Scientific-technical support activities to DG TAXUD-C-2 on the option to include e-cigarettes within the scope of excisable goods for the impact assessment on a possible revision of Directive 2011/64/EU : final report.
Date: 2018-01-18
Institutions: Joint Research Centre
Subjects: excise duty, flavouring, manufactured goods, research report, tobacco, tobacco industry

Scientific-technical  support  activities  to  DG 
include 
TAXUD-C-2  on 
e-cigarettes  within  the  scope  of  excisable 
goods for the Impact Assessment on a possible 
revision of Directive 2011/64/EU  

the  option 

to 

Administrative Arrangement 
TAXUD/2015/DE/329 

Final report 

Thomas  Wenzl,  Zuzana  Zelinkova,  Jorge 
Regueiro, Anupam Giri 

2017 

EUR 29062

 
 
 
 
 
 
 
 
 
 
This publication is a Technical report by the Joint Research Centre (JRC), the European Commission’s science and 
knowledge service. It aims to provide evidence-based scientific support to the European policy-making process. 
The scientific output expressed does not imply a policy position of the European Commission. Neither the 
European Commission nor any person acting on behalf of the Commission is responsible for the use which might 
be made of this publication. 

Contact information  
Name: Thomas Wenzl 
Address: JRC Geel, Retieseweg 111, B-2440 Geel 
E-mail: thomas.wenzl@ec.europa.eu 

JRC Science Hub 
https://ec.europa.eu/jrc 

JRC105825 

EUR 29062 EN 

PDF 

ISBN 978-92-79-77729-5 

ISSN 1831-9424 

doi:10.2760/663170 

Luxembourg: Publications Office of the European Union, 2017 

© European Union, 2017 

The  reuse  of  the  document  is  authorised,  provided  the  source  is  acknowledged  and  the  original  meaning  or 
message of the texts are not distorted. The European Commission shall not be held liable for any consequences 
stemming from the reuse. 

How  to  cite:  Wenzl,  T.;  Zelinkova,  Z.;  Regueiro,  J.;  Giri,  A.,  Scientific-technical  support  activities  to 
DG TAXUD-C-2  on  the  option  to  include  e-cigarettes  within  the  scope  of  excisable  goods  for  the  Impact 
Assessment  on  a  possible  revision  of  Directive  2011/64/EU,  EUR  29062  EN,  2017.  ISBN  978-92-79-77729-5, 
doi:10.2760/663170 

All images © European Union 2017 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table of contents 

Acknowledgements ................................................................................................ 3 

Executive summary ............................................................................................... 4 

Introduction ......................................................................................................... 5 

Determination of nicotine and nicotine related alkaloids in commercial e-liquids ............ 7 

1.1 

Samples ............................................................................................... 7 

1.1.1 

Sample preparation and analysis .......................................................... 7 

1.1.2 

Tobacco alkaloid content of liquids used in e-cigarettes ........................... 9 

Volatile profiles of tobacco and non-tobacco flavoured e-liquids ................................ 13 

1.2 

Samples ............................................................................................. 14 

1.2.1 

Sample preparation and analysis ........................................................ 14 

1.3 

Data Analysis ...................................................................................... 14 

1.3.1 

Classification of samples .................................................................... 15 

1.3.1.1 

Partial least square discrimination ................................................. 16 

1.3.1.2 

Decision tree ................................................................................. 18 

Conclusion.......................................................................................................... 20 

ANNEX 1 – Analytical method for the determination of tobacco alkaloids in e-liquids, test 
samples, and results ............................................................................................ 21 

A1.1. 

Analysis of tobacco alkaloids by HPLC-MS/MS in HILIC mode ..................... 21 

A1.2. 

Test samples ....................................................................................... 26 

A1.3. 

Contents of tobacco alkaloids determined in e-liquid test samples .............. 34 

ANNEX 2 – Analytical method for the evaluation of flavour profiles ............................ 44 

A2.1. 
thermal  desorption 
high-resolution gas chromatography QTOF mass spectrometry ............................... 44 

Analysis  of  volatile  constituents  of  e-liquids  by 

A2.2. 

Test samples ....................................................................................... 46 

A2.3. 

