Thesis: Are E-cigarettes safe? The Analysis of E-liquids using GC-MS & SPME GC-MS
1. Are E-Cigarettes Safe?
The Analysis of E-liquids using GC-MS
and SPME GC-MS
Aaron Jay Alcesto
School of Chemical and Pharmaceutical Sciences
TUD Kevin St.
April 2019
Thesis Submitted in Partial Fulfilment of
Examination Requirements Leading to the Award
BSc Analytical Chemistry
(Environmental, Forensic & Pharmaceuticals)
Technological University Dublin
Thesis Supervisor:
Dr. Γine Whelan
2.
3. 1
Table of Contents
Declaration.......................................................................................................................... i
Acknowledgments...............................................................................................................ii
List of Abbreviations...........................................................................................................iv
Abstract..............................................................................................................................vi
Chapter 1: Introduction...................................................................................................... 1
1 Introduction................................................................................................................ 2
1.1 Current Research................................................................................................ 3
1.2 Components....................................................................................................... 5
1.3 E-liquids.............................................................................................................. 6
1.3.1 Toxicants ........................................................................................................ 7
1.4 Rules & Regulations............................................................................................ 8
1.5 Methods of E-liquid Analysis.............................................................................. 9
1.5.1 Gas Chromatography Mass Spectrometry ..................................................... 9
1.5.2 Solid-phase Microextraction (SPME)............................................................ 10
1.6 Aims of the work .............................................................................................. 11
Chapter 2: Experimental .................................................................................................. 13
2 Experimental............................................................................................................. 14
2.1 Materials & Reagents....................................................................................... 14
2.2 Instrumentation ............................................................................................... 15
2.3 External Standard Calibration of Acetaldehyde Concentration ....................... 17
2.3.1 Preparation of Acetaldehyde Stock Solutions.............................................. 18
2.3.2 Preparation of Acetaldehyde Standard Solutions ........................................ 19
2.3.3 Preparation of the PFBHA Solution .............................................................. 19
2.3.4 Preparation of each of the Standards in a Vial............................................. 19
4. 2
2.3.5 SPME GC-MS Analysis of the Acetaldehyde Standards ................................ 20
2.4 Internal Standard Calibration of Acetaldehyde Concentration........................ 20
2.4.1 Preparation of the Capraldehyde Stock Solutions ....................................... 20
2.4.2 Preparation of Acetaldehyde Standard Solutions with the IS...................... 21
2.4.3 Preparation of the PFBHA Solution .............................................................. 21
2.4.4 SPME GC-MS Analysis of the Acetaldehyde Standards Spiked with IS......... 21
2.5 GC-MS Analysis of the Chemical Composition of E-liquids .............................. 22
2.5.1 Preparation of E-liquid Standard Solutions.................................................. 22
2.5.2 Preparation of Individual Substances Standard Solutions............................ 22
2.6 SPME GC-MS Analysis of Acetaldehyde in Vape .............................................. 22
2.6.1 Collection of the E-liquid Vapour ................................................................. 23
2.6.2 Preparation of the PFBHA Solution .............................................................. 23
2.6.3 Analysis of the Samples................................................................................ 24
2.7 Method Validation ........................................................................................... 24
Chapter 3: Results & Discussion....................................................................................... 25
3 Results & Discussion ................................................................................................. 26
3.1 PFHBA Derivatized vs Underivatized Acetaldehyde Solutions ......................... 26
3.2 Development of a Method for Quantification of Acetaldehyde in E-liquid
Vapours ........................................................................................................................ 27
3.2.1 External Standard Method........................................................................... 27
3.2.2 Internal Standard Method............................................................................ 31
3.2.3 Method Validation ....................................................................................... 33
3.3 GC-MS Analysis of the E-liquids........................................................................ 34
3.3.1 Analysis of the Chemical Composition of the E-liquids ................................ 34
3.3.2 Analysis of the Chemical Composition of the Standard Solutions ............... 42
3.4 SPME GC-MS Analysis of the E-liquid Vapours................................................. 44
7. i
Declaration
I declare that this thesis which I now submit for assessment on the award of Bachelor of
Science (Hons), is entirely my own work and has not been taken from the work of others,
save and to the extent that such work has been cited and acknowledged within the text
of my work.
Signed: __________________
Aaron Jay Alcesto
Date: __________________
8. ii
Acknowledgments
This thesis is dedicated to my beloved parents Jurgenne Salang Alcesto and Allan Paran
Alcesto. To a mother who have provided me with all out moral support, and to a father
who have bestowed significant guidance and perpetual motivation to keep me going
through the years. Without their love and support, I would not be to where I am today.
I would like to thank my supervisor Dr. Γine Whelan for her sincere guidance, buoyant
encouragement and impeccable support and co-operation throughout this thesis. This
thesis would not have been completed without her advice, assistance and excellent ability
to captivate me on the given title. I am with no doubt, extremely grateful for her role in
the completion of this thesis.
I would also like to express my gratitude to all the technical staff for their valuable
assistance and excellent patience throughout the course of this work, especially Grant
Morton, Brian Murphy and the Deirdre Sullivan.
9. iii
βTo raise new questions, new possibilities, to regard old problems from a new angle,
requires creative imagination and makes real advance in scienceβ
Albert Einstein
10. iv
List of Abbreviations
%RSD: Relative Standard Deviation (expressed as a percentage)
AMAH: 4-(2-aminooxyethyl)-morpholin-4-ium chloride coating
CDC: Centres for Disease Control & Prevention
CSM: Cigarette Smoking Machine
CV: Cardiovascular
DCM: Dichloromethane
E-cigarettes: Electronic Cigarettes
E-liquids: Electronic Cigarette Liquids
FDA: Food and Drug Administration
GC: Gas Chromatography
g: Grams
HPLC-MS: High Performance Liquid Chromatography-Mass Spectrometry
HSE: Health Service Executive
ICP-MS: Inductively Coupled Plasma-Mass Spectrometry
L: Litre
LC-MS: Liquid Chromatography-Mass Spectrometry
LOD: Limit of Detection
LOQ: Limit of Quantification
M: Molarity (mol/L)
m/z: Mass to charge ratio
ml: Millilitre
MS: Mass-Spectrometry
NG: Glycerol
nM: Nanomolar
NYT: National Youth Survey
OX/ROS: Oxidative Reactive Organic Species
PDMS/DVB: Polydimethylsiloxane/Divinylbenzene
PFHBA: O-(2,3,4,5,6-Pentafluorobenzyl)hydroxylamine hydrochloride
PG: Propylene Glycol
S: Seconds
12. vi
Abstract
The aim of this project was to analyse the chemical composition of six different e-liquid
samples using GC-MS and headspace SPME GC-MS with a PFBHA derivatized SPME fibre
to form detectable acetaldehyde oximes for analysis. Acetaldehyde in each e-liquid
sample were also analysed and its concentration was determined using an external
standard calibration curve. E-liquids in liquid form were analysed to examine their
composition and e-liquids in vapour form were analysed to examine any changes in the
composition from the generation of aerosols. The advertised ingredients were identified
in the chromatograms for the e-liquids in liquid form with the addition of other
compounds in low concentrations which function as flavour and fragrance agents.
Compounds identified in the chromatograms of the e-liquid analysis were identified and
confirmed through the GC-MS library search system, m/z fragmentation and retention
times. Acetaldehyde in 10 puff aerosols generated from the six e-liquids were found
significantly lower (0.042 ΞΌg - 0.123 ΞΌg for Oxime A and 0.098 ΞΌg - 0.195 ΞΌg for Oxime B)
than the acetaldehydes concentrations in 10 puffs from conventional cigarettes (1240.3
ΞΌg). The proposed method presented great precision as the %RSD results did not exceed
10% (0.411% - 1.107% for Oxime A and 0.424% - 0.738% for Oxime B). The estimated
LOD and LOQ for acetaldehyde determination ranged from 50 nM and 100 nM
respectively. The results demonstrates that the method is regarded as a reliable technique
in the determination of acetaldehyde concentrations.
14. 2
1 Introduction
E-cigarettes, also known as electronic nicotine delivery systems (ENDS), are electronic
devices designed to deliver nicotine in a vaping solution. The device was created as a safer
alternative to conventional cigarettes without the combustion of tobacco which leads to
damaging health effects such as cancer and heart disease. Tobacco cigarettes are
currently the leading preventable cause of death worldwide which causes approximately
6 million deaths annually and is expected to increase to 8 million per year by 2030
according to the Centers for Disease Control and Prevention (CDC)[1]
. The examination of
the concentrations of chemicals in e-cigarettes is crucial as there has been a significant
increase in its use in the past decade. According to a 2011 to 2018 survey by the CDCβs
National Youth Tobacco Survey (NYT), an estimate of 1.78 million high school and middle
school students in the US were reported to have used e-cigarettes. Between 2011 to 2012,
the use of e-cigarettes in high school students increased from 4.7% to 10% and between
2017 and 2018, the use of e-cigarettes by the same age group had increase by 78% (11.7%
to 20.8%)[2]
. Figure 1.1 exhibits a bar chart illustrating the increase in e-cigarette use and
a decrease in tobacco cigarette use by high school students from the CDCβs NYTS. It is
evident that the use in e-cigarettes especially in the youth is increasing rapidly. Therefore
it is important to assess the effects of e-cigarettes and evaluate the harmful substances it
contains.
Figure 1.1: Diagram illustrating the increase of e-cigarette use and the decrease of tobacco
cigarette use by high school students from 2011 to 2015[3]
15. 3
In this chapter, the use of e-cigarettes and the chemical compositions of the e-liquids will
be reviewed. This chapter will provide an overview of the different components in the e-
cigarette, the general ingredients in e-liquids, the toxicants generated from using e-
cigarettes, the e-cigarette regulations and the methods used to determine the e-liquid
composition.
