collected from

participating laboratories. Raw data plots left to right: Dual SPE (laboratory 1, laboratory 2,

laboratory 4) and MCX SPE (laboratory 2 and laboratory 3).

Figure E10. 3R4F intense mainstream cigarette smoke yields of 3-aminobiphenyl collected from














Figure E14. 3R4F intense mainstream cigarette smoke yields of 2,6-dimethylanilin collected from



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