Compound identification ....................................................................... 49 

References ......................................................................................................... 53 

List of figures ...................................................................................................... 55 

List of tables ....................................................................................................... 56 

 
 
 
 
 
Acknowledgements  

The authors wish to acknowledge the good collaboration with colleagues from DG 
TAXUD, particularly with Mrs Annerie Bouw.  

3 

 
 
 
 
Executive summary 

Electronic-cigarettes  are  electrical  devices  that  produce  an  inhalable  aerosol  from  a 
special liquid. The liquids (e-liquids) may consist of a solution of nicotine and flavourings 
in  an  appropriate  solvent.  Nicotine-free  products  are  available  as  well.  A  large  range  of 
differently  flavoured  e-liquids  is  offered  to  the  consumer.  The  market  of  electronic 
cigarettes  and  corresponding  e-liquids  experienced  in  recent  years  enormous  growth. 
Some  EU  Member  States  reacted  on  this  development  and  introduced  excise  duties  on 
e-liquids. 

The  partial  introduction  of  excise  duties  in  the  common  market  and  the  lack  of 
harmonisation of their magnitude might cause distortions of the market. For that reason, 
the Directorate-General Taxation and Customs Union (DG TAXUD) commissioned a study 
on  the  identification  and  characterisation  of  e-liquids,  which  should  be  based  on 
substances naturally occurring in tobacco.  

The Administrative Arrangement (AA) with DG  TAXUD  foresaw the determination of the 
concentrations  of  nicotine  and  related  tobacco  alkaloids  in  e-liquid  samples,  and  to 
evaluate the agreement of the measured concentrations with concentrations indicated on 
the product label.  
Additionally,  the  possibility  of  identifying  e-liquids  containing  tobacco  derived  flavours 
had to be studied with the aim to use this information for product classification. 

More  than  200  e-liquid  samples  acquired  in  different  countries  were  analysed  for  their 
tobacco  alkaloid  contents  by  liquid  chromatography  tandem  mass  spectrometry.  A  high 
proportion of the investigated products contained nicotine at a level significantly different 
from  the  indicated  concentration.  Mostly  lower  concentrations  were  found.  However, 
significant  amounts  of  nicotine  were  determined  in  some  products  declared  as  "zero-
nicotine". 

Volatile  profiles  of  108  e-liquid  samples  were  measured  by  gas  chromatography 
hyphenated to high-resolution quadrupole time-of-flight (QTOF) mass spectrometry. The 
data obtained from the analysis of was investigated with multivariate statistical methods 
for  possibilities  of  discriminating  tobacco-  and  non-tobacco  flavoured  e-liquids.  The 
developed statistical models allowed identification of tobacco derived products  with high 
probability.  

If  a  harmonized  taxation  of  e-liquids  were  to  be  proposed,  the  following  considerations 
should be taken into account in designing the tax base: 

•  The  declared  nicotine  content  often  does  not  match  with  the  measured 

nicotine content; 

•  Moreover,  in  products  declared  as  ''zero-nicotine''  significant  amounts  of 

nicotine can be found; 

•  Chemical analysis allows distinguishing tobacco and non-tobacco e-liquids with 

high probability. 

4 

 
 
 
 