1.1 Current Research
A wide range of methods and instrumentation have already been used to analyse the
nicotine concentration, chemical composition of e-liquids and potential toxicants
generated in e-liquid aerosols in previous studies[4,5,6]
. The most common instrumentation
used to analyse e-liquids is gas chromatography-mass spectrometry (GC-MS). The popular
use of GC-MS in the analysis of e-liquids is due to the volatile nature of the liquids, in
which they are designed to be volatilized into a vapour. A recent study analysed flavour
additives in e-liquid emissions using capture techniques of filter pads and methanol
impingers[7]
. Three e-liquid flavours (cinnamon, mango and vanilla) were analysed using
GC-MS and the results identified 13 compounds from cinnamon flavour, 31 compounds
from mango, and 19 compounds from vanilla. A study in the GC-MS analysis of toxic VOCs
in the aerosols of different e-liquid flavours used a purge-and-trap capture method to
examine and quantify VOC concentrations in the e-liquid samples[8]
. Figure 1.2 exhibits
the results from the quantification of the toxic VOCs.
Figure 1.2: Identified and quantified toxic VOCs using GC-MS[8]
16. 4
Other methods include different forms of liquid chromatography-mass spectrometry (LC-
MS) such as LC-MS-MS and HPLC-MS. One study analysed nicotine alkaloid contaminants
in e-liquids using ultra-high performance liquid chromatography-tandem mass
spectrometry (UHPLC-MS-MS) and identified alkaloids such as anabasine, myosmine and
cotinine and quantified nicotine concentrations in each e-liquid flavour[8]
. Figure 1.3
exhibits the results for the quantitation of nicotine in various e-liquid samples.
Figure 1.3: Quantitation of nicotine in different e-liquid samples[8]
Inductively coupled plasma-mass spectroscopy (ICP-MS) has also been used in the analysis
of e-liquids. The same study by Medana et al. quantified 14 different metal elements on
diluted e-liquid samples[8]
. Figure 1.4 exhibits the results for the quantitation of the heavy
metals in the e-liquid samples.
Figure 1.4: Quantitation of heavy metals in different e-liquid samples[8]
17. 5
Advanced methods such as Cigarette Smoking Machines (CSMs) which functions as
βpuffingβ machines that mimics the vapours released by an actual e-cigarette have been
recently introduced and used to perform analysis with e-liquid samples. One study
analysed the levels of oxidants or reactive oxygen species (OX/ROS) produced from e-
liquid aerosols and its effects on tissues and cells of the human lung using CSM
instrumentation[9]
. The results from the study determined produced OX/ROS levels in uM
concentrations. Another study controlled the generation e-cigarette emissions using a
CSM to examine the influence of e-liquid composition on aerosol composition [10]
. Figure
1.5 exhibits a diagram of a U-SAV CSM.
Figure 1.5: Schematic cross-section diagram of a CSM[10]
1.2 Components
E-cigarettes are long tubular battery powered devices which work by heating a liquid into
an aerosol that users inhale and exhale. The device resembles a cigarette and are mostly
reusable with cartridges which are replaceable and refillable. Most e-cigarettes consists
of a mouthpiece, cartridge, sensor, an atomizer, and a rechargeable battery. The first
component is the mouthpiece. It is a tube through which a user inhales the vapour. The
aerosolized solvent is also known as βvapeβ. The second component is the cartridge. It
contains the electronic cigarette liquid, also known as βe-liquidβ. The third component is
the atomizer. It is a battery powered heating element which vaporizes the e-liquid. The
fourth component is the sensor. It is triggered when a user puffs on the device which then
activates the heating coil. The fifth component is the rechargeable battery. It powers the
atomizer. Figure 1.6 below shows each of the components in an electronic cigarette.
18. 6
Figure 1.6: Diagram exhibiting the components of an e-cigarette[11]
1.3 E-liquids
The solutions in the cartridges of e-cigarettes are also known as βe-liquidsβ. They usually
contain nicotine, propylene glycol (PG), glycerol (NG), and flavourings. PG and NG are
humectants which acts as nicotine and flavourings carriers. Free-base nicotine is a viscous
liquid which canβt be vaporized and inhaled in pure form. Therefore PG and NG are used
in the e-liquids to dilute the nicotine into a solution of lower potency and lower boiling
point of the e-liquid which allows it to be vaporized and inhaled[12]
. PG and NG are clear,
colourless liquids at room temperature and atmospheric pressure. These compounds are
recognized as safe molecules for moderate oral consumption and are used in a number
of food, drug, and personal care applications due to their favourable physical properties.
Glycerol for example is an exceptionally viscous liquid of low volatility with a sweet taste.
When used as solvents, PG and NG form a highly thick protic environment due to the
occurrence of hydrogen bonding from their multiple hydroxyl groups. These strong
intermolecular associations makes the molecules viscous as liquids and bias them towards
aerosol droplet formation once vaporized[12]
.
Figure 1.7: Molecular structure of Glycerol and Propylene Glycol[12]
19. 7
1.3.1 Toxicants
E-liquids can be contaminated with nitrosamines, volatile organic compounds (VOCs) or
metals leached from the different metallic components of the e-cigarettes. Other
toxicants, which are generated from the thermal degradation of the heated e-liquid, are
referred to as βvaping toxicantsβ. One group of vaping toxicants are reactive oxygen
species that are generated from the cleavage of chemical bonds. Another group are
carbonyls such as formaldehyde, acetaldehyde and acrolein[13]
. When heated, NG
predominantly forms acrolein and formaldehyde while PG predominantly forms
acetaldehyde and formaldehyde[14]
. Figure 1.8 exhibits the oxidation of PG and NG in the
formation of formaldehyde and acetaldehyde. This project will focus on the vaping
toxicants, especially acetaldehyde in six different flavour e-liquids.
Figure 1.8: Diagram exhibiting the oxidation steps of Glycerol and Propylene glycol in the
formation of Formaldehyde and Acetaldehyde[15]
Formaldehyde, acetaldehyde and acrolein are considered the three most toxic aldehydes
due to their low molecular weight and are ranked by The Institute of Medicine to be the
most significant cardiovascular (CV) toxins in tobacco smoke[14]
. These aldehydes are
present in cigarette smoke, cigars, water pipes and e-cigarette aerosols. This project will
investigate the concentration of acetaldehydes in e-liquid aerosols and compare it to the
concentration of acetaldehydes in tobacco cigarette smoke (1240.3 Β± 17.7 ΞΌg/ 10 puffs)[14]
using headspace SPME-GC-MS.
20. 8
1.4 Rules & Regulations
In 2016, the Food and Drug Administration (FDA) implemented the regulatory scope of
the US Family Smoking Prevention and Tobacco Control Act 2009 against e-cigarettes. This
proposed e-cigarettes to be regarded as tobacco products, therefore producers of e-
cigarettes and e-liquids were to follow certain regulations and be approved before the
manufacture and marketing of new products. The regulation constrain manufacturers to
submit to inspections, disclose manufacturing details, and advertise health warnings on
the packages before product distribution[16]
. The administration of the regulation was
caused by the lack of sufficient information on e-cigarettes. The main concerns of the FDA
included the safety of the e-cigarette mechanical components and the composition of the
e-liquid ingredients. In 2016, the European Union (EU) administered the Tobacco Products
Directive 2014/14EU (TPD) for nicotine containing e-cigarettes and refill containers. The
TPD was transposed into the Irish legislation by the EU in 2016 and the provisions are
implemented in the Republic of Ireland by the Health Service Executive (HSE)[17]
. Figure
1.9 exhibits TPD regulations for e-cigarettes and refill containers.
Figure 1.9: TPD regulations for e-cigarettes and refill containers[17]
21. 9
1.5 Methods of E-liquid Analysis
The e-liquids will be analysed using headspace solid phase microextraction mass-
spectrometry (SPME-GC-MS). The method involves the preparation of a diluted e-liquid
sample solution into a glass vial and a O-(2,3,4,5,6-pentafluorobenzyl)- hydroxylamine
hydrochloride (PFBHA) derivatizing agent solution into another glass vial, allowing the
solutions to vaporize into headspace in the vial for fibre absorption. The PDMS/DVB SPME
fibre will then be exposed to the headspace of the PFBHA solution followed by an
exposure to the headspace of the e-liquid sample solution. The PFBHA and e-liquid
exposed SPME fibre will then be inserted into the GC injector for GC-MS analysis. For the
analysis of the e-liquid vapours, the e-liquid samples must be aerosolized into vapour
using the e-cigarette and collected into a glass vial using a pump apparatus. The SPME
fibre is then exposed to the PFBHA solution followed by an exposure to the e-liquid vapour
vial before being inserted into the GC injector for analysis.
1.5.1 Gas Chromatography Mass Spectrometry
Mass spectrometry coupled with gas chromatography will be used as the technique to
analyse the e-liquid samples. The instrument will enable the detection of the analytes in
the sample through the retention times and the mass of each main fragment generated
and the ratio between their intensities, which both ensures that the signal is related to
the analyte. Gas Chromatography Mass Spectrometry (GC-MS) is one of the most
common analytical techniques used for environmental analysis due to its ability to obtain
accurate and reliable results from the analysed sample. It is an analytical technique used
to identify and quantify compounds in a mixture. The GC component separates the volatile
organic compounds in a sample mixture and the MS component identifies the compounds
at a molecular level. In GC-MS, the sample is eluted and analysed in the gas phase. The
compounds must be volatile and must not decompose upon heating for analysis to be
possible. The sample mixture is vaporized and the heated gases are carried through a
column with an inert gas such as helium. The sample mixture is separated into individual
compounds and flows into the MS where they are identified by the mass of the molecule.
The separation of the compounds is based on the strength of interaction between the
compounds and the stationary phase. The stronger the interaction, the longer the
compound will interact with the stationary phase and migrate through the column which
results in a longer retention time. The strength of the interaction is influenced by various
22. 10
factors such as vapour pressure, polarity of the compounds and the stationary phase,
column temperature, column length, carrier gas flow rate and the injection volume
Figure 1.10: Schematic diagram of a GC-MS apparatus[19]
1.5.2 Solid-phase Microextraction (SPME)
SPME uses a fibre that is coated with an extraction phase comprising of an adsorptive
polymer coating which extracts the compounds from the sample to be analysed[20]
. In this
project, the SPME PDMS/DVB fibre will be exposed to the headspace of the PFBHA
derivatizing agent followed by the exposure to the headspace of the e-liquid sample. The
SPME process will cause an interaction between the fibre and the analytes in the
headspace of the sample. The SPME fibre containing the adsorbed analytes will then be
thermally desorbed in the GC injector and will be rapidly transferred to the GC column[20]
.
