Critical care analytes: pre-analytical factors affecting result
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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
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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.
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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
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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.
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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)
Dual SPE (2 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
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Table 5. Mean yields for intense mainstream smoke across all participating laboratories
Analyte
Dual SPE (3 cigarettes)
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.
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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.
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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
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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.
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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
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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.
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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.
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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.
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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).
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 17/35
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)
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 18/35
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
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 19/35
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
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 20/35
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.
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 21/35
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.
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 22/35
Figure 2. Examples of chromatograms of aromatic amines in 3R4F mainstream cigarette smoke
generated under ISO regime.
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 23/35
Figure 3. Chromatograms for aromatic amines in 1R5F mainstream cigarette smoke generated
under ISO regime.
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 24/35
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
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 25/35
Table C2. Mean 3R4F intense mainstream smoke yields
Analyte
(ISO intense smoking regime)
Laboratory 1 Laboratory 2 Laboratory 3 Laboratory 4
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
2,6-dimethyl aniline
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
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 26/35
Appendix D: Full Data Sets
Table D1: 3R4F smoke data
Experiment:
Smoking regime/product and
a number of cigarettes
smoked/replicate
Laboratory 1 Laboratory 2 Laboratory 3 Laboratory 4
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
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 27/35
Table D2: 3R4F ISO mainstream smoke yields
Replicate Analyte
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
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
2,6-dimethyl aniline
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
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 28/35
Table D3: 3R4F intense smoking regime mainstream smoke yields
Replicate Analyte
Laboratory 1 Laboratory 2 Laboratory 3 Laboratory 4
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
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 29/35
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
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).
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 30/35
Figure E3. 3R4F ISO mainstream cigarette smoke yields of 3-aminobiphenyl 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 E4. 3R4F ISO mainstream cigarette smoke yields of 4-aminobiphenyl 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).
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 31/35
Figure E5. 3R4F ISO mainstream cigarette smoke yields of o-anisidine 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 E6. 3R4F ISO mainstream cigarette smoke yields of o-toluidine 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).
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 32/35
Figure E7. 3R4F ISO mainstream cigarette smoke yields of 2,6-dimethylanilin 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 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).
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 33/35
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
participating laboratories. Raw data plots left to right: Dual SPE (laboratory 1, laboratory 2,
laboratory 4) and MCX SPE (laboratory 2 and laboratory 3).
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 34/35
Figure E11. 3R4F intense mainstream cigarette smoke yields of 4-aminobiphenyl 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 E12. 3R4F intense mainstream cigarette smoke yields of 3-aminobiphenyl 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).
SMA-048-3-CTR Aromatic Amines-LC-MS/MS – November 2019 35/35
Figure E13. 3R4F intense mainstream cigarette smoke yields of 3-aminobiphenyl 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 E14. 3R4F intense mainstream cigarette smoke yields of 2,6-dimethylanilin 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).