 
Introduction 

General information about e-cigarettes 

Electronic  (e-)  cigarettes  are  battery-powered  hand-held  devices  that  convert  a  liquid 
into  an  inhalable  aerosol.  Attempts  to  produce  and  commercialise  such  aerosol 
dispensers are known for a long time (Robinson, 1930).  
However,  the  currently  marketed  e-cigarettes  are  based  on  an  invention  made  in  2004 
by the Chinese pharmacist Lik Hon (Hon, 2004). He designed a battery-powered device 
that aimed to supply nicotine to smokers without exposing them to the tar component of 
conventional  smoking.  E-cigarettes  typically  consist  of  a  mouthpiece,  an  atomizer,  a 
battery,  and  a  tank  that  contains  the  liquid  for  vaporization  ("e-liquid")(Famele  et  al., 
2015).  The  atomizer  is  a  heating  element  forming  a  fine  mist  from  the  e-liquid  which 
carries the flavour, and which provides a sensation of smoking to the user. E-cigarettes 
might  be  manufactured  for  single  use  only,  or  might  contain  a  replaceable  or  refillable 
e-liquid tank, and a rechargeable battery. 
The  lack  of  standardization  has  led  to  various  designs  of  e-cigarettes.  Some  of  the 
e-cigarettes  are  in  size  close  to  tobacco  containing  cigarettes,  while  others  are  much 
larger and heavier. Similarly, the number and composition of refill solutions grew in the 
last  decade  exponentially.  In  contrast  to  tobacco  containing  cigarettes,  for  which  EU 
legislation prohibits characterising flavours, e-liquids are marketed with a broad range of 
flavours  and  each  of  them  is  usually  available  with  different  levels  of  nicotine  content 
(EU, 2014). It should be stressed that only a part of the commercialized e-liquids smells 
of tobacco. A big proportion of the market comprises e-liquids with non-tobacco flavours. 
These  might  taste  like  alcoholic  and  non-alcoholic  beverages  (coffee,  wine,  black  tea, 
brandy, lemonade...), sweets (candy, cookies, chocolate, muffins...), or all kinds of fruits 
(cherry, strawberry, apple...).  
The  non-tobacco  flavoured  e-liquids  are  usually  manufactured  from  flavourings  that  are 
GRAS  (generally  recognized  as  safe)  certified  by  the  Flavour  &  Extract  Manufacturers 
Association  (FEMA).  However,  it  has  to  be  noted  that  this  certificate  relates  only  to  the 
use of the flavour as a food additive, and to exposure via food.  
Tobacco  flavours  are  mainly  produced  via  the  extraction  of  tobacco.  Residues  of  the 
manufacturing of cigarettes and other tobacco products, such as leaf stems, may be used 
for  that  purpose.  Technologies  applied  for  the  extraction  of  tobacco  flavour  comprise 
amongst others liquid extraction, supercritical fluid extraction, and steam distillation. 
Nicotine  in  e-liquids  is  extracted  from  tobacco  and  this  process  may  also  extract  other 
minor  tobacco  alkaloids,  such  as  nornicotine,  anatabine,  anabasine  and  myosmine  and 
cause  the  formation  of  degradation  products  of  nicotine  such  β-nicotyrine,  cotinine  and 
nicotine-N-oxide  (Etter  et  al.,  2013).  Oxidative  degradation  of  nicotine  can  also  occur 
during the manufacturing processes of e-liquids, and therefore, high amounts of nicotine-
related tobacco alkaloids can indicate the inadequate handling and storage of the product 
(Famele  et  al.,  2015).  In  some  cases,  tobacco  absolute  extracts  (also  called  naturally 
extracted  tobacco  (NET))  are  used  as  additives  in  tobacco-flavoured  e-liquids  to  get  a 
more  "authentic"  tobacco  flavour.  NETs  comprise  tobacco  extracts  that  are  obtained  by 
maceration  in  usually  propylene  glycole.  This  production  process  can  result  in  higher 
amounts  of  nicotine-related  impurities  (Farsalinos  et  al.,  2015).  However,  different 
research groups observed significant differences between the nicotine contents measured 
and  declared  on  the  product  label  (Cheng,  2014;  Davis  et  al.,  2015).  This  can  be 
reasoned  by  a  lack  of  regulations  on  the  composition  of  e-liquids.  The  deviations  might 

5 

 
 
 
 
also find their origin in the large number of small and medium scale producers, who not 
always  have  the  necessary  tools  and  procedures  for  a  proper  quality  control  of  the 
production process.  

Use of e-cigarettes 
E-cigarettes  continued  arising  curiosity  among  European  citizens.  According  to  the 
Special Eurobarometer 429, 12% of the EU 28 population above the age of 15 years use 
or  have  used  e-cigarettes,  which  is  more  than  was  recorded  in  a  survey  conducted  in 
2012  (European  Commission,  2015).  Four  percent  of  smokers  and  three  percent  of  ex-
smokers used at the time  of the  survey (end  2014) e-cigarettes.  Nicotine containing e-
cigarettes are preferred by users  
However,  Eurobarometer  429  does  not  allow  drawing  conclusions  on  the  types  of 
flavours preferred by consumers (Giovenco et al., 2015). They reported that unflavoured 
and  menthol  flavoured  e-cigarettes  dominated  the  US  e-cigarette  market  in  2012  and 
2013. E-cigarettes sales via traditional retail channels reached within this period the level 
of  about  $636  million.  Unclear  is  the  volume  of  online-traded  electronic  cigarettes  and 
refill liquids.  