Figure 1.11 exhibits a schematic diagram of the SPME extraction and GC injector
desorption process
Figure 1.11: Schematic diagram of the SPME extraction and GC injector desorption process[21]
23. 11
The purpose of the derivatizing agent is to trap the low molecular weight aldehydes in
oxime adducts. This is to allow the aldehydes to be retained and detected for analysis by
increasing its molecular weight[22,23]
. The derivatization of the acetaldehyde with the
PFBHA is a fast reaction and forms the oximes in seconds. SPME derivatization with PFBHA
has previously been used to extract aldehydes from environmental and biological samples
such as in air, water and body fluids. This project will involve the use of SPME
derivatization with PFBHA to determine the aldehyde concentrations in electronic
cigarette aerosols of different flavour e-liquids. Figure 1.12 exhibits the reaction scheme
in the formation of the two acetaldehyde oxime isomers.
Figure 1.12: Reaction of PFBHA with an aldehyde to form cis and trans oxime derivatives[23]
1.6 Aims of the work
The aim of this project is to develop a reliable and cost-effective method to detect the
presence and quantify the concentrations of acetaldehyde in the aerosols of six different
flavoured e-liquids. The determined acetaldehyde concentrations will then be compared
to a reference acetaldehyde concentration in a traditional tobacco cigarette to establish
whether e-cigarettes are safer than tobacco cigarettes. The correlation between e-liquid
flavourings will also be investigated.
Research involving SPME GC-MS analysis of nicotine, flavour additives, and carbonyl
compounds in e-cigarette aerosols from different brands have already been assessed in
previous studies. A 2009 study by D.Poli et al. determined C3 to C9 aldehydes in exhaled
breath of patients with lung cancer using a PFBHA derivatized SPME GC-MS analysis
method[23]
. Advanced methods and instrumentation such as cigarette smoking machines
24. 12
(CSMs) have been developed in recent years to improve the analysis of e-cigarette
vapours. This project will investigate the repeatability and precision of a more cost-
effective SPME GC-MS method but with the determination of a C2 aldehyde in the
aerosols of different flavoured e-liquids. A recent study by Ogunwale et al. tested 6
different flavoured e-liquids to examine the variation of aldehyde concentrations in the
e-cigarette aerosols of the e-liquids[14]
. This project will also examine the correlation
between flavour and acetaldehyde concentration in e-liquid aerosols of different flavours.
The aerosols will be captured using a pump apparatus and analysed using PFBHA
derivatized SPME GC-MS analysis. The ingredients labelled on the package for each e-
liquid flavour will then be compared and confirmed with the obtained results through GC-
MS analysis.
26. 14
2 Experimental
This chapter will outline the methodology carried out in this project. The materials,
reagents and instruments used are tabulated throughout the chapter.
2.1 Materials & Reagents
All reagents used in this experimental such as dichloromethane (DCM) and methanol were
purchased from Sigma Aldrich and used without further purifications. The Logic brand
LQD e-liquids Watermelon, Vanilla, Roast Tobacco, Blueberry, Berry Mint and Menthol
were provided by Dr. Aine Whelan. Table 2.1 exhibits the different reagents used in the
experimental. Table 2.2 exhibits the different flavours of e-liquids provided.
Table 2.1: Reagents used and their use in the experimental
Chemical Purity (%) Use
Deionised Water N/A Solvent for standard preparation
Methanol 99.8 Solvent for standard preparation
Acetaldehyde (Ethanal) 99.5 Standard for SPME GCMS analysis
Dichloromethane (DCM) 99.5 Solvent for standard preparation
Pentafluorobenzylhydroxylamine
(PFBHA)
N/A Standard for SPME GCMS analysis
Nicotine 98 Standard for GCMS autosampler analysis
Propylene Glycol 99.5 Standard for GCMS autosampler analysis
Glycerol 99.5 Standard for GCMS autosampler analysis
Menthol 99.5 Standard for GCMS autosampler analysis
Vanillin 99.0 Standard for GCMS autosampler analysis
Cinnamaldehyde 98.0 Standard for GCMS autosampler analysis
Carvone 98.0 Standard for GCMS autosampler analysis
27. 15
Table 2.2: The different flavour e-liquids and their labelled ingredients
E-liquid flavour Labelled ingredients
Watermelon Nicotine, Propylene Glycol, Glycerol, Anethole, Ethyl Butyrate, Piperonal, Flavours
Vanilla Nicotine, Propylene Glycol, Glycerol, Vanillin, Flavours
Roast Tobacco Nicotine, Propylene Glycol, Glycerol, Phenyl Carbinol, Flavours
Blueberry Nicotine, Propylene Glycol, Glycerol, Isoamyl Acetate, Flavours
Berry Mint Nicotine, Propylene Glycol, Glycerol, Menthol, Spearmint, Ethyl Butyrate, Flavours
Menthol Nicotine, Propylene Glycol, Glycerol, Menthol, Nicotine, Flavours
Figure 2.1: The different flavour e-liquids
2.2 Instrumentation
A calibrated 100-1000 ΞΌl micropipette (Sartorius Proline Plus Mechanical Pipette) was
used to measure miniscule amounts of solvents used such as acetaldehyde for the
preparation of acetaldehyde standards solution. An analytical balance was used to
measure masses of solid compounds such as PFBHA for the preparation of the PFBHA
solution. Grade A glass pipettes and volumetric flasks were used to measure and contain
the solutions. All glassware were washed three consecutive times with the specific solvent
before use. A sonicator was used to agitate particles and solutions into a homogenous
mixture. A PFBHA derivatized SPME fibre (SupelcoTM PDMS/DVB Solid Phase
Microextraction Fibre) was used to analyse the acetaldehyde solution in headspace for
GC-MS analysis. The Varian GCMS system was used to conduct an SPME analysis of the
acetaldehyde standards and of the vaporised e-liquid samples in headspace. The Agilent
Technologies GCMS system was used to analyse each of the different flavoured e-liquids.
28. 16
Table 2.3 and Table 2.4 exhibits the GCMS parameters for the Varian and the Agilent
Technologies GCMS system respectively. Figure 2.2 exhibits a schematic diagram of the
SPME GC-MS process.
Figure 2.2: Schematic Diagram of SPME GCMS process[20]
Table 2.3: Varian GCMS Parameters
Parameter Setting
Total Run Time (min) 13 minutes
Oven Temperature (β) Held at 80 β for 1 minute,
then increased by 25 β per minute until 220 β then held at 220 β for 1 minute
Column Type Chrompack Capillary Column CP-Sil 8 CB:
25 m, 250 ΞΌm x 0.25 ΞΌm
Injection Port Temperature (β) 230 β
Carrier Gas Helium
Flow Rate of Carrier Gas (mL/min) 1 ml/min
Injection Volume (uL) 1 ΞΌl
Split Ratio Splitless
29. 17
Table 2.4: Agilent Technologies GCMS Parameters
Parameter Setting
Total Run Time (min) 21 minutes
Oven Temperature (β) Held at 40 β for 1 minute,
then increased by 10 β per minute until 220 β then held at 220 β for 1 minute
Column Type Thermo TG-SQC 26070:
330 β, 30 m, 250 um x 0.25 ΞΌm
Injection Port Temperature
(β)
250 β
Carrier Gas Helium
Flow Rate of Carrier Gas
(mL/min)
1 ml/min
Injection Volume (uL) 1 ΞΌl
Split Ratio 50:1
Detection Parameters Solvent
Delay
(min)
Start and End Mass
(m/z)
Scan Speed
(min)
Frequency
(scan/sec)
2.5 40 - 400 1.5 4.0
2.3 External Standard Calibration of Acetaldehyde Concentration
In an External Standardization, a calibration plot is constructed. This is done by making a
series of calibration solutions containing known concentrations of reference standard
from a stock solution. The analysis of the Acetaldehyde standards was an example of
External Standardization. A calibration plot was constructed using the made series of
calibration solution of concentrations 200 nM, 400 nM, 600 nM, 800 nM and 1000 nM of
the Acetaldehyde reference standard.
30. 18
2.3.1 Preparation of Acetaldehyde Stock Solutions
1M of Acetaldehyde was made up in a 10 ml volumetric flask with DCM. Exhibited below
is the calculated value of the amount of Acetaldehyde needed to make up the 1M stock
solution of Acetaldehyde. The volumes were measured using a calibrated micropipette.
πΆππππ’πππ‘πππ:
(1 π)(0.01 π) = 0.01 πππππ
(0.01 πππππ )(44.05 π/πππ) = 0.4405 π
ππππ’ππ =
πππ π
ππππ ππ‘π¦
=
0.4405 π
0.785 π/ππ
= 0.561 ππ = 560 π’π
The 1M stock solution was diluted to 0.01M using a 100 ml volumetric flask with DCM.
Exhibited below are the calculations for the dilution.
πΆππππ’πππ‘πππ:
(1 π)(0.001 πΏ)
(0.1 πΏ)
= 0.01 π
The 0.01M stock solution was diluted to 0.0001M using a 100 ml volumetric flask with
DCM. Exhibited below are the calculations for the dilution.
πΆππππ’πππ‘πππ:
(0.01 π)(0.001 πΏ)
(0.1 πΏ)
= 0.0001 π
31. 19
2.3.2 Preparation of Acetaldehyde Standard Solutions
Five standards of 200 nM, 400 nM, 600 nM, 800nM and 1000 nM concentrations were
prepared from the 0.0001M stock solution respectively. 100 ΞΌL of stock solution was
added to a 50 ml volumetric flask and was made up to the mark with DCM for standard 1.
πΈπ₯πππππ πππππ’πππ‘πππ πππ ππ‘ππππππ 1:
(2 π₯ 10IJ
π)(50 ππ)
(0.0001 πΏ)
= 0.1 ππ = 100 π’π
Table 2.5: Tabulated data of each of the standards and their concentration and volume of stock
solution used.
Standard no. Concentration (nM) Volume (ml) of stock solution used
1 200 0.1
2 400 0.2
3 600 0.3
4 800 0.4
5 1000 0.5
2.3.3 Preparation of the PFBHA Solution
10 mg of PFBHA and 1 ml of deionised water was added to a 20 ml glass vial. Parafilm was
placed on the vial. The vial was sonicated to agitate the mixture and was left for 30
minutes to allow the PFBHA solution to vaporize into headspace.
2.3.4 Preparation of each of the Standards in a Vial
1 mL of each standard were aliquoted into 20 ml glass vials. A cap containing a septum
was placed on the vials. The vial was sonicated to agitate the mixture and was left for 30
minutes to allow the acetaldehyde solution to vaporize into headspace.
32. 20
2.3.5 SPME GC-MS Analysis of the Acetaldehyde Standards
An SPME fibre was blanked in the injection port for 2 minutes to eliminate carryovers
between runs. The blanked fibre was then exposed to the PFBHA solution for 10 minutes
followed by another 10 minutes exposure to a standard solution. The fibre was inserted
into the injection port for 2 minutes while the analysis ran for a total of 13 minutes. This
process was repeated for each standard solution of 200 nM, 400 nM, 600 nM, 800 nM,
and 1000 nM concentrations respectively.
The procedure was also carried out with the exposure of the blanked fibre to the PFBHA
solution for 10 minutes followed by a 15 minute exposure to the standard solution.
2.4 Internal Standard Calibration of Acetaldehyde Concentration
For Internal Standardization, a known amount of the internal standard (IS) is added to
every sample. For this experiment, Capraldehyde is chosen as the IS. Instead of basing the
calibration on the absolute response of the analyte, the calibration uses the ratio of
response between the analyte and the IS.
2.4.1 Preparation of the Capraldehyde Stock Solutions
2M of Capraldehyde was made up in a 10 ml volumetric flask with DCM. Exhibited below
is the calculated value of the amount of Capraldehyde needed to make up the 1M stock
solution of Capraldehyde. The volumes were measured using a calibrated micropipette.
πΆππππ’πππ‘πππ:
(2 π)(0.01 π) = 0.02 πππππ
(0.02 πππππ )(100.16 π/πππ) = 2.0032 π
ππππ’ππ =
πππ π
ππππ ππ‘π¦
=
2.0032 π
0.815 π/ππ
= 2.458 ππ = 2458 π’π
The 2M stock solution was diluted to 0.002M using a 100 ml volumetric flask with DCM.
Exhibited below are the calculations for the dilution.
33. 21
πΆππππ’πππ‘πππ:
(2 π)(0.0001 πΏ)
(0.1 πΏ)
= 0.002 π
The 0.002M stock solution was diluted to 0.000002M using a 100 ml volumetric flask with
DCM. Exhibited below are the calculations for the dilution.
πΆππππ’πππ‘πππ:
(0.002 π)(0.0001 πΏ)
(0.1 πΏ)
= 0.000002 π
2.4.2 Preparation of Acetaldehyde Standard Solutions with the IS
Capraldehyde (2 M stock solution, 100 ΞΌl) and 1 ml of the acetaldehyde standard solution
was added to a 20 ml vial. A cap consisting of a septum was placed on the vials. The vials
were sonicated to agitate the mixture and was left for 30 minutes to allow the solutions
to vaporize into headspace. This process was repeated for each standard solution of 200
nM, 400 nM, 600 nM, 800 nM and 1000 nM concentrations respectively.
2.4.3 Preparation of the PFBHA Solution
10 mg of PFBHA and 1 ml of deionised water was added to a 20 ml glass vial. Parafilm was
placed on the vial. The vial was sonicated to agitate the mixture and was left for 30
minutes to allow the PFBHA solution to vaporize into headspace.
2.4.4 SPME GC-MS Analysis of the Acetaldehyde Standards Spiked with IS
An SPME fibre was blanked in the injection port for 2 minutes to eliminate carryovers
between runs. The blanked fibre was then exposed to the PFBHA solution for 10 minutes
followed by another 10 minutes exposure to a standard solution. The fibre was inserted
into the injection port for 2 minutes while the analysis ran for a total of 13 minutes. This
process was repeated for each standard solution of 200 nM, 400 nM, 600 nM, 800 nM,
and 1000 nM concentrations respectively.
34. 22
2.5 GC-MS Analysis of the Chemical Composition of E-liquids
Each of the flavours of the Logic brand LQD e-liquids were analysed using the Agilent
Technologies GCMS system to examine their chemical composition and confirm that it
matched the stated ingredients on the product label. Substances identified in the
chromatograms of the e-liquids were further confirmed by GCMS analysis of standard
solutions. The retention times were then compared to confirm the identification.
2.5.1 Preparation of E-liquid Standard Solutions
1:10 dilutions of the original e-liquid solutions were prepared using methanol. The
solutions were analysed using the Agilent Technologies GCMS.
2.5.2 Preparation of Individual Substances Standard Solutions
Nicotine, Propylene Glycol, Glycerol, Cinnamaldehyde and Carvone were available in pure
liquid form. The substances were diluted with Methanol at a 1:100 dilution ratio in a 10
ml volumetric flask. 2000 ΞΌl of each standard solution were transferred into a 2 ml glass
vial. The solutions were analysed using the Agilent Technologies GCMS. Vanillin and
Menthol were available in solid form. A 0.01M solution of both substances were prepared
for analysis. The calculations are shown below.
ππππππ πππππ’πππ‘πππ πππ π‘βπ ππππππππ‘πππ ππ π‘βπ ππππππππ π π‘ππππππ:
(1π)(0.01π) = 0.01 πππππ
(0.01 πππππ )(152.15 π/πππ) = 1.5215 π ππ ππππππππ π€ππ π’π ππ
(1π)(0.001πΏ)
(0.1πΏ)
= 0.01π ππ ππππππππ π€ππ ππππππππ
2.6 SPME GC-MS Analysis of Acetaldehyde in Vape
Each of the different flavoured e-liquids were vaporized and the aerosols generated were
collected into a 20 ml glass vial using a pump apparatus. Figure 2.3 exhibits the pump
apparatus used to collect the vapours. The vapour samples were then analysed in
headspace using SPME GCMS.
35. 23
Figure 2.3: The pump apparatus
2.6.1 Collection of the E-liquid Vapour
A 20 ml glass vial was connected to the outer tube of the pump apparatus using parafilm.
The mouthpiece of the e-cigarette was connected to the inner tube of the pump
apparatus. The pump apparatus was turned on. The 4.7 ohm activation button was held
for 5 seconds and was released for 5 seconds. This was repeated 20 times. A cap
containing a septum was placed on the vial. This process was repeated in triplicate for
each of the vapour samples.
2.6.2 Preparation of the PFBHA Solution
10 mg of PFBHA and 1 ml of deionised water was added to a 20 ml glass vial. Parafilm was
placed on the vial. The vial was sonicated to agitate the mixture and was left for 30
minutes to allow the PFBHA solution to vaporize into headspace.
36. 24
2.6.3 Analysis of the Samples
An SPME fibre was blanked in the injection port for 2 minutes to eliminate carryovers
between runs. The blanked fibre was then exposed to the PFBHA solution for 10 minutes
followed by another 10 minutes exposure to a vapour sample. The fibre was inserted into
the injection port for 2 minutes while the analysis ran for a total of 13 minutes. This
process was repeated in triplicate for each of the vapour samples to determine the
precision of the method.
2.7 Method Validation
200 nM, 100 nM and 50 nM acetaldehyde standard solutions were prepared for the
determination of the LOD and LOQ. A 10 mg PFBHA with 1 ml deionised water solution
was prepared in a 20 ml glass vial to function as a derivatizing agent for the acetaldehyde
in the acetaldehyde standard solutions. An SPME fibre was blanked in the injection port
for 2 minutes to eliminate carryovers between runs. The blanked fibre was then exposed
to the PFBHA solution for 10 minutes followed by another 10 minutes exposure to an
acetaldehyde standard sample. The fibre was inserted into the injection port for 2 minutes
while the analysis ran for a total of 13 minutes. The analysis was performed for the 200
nM, 100 nM and 50 nM acetaldehyde standard solutions.
38. 26
3 Results & Discussion
3.1 PFHBA Derivatized vs Underivatized Acetaldehyde Solutions
The purpose of the derivatizing agent was to trap the low molecular weight aldehydes in
oxime adducts. This was to allow the aldehydes to be retained and detected for analysis
by increasing its molecular weight. Without the use of a derivatizing agent, the aldehyde
would not be retained for analysis. Exhibited below in Figure 3.1, is a chromatogram
comparing the analysis of the 1000 nM acetaldehyde standard with and without the use
of the PFBHA derivatizing agent. The red peaks represents the analysis with the
derivatizing agent and the green peaks represents the analysis without the derivatizing
agent. It can be observed that when the derivatizing agent was used, both the PFBHA
(PFBHA at 7.6 minutes) and the acetaldehyde oximes (Oxime A at 7.7 minutes, Oxime B
at 7.8 minutes) were present.
Figure 3.1: 1000 nM Acetaldehyde standard chromatogram with (red line) and without (green
line) the use of the PFBHA Derivatizing Agent
39. 27
3.2 Development of a Method for Quantification of Acetaldehyde in E-liquid Vapours
3.2.1 External Standard Method
A calibration curve was constructed to understand the instrumental response of the
acetaldehyde analyte and predict the unknown concentrations of acetaldehyde in the e-
liquid vapour samples. The calibration curve concentration used ranged from 200 nM to
1000 nM as this concentration range was used in previous papers in the analysis of
aldehydes in e-cigarette aerosols. The standard solutions and the e-liquid vapour samples
were ran in the same environment to ensure accuracy of the results. This involved the use
of DCM as the solvent and an equal amount of headspace exposure duration. In this
section, the results obtained for the quantitative analysis of the acetaldehyde are shown.
Table 3.1 and 3.2 exhibits the results obtained for the analysis of each standards when
the fibre was exposed to PFBHA for 10 minutes and Acetaldehyde for 10 minutes. Figures
3.2 and 3.3 exhibits the calibration curves for both oxime A and B. Figure 3.4 exhibits a
sample chromatogram to illustrate identity of the peaks obtained in the chromatogram.
Table 3.1 : Tabulated data of results of Oxime A
Concentration (nM) Retention Time Peak Area
0 0 0
200 7.797 101875
400 7.804 176253
600 7.789 244171
800 7.787 296296
1000 7.777 391091
40. 28
Table 3.2: Tabulated data of results of Oxime B
Concentration (nM) Retention Time Peak Area
0 0 0
200 7.891 78248
400 7.891 137034
600 7.882 206422
800 7.880 296329
1000 7.879 344390
Figure 3.2: Calibration curve of Oxime A for 10 minute Acetaldehyde exposure
Figure 3.3 Calibration curve of Oxime B for 10 minute Acetaldehyde exposure
41. 29
Figure 3.4: 1000 nM Acetaldehyde standard chromatogram. The first peak represents PFBHA,
the second peak represents Oxime A, and the third peak represents Oxime B
Each of the peaks in the chromatogram in Figure 3.4 represents the signal created when
a compound elutes from the GC column into the detector. 2 peaks following the PFBHA
peak at 7.6 minutes was observed in the gas chromatogram for each of the Acetaldehyde
standards. As PFBHA bonds with Acetaldehyde to form an oxime-adduct in order for
analysis to be possible, two possible oxime isomers were formed (cis-pentafluorobenzyl
oxime and trans-pentafluorobenzyl oxime). This results in the observation of 2 peaks in
the chromatogram which represents Oxime A at 7.7 minutes and Oxime B at 7.8 minutes.
The analysis was repeated with an increased acetaldehyde standard solution exposure
from 10 minutes to 15 minutes to investigate the difference in the results obtained with
the alteration of exposure duration. Although an increase in the acetaldehyde exposure
resulted in the increase in peak area, the 15 minute exposure derived a less linear
calibration curve of 0.962 for Oxime A and 0.946 for Oxime B, whereas the 10 minute
exposure acquired a significantly linear calibration curve of >0.99 for both Oxime A and B
as shown in Figure 3.2 and Figure 3.3. Thus the results from the 10 minute exposure was
used to determine the concentration of the e-liquid vapour samples. Figure 3.5 and Figure
3.6 exhibits the calibration curve for Oxime A and Oxime B for the 15 minute exposure.
Figure 3.7 exhibits the reaction scheme in the formation of the two oxime isomers.
42. 30
Figure 3.5: Calibration curve of Oxime A for 15 minute Acetaldehyde exposure
Figure 3.6: Calibration curve of Oxime B for 15 minute Acetaldehyde exposure
Figure 3.7: Diagram exhibiting the formation of the two oxime isomers from the reaction of
PFBHA with acetaldehyde
43. 31
3.2.2 Internal Standard Method
The purpose of the internal standard (IS) is to behave similarly to the analyte but to
provide a signal that can be distinguished from that of the analyte. Capraldehyde is chosen
as the IS as the compound is similar but not identical to acetaldehyde. The intentionally-
added compound is used to quantitate the unknown. The IS also compensates for the
varying injection amounts that can occur during the manual injection of the fibre in the
GC-MS injection port[25]
. A known amount of the capraldehyde IS was added to each of
the acetaldehyde standard solutions and was analysed using headspace SPME GCMS.
Figure 3.8 exhibits a sample chromatogram to illustrate the identity of the peaks obtained
in the chromatogram. A study in the SPME GC-MS analysis of aldehyde levels in exhaled
breath used 2-methylpentanale as the IS due to its similarity to the compounds being
examined[24]
.
Figure 3.8: Graph exhibiting the Capraldehyde spiked chromatogram at 1000 nM. The first peak
represents PFBHA, the second peak represents Oxime A, the third peak represents Oxime B and
the fourth peak represents Capraldehyde
The capraldehyde spiked chromatogram illustrates similar peaks to that of the
chromatogram from the non-spiked acetaldehyde standard solutions. The chromatogram
shows the PFBHA peak at 7.6 minutes, followed by Oxime A at 7.7 minutes and Oxime B
at 7.8 minutes. The capraldehyde internal standard peak is observed at 8.4 minutes.
44. 32
The external standard method was used to determine the concentration of the
acetaldehyde as the method deemed a better and more linear result represented by the
R2
values as shown in Figure 3.2 and Figure 3.3 in comparison to the results obtained from
the internal standard method shown in Figure 3.9 and Figure 3.10 below.
Figure 3.9: Calibration curve of Oxime A for the 10 minute exposure IS spiked Acetaldehyde
standards
Figure 3.10: Calibration curve of Oxime B for the 10 minute exposure IS spiked Acetaldehyde
standards
45. 33
3.2.3 Method Validation
The Limit of Detection (LOD) is the point at which analysis is just practical. It is determined
by a statistical approach which involves measuring the replicate blank samples or an
empirical approach by measuring progressively more dilute concentrations of the target
analyte. The Limit of Quantification (LOQ) is the point at which the quantitative results
obtained can be reported with a high degree of confidence and can be deduced by either
the statistical or the empirical approach[26]
. The LOQ and LOD for the acetaldehyde
analytes were determined from acetaldehyde standards yielding a signal-to-noise ratio of
at least 3:1 for the LOD and 10:1 for the LOQ.
A 200 nM, 100 nM and a 50 nM acetaldehyde standard solution was examined for the
LOD and LOQ. The peak to peak noise of a blank and an acetaldehyde peak were measured
for each standard solution using the Varian GCMS software. The result obtained for the
acetaldehyde peak was divided by the result obtained for the blank. Results which deduce
a peak to peak noise ratio of at least 3:1 was determined the LOD and a peak to peak noise
ratio of at least 10:1 was determined the LOQ. Figure 5.0 exhibits the LOD and LOQ results
for both Oxime A and B. 100 nM was established the LOQ and 50 nM was established the
LOD as the peak to blank noise ratio was 10:1 and 3:1 respectively.
Figure 3.11: Concentration vs Peak noise chart for the peak noise to blank noise ratio of Oxime A
and B
46. 34
3.3 GC-MS Analysis of the E-liquids
3.3.1 Analysis of the Chemical Composition of the E-liquids
Six different e-liquids were analysed with the Agilent Technologies GCMS using the
programme enlisted in section 2. The purpose of analysing the e-liquids is to identify and
compare its chemical composition with the e-liquid ingredients labelled on the package.
Standards solutions of Nicotine, Propylene Glycol, Glycerol, Cinnamaldehyde, Carvone,
Vanillin and Menthol were ran under the same conditions and the retention times were
compared to confirm the identification of the compounds in the e-liquids. Table 3.3
exhibits each of the e-liquid samples with their corresponding labelled ingredients and
the ingredients identified from the GC-MS analysis. The ingredients highlighted in red are
compounds found in the GC-MS that are not mentioned on the packaging.
Table 3.3: Tabulated data of the different e-liquid flavours and their labelled and identified
ingredients.
Sample Flavour Labelled Ingredients from package Identified Ingredients from analysis
1 Watermelon Nicotine
Propylene Glycol
Glycerol
Trans-anethole
Ethyl Butyrate
Piperonal
Flavours
Nicotine
Propylene Glycol
Glycerol
Trans-anethole
Ethyl Butyrate
Piperonal
Nonen-1-ol
Ethyl Vanillin
Nerolidol
Heliotropine
2 Vanilla Nicotine
Propylene Glycol
Glycerol
Vanillin
Flavours
Nicotine
Propylene Glycol
Glycerol
Vanillin
Ethyl Maltol
Piperonal
Heliotropine
3 Roast Tobacco Nicotine
Propylene Glycol
Glycerol
Nicotine
Propylene Glycol
Glycerol
48. 36
Figure 3.12: Chromatogram exhibiting the chemical composition of the Watermelon e-liquid
The Watermelon flavour was observed to have a marmalade orange colour and had the
sweet scent of watermelon. It contains the usual Propylene Glycol, Glycerol and Nicotine
ingredients which are observed at peaks 1, 3 and 6 respectively. A broad nonsymmetric
peak was observed for Glycerol due to the analysis of the polar compound containing
three OH groups in a non-polar stationary phase[27]
. The GC-MS analysis of high polar
compounds results in poor detection limits and reproducibility of retention indices in
comparison to compounds of low polarity. Less polar compounds would produce
narrower chromatographic peaks which provide a better signal-to-noise ratio and lower
detection limits[27]
. Ethyl Butyrate, Piperonal and Anethole were observed at peaks 2, 4
and 5 respectively. These substances function as flavour and fragrance agents. Anethole
is a terpene and is found is essential oils such as anise and fennel. It is used as a flavouring
agent in products such as toothpaste and medicinal candies. It has a very sweet smell and
flavour reminiscent of liquorice[28]
. Ethyl Butyrate has a fruity odour, similar to pineapple
and is used as a flavour enhancer in processed orange juices and beers[28]
. Piperonal has
a floral odour which is commonly described as being similar to vanillin or cherry. It is
commonly used in fragrances and artificial flavours[28]
. Nonen-1-ol, Ethyl Vanillin,
Nerolidol and Heliotropin propylene glycol acetal were present in the chromatogram at
low concentrations. These compounds also function as flavour and fragrance agents.
49. 37
Table 3.5: Compounds identified in the Vanilla flavour e-liquid
Retention Time Peak No. ID % Match in Library M/Z Fragments
3.610 1 Propylene Glycol 90 40, 45, 61, 77
8.740 2 Glycerol 83 41, 43, 61, 75
12.879 3 Nicotine 95 42, 84, 133, 162
13.556 4 Vanillin 96 41, 81, 109, 153
Figure 3.13: Chromatogram exhibiting the chemical composition of the Vanilla e-liquid
The Vanilla flavoured e-liquid contains the usual Propylene Glycol, Glycerol and Nicotine
which are observed at peaks 1, 2 and 3 respectively. The e-liquid has a fire orange colour
and has a sweet vanilla-like aroma which may be due to the observed Vanillin peak
present in the liquid. Vanillin elutes at 13.6 minutes and is present at a medium peak
height which suggests that a moderate amount of Vanillin is present in the Vanilla
flavoured e-liquid. Peak 4 is confirmed Vanillin as an individual run of the Vanillin standard
had a similar elution time of 13.6 minutes and an identical m/z fragments of 41, 81, 109,
and 153. The GCMS Library search identified peak 4 as Vanillin and also gave identical m/z
fragments. Ethyl Maltol, Piperonal and Heliotropin propylene glycol acetal were present
in the chromatogram at low concentrations. These compounds also function as flavour
and fragrance agents.
50. 38
Table 3.6: Compounds identified in the Roast Tobacco flavour e-liquid
Retention Time Peak No. ID % Match in Library M/Z Fragments
3.610 1 Propylene Glycol 90 43, 71, 88, 116
8.740 2 Glycerol 83 41, 43, 61, 75
10.351 3 Menthol 90 41, 71, 81, 154
12.884 4 Nicotine 95 42, 84, 133, 162
13.556 5 Vanillin 91 41, 81, 109, 153
13.907 6 Caryophyllene 94 4, 93, 133, 204
Figure 3.14: Chromatogram exhibiting the chemical composition of the Roast Tobacco e-liquid
The Roast Tobacco flavoured e-liquid contains the usual Propylene Glycol, Glycerol and
Nicotine which are observed at peaks 1, 2 and 4 respectively. The e-liquid has an apricot
orange colour and has a sweet almond scent with a hint of floral and vanilla-like aroma.
These mixtures of scents are due to the ingredients observed at peaks 3, 5 and 6 which all
function as flavour and fragrance agents. The peaks were identified using the GCMS
Library search and are supported by a GC-MS analysis of each individual peaks. The m/z
fragments results of the individual analysis were identical to the m/z fragments results of
the Library search. These are exhibited in the table above. Vanillin propylene glycol acetal
is present in the chromatogram at low concentrations. The compounds also function as a
flavour and fragrance agent.
51. 39
Table 3.7: Compounds identified in the Blueberry flavour e-liquid
Retention Time Peak No. ID % Match in Library M/Z Fragments
3.610 1 Propylene Glycol 90 43, 71, 88, 116
4.375 2 Butanoic acid ethyl ester 97 43, 71, 88, 116
5.547 3 Isoamyl acetate 90 41, 55, 70, 115
8.794 4 Glycerol 83 41, 43, 61, 75
10.346 5 Menthol 91 41, 71, 81, 154
12.883 6 Nicotine 95 42, 84, 133, 162
Figure 3.15: Chromatogram exhibiting the chemical composition of the Blueberry e-liquid
The Blueberry flavoured e-liquid contains the usual Propylene Glycol, Glycerol and
Nicotine which are observed at peaks 1, 4 and 6 respectively. The e-liquid has a colourless
colour and has a sweet fruity smell. Peaks 2, 3 and 5 contribute to this fruity scent. Ethyl
Butyrate has a fruity odour, similar to pineapple and is a key ingredient used as a flavour
enhancer in processed orange juices[28]
. Isoamyl acetate has a strong odour that is similar
to Juicy Fruit or a pear drop, which is reminiscent of the smell of both banana and pear[28]
.
The peaks were identified using the GCMS Library search and are supported by a GC-MS
analysis of each individual peaks. The m/z fragments results of the individual analysis were
identical to the m/z fragments results of the Library search and are exhibited in Table 4.2
above. Butanoic acid, 2-methyl ethyl ester, 3-Hexen-1-ol and 1,3-Dioxolane, 4-methyl-2
52. 40
phenyl were present in the chromatogram at low concentrations. These compounds also
function as flavour and fragrance agents.
Table 3.8: Compounds identified in the Berry Mint flavour e-liquid
Retention Time Peak No. ID % Match in Library M/Z Fragments
3.610 1 Propylene Glycol 90 43, 71, 88, 116
4.371 2 Butanoic acid ethyl ester 97 43, 71, 88, 116
5.128 3 Butanoic acid 2-methyl ethyl ester 83 43, 71, 88, 116
5.179 4 Butanoic acid 3-methyl ethyl ester 86 43, 71, 88, 116
5.547 5 Isoamyl acetate 83 41, 55, 70, 115
8.773 6 Glycerol 83 41, 43, 61, 75
10.346 7 Menthol 91 41, 71, 81, 154
11.416 8 Carvone 97 41, 82, 108, 150
11.805 9 Cinnamaldehyde 91 44, 78, 103, 133
12.884 10 Nicotine 94 42, 84, 133, 162
13.556 11 Vanillin 94 41, 81, 109, 153
Figure 3.16: Chromatogram exhibiting the chemical composition of the Berry Mint e-liquid
The Berry Mint flavoured e-liquid contains the usual Propylene Glycol, Glycerol and
Nicotine which are observed at peaks 1, 6 and 10 respectively. The e-liquid has an amber
53. 41
orange colour and has a sweet odour. Peaks 2, 3, 4, 5, 7, 8, 9 and 11 contribute to this
fragrance. Carvone produces a sweet caraway and spearmint smell while Menthol
produces a fresh, minty smell[28]
. Both of these compounds were observed to have high
peak heights. Cinnamaldehyde and Vanillin were also observed during the analysis but are
present at low peak heights which concludes the presence of the compounds at low
concentrations in the e-liquid. The peaks were identified using the GC-MS Library search
and were supported by a GC-MS analysis of standard solutions. The m/z fragments results
of the individual analysis were identical to the m/z fragments results of the Library search
and are exhibited in Table 4.3 above. 4H-Pyran-4-one, 2-ethyl-3-hydroxy, 2(3H)-Furanone,
5-hexyldihydro, 3-Buten-2-one, 2,6,6-trimethyl-cyclohexen-1-yl and 4-(4-Hydroxyphenyl)-
2-butanone were present in the chromatogram at low concentrations. These compounds
also function as flavour and fragrance agents.
Table 3.9: Compounds identified in the Menthol flavour e-liquid
Retention Time Peak No. ID % Match in Library M/Z Fragments
3.610 1 Propylene Glycol 90 43, 71, 88, 116
8.731 2 Glycerol 83 41, 43, 61, 75
10.351 3 Menthol 91 41, 71, 81, 154
12.879 4 Nicotine 94 42, 84, 133, 162
Figure 3.17: Chromatogram exhibiting the chemical composition of the Menthol e-liquid
54. 42
The Menthol flavoured e-liquid contains the usual Propylene Glycol, Glycerol and Nicotine
which are observed at peaks 1, 2 and 4 respectively. The e-liquid has a colourless colour
and has a fresh, minty odour. This fragrance is due to peak 4 which was identified as
Menthol. The peaks were identified using the GCMS Library search and are supported by
a GC-MS analysis of standard solutions. The m/z fragments results of the individual
analysis were identical to the m/z fragments results of the Library search and are exhibited
in Table 4.4 above.
3.3.2 Analysis of the Chemical Composition of the Standard Solutions
Standard solutions of Nicotine, Propylene Glycol, Glycerol, Cinnamaldehyde and Carvone
were each analysed using the Agilent Technologies GCMS. Vanillin and Menthol were later
analysed. The separate analysis of the substances identified in the e-liquids was
performed for confirmation purposes. Although the substances were compared and
confirmed using the GCMS βlibrary researchβ and m/z results, an analysis of the
substances was carried out to further confirm the identity of the e-liquid ingredients.
Figure 4.8 exhibits an overlaid chromatogram of the substances. Table 4.0 exhibits each
substance with their corresponding retention times. The retention times of each
substance were compared to the retention times of the substances observed from the
analysis of the e-liquid composition using the Agilent Technologies GCMS. Table 4.1
exhibits the retention times for each substance obtained from the e-liquid composition
analysis. The % difference between the retention times are relatively low for the
substances which concluded that the substances are identically similar. The 21% retention
time difference for Glycerol is due to the broadness of the peak which leads to
irreproducible retention times.
55. 43
Figure 3.18: Overlaid chromatogram of the standards
Table 4.0: Tabulated data of each standard with their corresponding retention time
Substance Retention Time
(mins)
Nicotine 12.905
Propylene Glycol 3.568
Glycerol 7.923
Cinnamaldehyde 11.826
Carvone 11.065
Vanillin 13.552
Menthol 10.351
56. 44
Table 4.1: Tabulated data of each standard with their corresponding retention time from the e-
liquid composition analysis
Substance E-liquid sample Average Retention Time
(mins)
Difference
(%)
Nicotine All Flavours 12.883 2.2
Propylene Glycol All Flavours 3.610 4.2
Glycerol All Flavours 8.133 21
Cinnamaldehyde Berry Mint 11.805 2.1
Carvone Berry Mint 11.416 3.5
Vanillin Vanilla, Roast Tobacco 13.556 0.4
Menthol Menthol, Roast Tobacco, Berry Mint 10.349 0.2
3.4 SPME GC-MS Analysis of the E-liquid Vapours
The e-liquid vapours were analysed using headspace SPME GC-MS to analyse the presence
of acetaldehyde and to examine the difference between the e-liquids in liquid form and
in aerosol form. A pump apparatus was used to collect the e-liquid vapours which mimic
the smoking action of a regular smoker. The chemical composition of the e-liquid in liquid
form was previously examined using the Agilent Technologies GCMS system. The results
below exhibits the results for the e-liquid vapour analysis using the Varian GCMS. The
peak area of the acetaldehyde isomers for each e-liquid sample was obtained and the
acetaldehyde concentration in each sample was determined using the external standard
calibration curve. Figure 3.19 exhibits a chromatogram illustrating the obtained
acetaldehyde oxime peak area values for the Berry Mint e-liquid vapour sample. Tables
4.2 - 4.7 exhibits the results for the e-liquid vapour analysis using the Varian GCMS.
57. 45
Figure 3.19: Sample chromatogram of the Berry Mint sample illustrating the retention times and
the peak areas for each acetaldehyde oxime peak
Table 4.2: Concentration of Acetaldehyde detected in the vapour from the Watermelon
flavoured e-liquid
Oxime Concentration (nM) Mean
of Peak Area
Standard Deviation
of Peak Area
%RSD ΞΌg/ 10 puffs
A 688.35 271756 1118.798 0.411 0.123
B 1082.67 380641 1622.967 0.426 0.194
Table 4.3: Concentration of Acetaldehyde detected in the vapour from the Vanilla flavoured e-
liquid
Oxime Concentration (nM) Mean
of Peak Area
Standard Deviation
of Peak Area
%RSD ΞΌg/ 10 puffs
A 641.33 245247.33 1355.43 0.552 0.115
B 1088.86 382803.33 1622.37 0.424 0.195
58. 46
Table 4.4: Concentration of Acetaldehyde detected in the vapour from the Roast Tobacco
flavoured e-liquid
Oxime Concentration (nM) Mean
of Peak Area
Standard Deviation
of Peak Area
%RSD ΞΌg/ 10 puffs
A 359.06 149132.66 1084.82 0.727 0.064
B 654.56 231070.66 1392.45 0.602 0.117
Table 4.5: Concentration of Acetaldehyde detected in the vapour from the Blueberry flavoured
e-liquid
Oxime Concentration (nM) Mean
of Peak Area
Standard Deviation
of Peak Area
%RSD ΞΌg/ 10 puffs
A 418.4 140380 1268.70 0.903 0.075
B 625.45 220900 1630.75 0.738 0.112
Table 4.6: Concentration of Acetaldehyde detected in the vapour from the Berry Mint flavoured
e-liquid
Oxime Concentration (nM) Mean
of Peak Area
Standard Deviation
of Peak Area
%RSD ΞΌg/ 10 puffs
A 416.99 170707 741.64 0.434 0.074
B 705.18 248814 1534.35 0.616 0.126
Table 4.7: Concentration of Acetaldehyde detected in the vapour from the Menthol flavoured e-
liquid
Oxime Concentration (nM) Mean
of Peak Area
Standard Deviation
of Peak Area
%RSD ΞΌg/ 10 puffs
A 234.81 102867 1138.87 1.107 0.042
B 549.8 194470 1203.2 0.618 0.098
60. 48
πΆππππππ‘πππ‘πππ ππ π πππππ
372.38π₯ + 15426 = π₯Μ
372.38π₯ + 15426 = 170707
π₯ =
170707 β 15426
372.38
π₯ = 416.99 ππ
π’π πππ 10 ππ’πππ
πππ/1.5 = _
416.99 π₯ 10I`
1000
a (1.5 ππ) = 6.25 π₯ 10Ibc
(6.25 π₯ 10Ibc
πππ/1.5)(239.145 π/πππ) = 1.495 π₯ 10IJ
π
π’π πππ 20 ππ’πππ = (1.495 π₯ 10IJ
π)(10d) = 0.1495 π’π
π’π πππ 1 ππ’ππ =
0.1495 π’π
20
= 0.0074 π’π
π’π πππ 10 ππ’πππ = (0.00747 π’π)(10) = 0.074 π’π
Variabilities in acetaldehyde concentrations can be explained by factors such as the type
of e-cigarette coil, the power output of the e-cigarette, the composition of the e-liquid
and the βdry puffβ phenomenon[29,30,31]
. In this project, the occurrence of the dry puff
phenomenon and the insufficient encapsulation of the e-liquid vapours were observed to
contribute in the increase and decrease in acetaldehyde concentrations. Therefore it is
important to perform corrective measures to eliminate or minimize this problem for
method reproducibility. Farsalinos et al have found that high aldehyde concentrations in
e-cigarette aerosols occur mainly during dry puff conditions. This phenomenon occurs
when the power output of the e-cigarette is at a high setting and also when there is
insufficient supply of e-liquid in the wick thus causing an increase in temperature[31]
. This
βdry puffβ phenomenon was observed during the experiment where following 10
consecutive puffs of 5 second activation 5 second rest method for 20 repetitions, the
vapours captured in the vial appeared clear in contrast to being cloudy. The insufficient
61. 49
amounts of e-liquid in the cartridge throughout each vapour collection was one of the
reasons to why there was a significant increase in acetaldehyde concentration as it
contributed to the occurrence of the dry puff phenomenon. The unsecure entrapment of
the generated vapours into the glass vials with parafilm during the vapour collection
process lead to the decrease in acetaldehyde concentration. These propositions were
investigated by performing a repeated analysis of the e-liquid vapour collection. The SPME
GC-MS analysis of the e-liquid vapours was repeated to eliminate or minimize the factors
by ensuring that full amounts of e-liquid was contained in the e-cigarette cartridge and by
improving the confinement of the vapours with the use of excess parafilm to fully cover
vapour escape routes. The results for the corrected analysis is exhibited in Table 4.2 to 4.7
above. An increase in peak areas and the presence of new peaks were observed following
the corrective measures. An example of the improvements are exhibited in Figure 3.20
and Figure 3.21.
Figure 3.20: Chromatogram of Watermelon sample before improvements
62. 50
Figure 3.21: Chromatogram of Watermelon sample after improvements
The phenomenon can also be prevented by reducing the power levels and puff duration
or by increasing interpuff interval[31]
. The power levels can be reduced by the use of the
3.7 W setting for activation instead of 4.2 W which was the power output used in the
experiment. The puff duration can be reduced by decreasing the duration of activation
from 5 to 10 seconds and the interpuff interval can be increase by prolongating the
deactivation from 5 to 10 seconds. If the experiment were to be repeated, these
alterations would be investigated to determine if the changes would result in a decrease
in the acetaldehyde concentration as the size of the peak area is proportional to the
concentration of the acetaldehyde analyte.
Acetaldehyde in 10 puff aerosols generated from the six e-liquids ranged from 0.042 ΞΌg -
0.123 ΞΌg for Oxime A and 0.098 ΞΌg - 0.195 ΞΌg for Oxime B at a power output of 4.2W.
These results are significantly lower than the acetaldehydes concentrations determined
from a study by Counts et al. exhibited in Table 5.2 below. The difference in the
acetaldehyde concentrations is mainly due to the difference in the e-cigarette power
output where the study used a higher power output of 4.6W. In the study, acetaldehyde
concentrations were observed to vary on the power output of the e-cigarette. At a power
output of 4.6 W, acetaldehyde concentrations in 10 puffs ranged from 0.15 ΞΌg to 0.57 ug
63. 51
whereas at a power output of 9.1 W, it ranged from 13.3 ΞΌg to 63.1 ΞΌg[32]
. Although
aldehyde concentrations also depend on different features such as the type of e-cigarette
and the chemical composition of the e-liquid, the comparison of both the experiment and
the study from Counts et al. affirms that power output is a major characteristic in the
generation of aldehydes. It can also be concluded that e-cigarettes are a much safer
alternative to traditional cigarettes in terms of toxicant production as the concentrations
of acetaldehydes generated in e-cigarettes are significantly lower than that of
conventional cigarettes (1240.3 ΞΌg per 10 puffs)[32]
.
Other carbonyl compounds in aerosols of e-cigarettes such as formaldehyde and acrolein
were found in concentrations that relate to the battery power output of the device and
the composition of the e-liquids according to previous studies from Sleiman et al.[33]
and
Bekki et al.[15]
A recent study of carbonyl emissions produced from 27 e-cigarette products
observed that characteristics of the e-liquid such as propylene glycol (PG) and glycerol
(NG) ratios contribute to the variance in aldehyde emissions[34]
. A similar study also found
that different e-liquid flavouring lead to variabilities in aldehyde concentrations. It was
deduced that the presence of either vanillin or cinnamaldehyde in e-liquids were
associated with higher aldehyde toxicity values[35]
. This conclusion can be observed and
supported by the results of the project as the top three most acetaldehyde concentrated
e-liquid flavours (Watermelon, Vanilla and Berry Mint) contained either vanillin or
cinnamaldehyde.
Figure 3.22: Carbonyl Compounds formed with their corresponding concentrations per 10 puffs
from Counts et al.[32]
64. 52
The precision of an analytical method represents the proximity of individual measures of
an analyte when the method is constantly applied to various aliquots of a single matrix.
Precision was determined by the relative standard deviation (%RSD) of the peak area of
the e-liquid samples repeated in triplicate. The %RSD was expected to not exceed the
acceptance limit of 10% for an analytical method[36]
as SPME GC-MS is regarded to have
exceptional precision since SPME is a single step process where there are minimum
sources of error in the transfer of analytes[37]
. The excellent precision of the method is
exhibited in Table 4.2 to Table 4.7 with a %RSD of 0.411% - 1.107% for Oxime A and
0.424% - 0.738% for Oxime B for all of the 6 different flavour e-liquid samples.
The results can be improved by the use of more advanced aerosol capturing methods. In
the study of aldehyde detection in e-cigarette aerosols, a microreactor capture approach
with an 4-(2-aminooxyethyl)-morpholin-4-ium chloride (AMAH) coating was used[14]
. In
the study of nicotine concentration and flavourings in e-cigarette aerosols, a single port
piston-operated smoking machine was used[38]
. A change in the aerosol generation
condition could also improve the results. Leigh et al demonstrated the generation and
capture of e-cigarette aerosol using conditions of 3 s puff duration with a 30 s interpuff
interval. The results from the experiment achieved a variability of 0.05[38]
.
66. 54
4 Conclusions & Future Work
Acetaldehydes were generated from the oxidation of propylene glycol and glycerol in the
e-liquids by the atomizer in e-cigarettes. This was evident when comparing the
chromatograms between the GC-MS analysis of the e-liquid chemical composition and the
SPME GC-MS analysis of the e-liquid vapour results. The external standard calibration
curve was chosen to determine the acetaldehyde concentrations as the calibration curve
was deemed more linear (R2
= >0.99) in comparison to the internal standard calibration
curve. The chemical composition of the e-liquid such as flavourings, was found to affect
the variability in the concentration of acetaldehyde. This was supported by the differences
in acetaldehyde concentrations between the different flavours. It was also found that the
power output control the levels of acetaldehyde concentrations by the comparison
between the results obtained from the project and the results from other studies. The
method is deemed to be of high precision and reliability as the %RSD did not meet over
the criteria of 10% (0.411% - 1.107% for Oxime A and 0.424% - 0.738% for Oxime B). E-
cigarettes are concluded safer that conventional cigarettes as the acetaldehyde
concentrations in 10 puff aerosols were significantly lower (0.042 ΞΌg - 0.123 ΞΌg for Oxime
A and 0.098 ΞΌg - 0.195 ΞΌg for Oxime B) than that of conventional cigarettes (1240.3 ΞΌg).
The analysis of a larger sample size of different flavours and brands would be of interest
to determine if similar flavours from different brands would produce similar
concentrations of acetaldehyde obtained in this project. An analysis using e-cigarettes of
different generations (first and third generation) would be of interest to investigate the
influences of the variable in acetaldehyde concentrations. Figure 4.1 exhibits the
differences between first, second and third generation e-cigarettes.
Figure 4.1: Difference between a first, second and third generation e-cigarette[39]
67. 55
It would also be of interest to analyse the same e-liquid samples with the use of more
advanced aerosol capture and collection methods such as CSMs to compare the levels of
acetaldehyde concentrations produced. Further research in alternatives to the nicotine
and flavouring bases of propylene glycol (PG) and glycerol (NG) would also be of interest
as the two humectants are the main contributors to the production of the toxic aldehydes.
69. 57
5 References
1. Prasad S, Kaisar MA, Cucullo L. Unhealthy smokers: scopes for prophylactic
intervention and clinical treatment. BMC Neurosci. 2017;18(1):70. Published 2017
Oct 4. doi:10.1186/s12868-017-0388-6
2. Durmowicz EL. The impact of electronic cigarettes on the paediatric population.
Tob Control. 2014;23 Suppl 2(Suppl 2):ii41βii46. doi:10.1136/tobaccocontrol-
2013-051468
3. Singh T, Arrazola RA, Corey CG, et al. Tobacco Use Among Middle and High School
Students β United States, 2011β2015. MMWR Morb Mortal Wkly Rep
2016;65:361β367. DOI: http://dx.doi.org/10.15585/mmwr.mm6514a1External
4. Liu, Xinyu & Joza, Peter & Rickert, Bill. (2017). Analysis of Nicotine and Nicotine-
Related Compounds in Electronic Cigarette Liquids and Aerosols by Liquid
Chromatography-Tandem Mass Spectrometry. BeitrΓ€ge zur Tabakforschung
International/Contributions to Tobacco Research. 27. 10.1515/cttr-2017-0016.
5. Chemical Composition of Aerosol from an E-Cigarette: A Quantitative Comparison
with Cigarette Smoke Jennifer Margham, Kevin McAdam, Mark Forster, Chuan Liu,
Christopher Wright, Derek Mariner, and Christopher Proctor, Chemical Research
in Toxicology 2016 29 (10), 1662-1678 DOI: 10.1021/acs.chemrestox.6b00188
6. Flora, J. W., Meruva, N., Huang, C. B., Wilkinson, C. T., Ballentine, R., Smith, D. C.,
et al. (2016). Characterization of potential impurities and degradation products in
electronic cigarette formulations and aerosols. Regul. Toxicol. Pharmacol. 74, 1β
11. doi: 10.1016/j.yrtph.2015.11.009
7. Eddingsaas N, Pagano T, Cummings C, Rahman I, Robinson R, Hensel E. Qualitative
Analysis of E-Liquid Emissions as a Function of Flavor Additives Using Two Aerosol
Capture Methods. Int J Environ Res Public Health. 2018;15(2):323. Published 2018
Feb 13. doi:10.3390/ijerph15020323
8. Medana, C., et al. (2016) Analysis of nicotine alkaloids and impurities in liquids for
e-cigarettes by LC-MS, GC-MS, and ICP-MS. Current Trends in Mass Spectrometry,
5 (29), 20-28.
9. Chastain Anderson, Andrew Majeste, Jakub Hanus, Shusheng Wang, E-Cigarette
Aerosol Exposure Induces Reactive Oxygen Species, DNA Damage, and Cell Death
71. 59
20. Zhang S., Raftery D. (2014) Headspace SPME-GC-MS Metabolomics Analysis of
Urinary Volatile Organic Compounds (VOCs). In: Raftery D. (eds) Mass
Spectrometry in Metabolomics. Methods in Molecular Biology (Methods and
Protocols), vol 1198. Humana Press, New York, NY
21. Kusch, Peter. (2012). Pyrolysis-Gas Chromatography/Mass Spectrometry of
Polymeric Materials. 10.5772/32323.
22. Li, Zhong et al. βDetection and quantification of low-molecular-weight aldehydes
in pharmaceutical excipients by headspace gas chromatography.β Journal of
chromatography. A 1104 1-2 (2006): 1-10 .
23. Svensson, Sophie & LΓ€rstad, Mona & Broo, Klas & Olin, Anna-Carin. (2008).
Determination of aldehydes in human breath by on-fibre derivatization, solid-
phase microextraction and GC-MS. Journal of chromatography. B, Analytical
technologies in the biomedical and life sciences. 860. 86-91.
10.1016/j.jchromb.2007.10.021.
24. Diana Poli, Matteo Goldoni, Massimo Corradi, Olga Acampa, Paolo Carbognani,
Eveline Internullo, Angelo Casalini, Antonio Mutti, Determination of aldehydes in
exhaled breath of patients with lung cancer by means of on-fiber-derivatization
SPME-GC/MS, J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Oct 1; 878(27):
2643β2651. Published online 2010 Jan 25. doi: 10.1016/j.jchromb.2010.01.022
25. Dettmer-Wilde, Katja & Engewald, Werner. (2014). Practical Gas Chromatography:
A Comprehensive Reference. 10.1007/978-3-642-54640-2.
26. Armbruster DA, Pry T. Limit of blank, limit of detection and limit of quantitation.
Clin Biochem Rev. 2008;29 Suppl 1(Suppl 1):S49βS52.
27. J.Cazes, Encyclopedia of Chromatography Vol. 1, Published by Taylor & Francis,
Boca Raton, Florida, U.S.A. (2005)
28. The Good Scents Company (2009). Flavor and fragrance information catalog.
<http://www.thegoodscentscompany.com/allprod.html> (Accessed on 10.04.19)
29. Geiss, O., Bianchi, I., and Barrero-Moreno, J. (2016). Correlation of volatile
carbonyl yields emitted by e-cigarettes with the temperature of the heating coil
and the perceived sensorial quality of the generated vapours. Int. J. Hyg. Environ.
Health. 219, 268β277. doi: 10.1016/j.ijheh.2016.01.004
72. 60
30. Gillman, I. G., Kistler, K. A., Stewart, E. W., and Paolantonio, A. R. (2016). Effect of
variable power levels on the yield of total aerosol mass and formation of aldehydes
in e-cigarette aerosols. Regul. Toxicol. Pharmacol. 75, 58β65. doi:
10.1016/j.yrtph.2015.12.019
31. Farsalinos, K. E., Voudris, V., and Poulas, K. (2015). E-cigarettes generate high
levels of aldehydes only in βdry puffβ conditions. Addiction 110, 1352β1356.
32. Counts, M. E., Morton, M. J., Laffoon, S. W., Cox, R. H., and Lipowicz, P. J. (2005).
Smoke composition and predicting relationships for international commercial
cigarettes smoked with three machine-smoking conditions. Regul. Toxicol.
Pharmacol. 41, 185β227. doi: 10.1016/j.yrtph.2004.12.002
33. Sleiman, M., Logue, J. M., Montesinos, V. N., Russell, M. L., Litter, M. I., Gundel, L.
A., et al. (2016). Emissions from electronic cigarettes: key parameters affecting the
release of harmful chemicals. Environ. Sci. Technol. 50, 9644β9651. doi:
10.1021/acs.est.6b01741
34. DeVito EE, Krishnan-Sarin S. E-cigarettes: Impact of E-Liquid Components and
Device Characteristics on Nicotine Exposure. Curr Neuropharmacol.
2018;16(4):438β459. doi:10.2174/1570159X15666171016164430
35. Sassano MF, Davis ES, Keating JE, Zorn BT, Kochar TK, Wolfgang MC, et al. (2018)
Evaluation of e-liquid toxicity using an open-source high-throughput screening
assay. PLoS Biol 16(3): e2003904.
36. Gupta A, Singh Y, Srinivas KS, Jain G, Sreekumar VB, Semwal VP. Development and
validation of a headspace gas chromatographic method for the determination of
residual solvents in arterolane (RBx11160) maleate bulk drug. J Pharm Bioallied
Sci. 2010;2(1):32β37. doi:10.4103/0975-7406.62706
37. J. Havenga, Willem & R. Rohwer, Egmont. (2000). The Use of SPME and GC-MS for
the Chemical Characterisation and Assessment of PAH Pollution in Aqueous
Environmental Samples. International Journal of Environmental Analytical
Chemistry. 78. 205-221. 10.1080/03067310008041342.
38. Leigh NJ, Lawton RI, Hershberger PA, et al. Flavourings significantly affect
inhalation toxicity of aerosol generated from electronic nicotine delivery systems
(ENDS), Tobacco Control 2016;25:ii81-ii87
39. https://www.smokefreehackney.org/ecigarette-guidance (Accessed on 10.04.19)
75. 6βA
6 Appendices
Appendix 6.1: Sample results obtained from the GC-MS analysis of e-liquid chemical
composition
Figure 6.1: GC Chromatogram of Propylene Glycol standard
Figure 6.2: m/z spectra of Propylene Glycol standard
Figure 6.3: GC-MS Library Search results of Propylene Glycol standard
76. 6βB
Appendix 6.2: Sample results obtained from the SPME GC-MS analysis of e-liquid vapour
Figure 6.4: SPME GC-MS Chromatogram results of Watermelon flavour e-liquid
Appendix 6.3: Sample results obtained from the Method Validation analysis
Figure 6.5: Peak to Peak Noise results of Oxime A