Legal and fiscal background 

Electronic  cigarettes  and  refill  containers  are  regulated  in  the  EU  by  Directive 
2014/40/EU  of  the  European  Parliament  and  of  the  Council  (tobacco  products  directive 
"TPD").  

The TPD mandates the European Commission to detail the provisions for the notification 
of electronic cigarettes and refill containers as well as of technical standards for the refill 
mechanism of these products. The power for  authorisation for placing on the market of 
e-liquids remains with the Member States.  

Е-cigarettes  are  currently  not  excisable  tobacco  products,  as  defined  in  Directive 
2011/64/EU.  

However, some EU Member States introduced or are in the process of introducing excise 
duties on e-liquid refills. Excise duties range from €0.01 per ml e-liquid to €0.60 per ml 
e-liquid. The different approaches taken in taxation of e-cigarettes and different levels of 
excise  duties  might  lead  to  a  fragmentation  of  the  market,  and  might  have  negative 
impact on the functioning of the internal market. This was also expressed in a study on 
"measuring  and  reducing  of  administrative  costs  for  economic  operators  and  tax 
authorities and obtaining in parallel a higher level of compliance and security in imposing 
excise  duties  on  tobacco  products"  commissioned  by  DG  TAXUD  (Stener  et  al.,  2014). 
Moreover,  in  view  of  the  increasing  market  share  of  e-cigarettes,  replacing  partially 
traditional tobacco products, a loss of revenue of Member States can be expected. 

On 16 March 2016, the Council agreed that the market of e-cigarettes should continue to 
be monitored and when appropriate, the efforts to develop an efficient taxation method 
for such products should be intensified. 

6 

 
 
 
 
 
 
 
Terms of reference 

In the light of the possible need to develop an efficient taxation method and the potential 
inclusion of e-liquids in the scope of excisable products, the JRC was asked by DG TAXUD 
to  identify  and  characterise  components  of  liquids  used  in  e-cigarettes  to  provide  the 
scientific  information  needed  for  the  potential  harmonised  implementation  of  fiscal 
measures  in  the  Member  States  related  to  e-cigarettes.  The  definition  of  the 
'characterising  element'  of  e-cigarettes  should  be  based  on  substances  naturally 
occurring in tobacco, irrespective whether they have been extracted from tobacco leaves, 
synthesised or obtained from other natural sources. Data on the content of nicotine and 
related alkaloids of at least 150 samples had to be provided. Additionally, flavour profiles 
of  tobacco  flavoured  and  non-tobacco  flavoured  e-liquids  had  to  be  studied.  Details  on 
the  implementation  of  the  scope  were  discussed  and  agreed  upon  during  the  kick-off 
meeting. 

Determination  of  nicotine  and  nicotine  related  alkaloids  in 

commercial e-liquids 

1.1  Samples 

DG  TAXUD  supplied  the  JRC  with  22  e-liquid  samples.  The  rest  of  the  in  total  216 
samples  were  acquired  from  local  markets  and  internet  shops  between  June  2015  and 
February 2016.  The test samples originated from 12 EU Member States (in alphabetical 
order:  Belgium,  Croatia,  Czech  Republic,  Finland,  France,  Germany,  Hungary,  Italy, 
Lithuania, Netherlands, Poland, Romania, and United Kingdom). According to labels, the 
e-liquids  were  manufactured  in  EU  Member  States  (Croatia,  Czech  Republic,  France, 
Germany, Hungary, Italy, Netherlands, and Spain), as well as in third-countries such as 
China  and  USA.  Nicotine  concentration  ranged  from  0  to  48  mg/mL  as  declared  by 
manufacturers.  For  comparison  purposes,  several  traditional  cigarettes  from  major 
brands were acquired in EU local markets, as well as 3R4F research cigarettes from the 
University of Kentucky (Kentucky Tobacco Research & Development Center, USA). 

It has to be stressed that, due to the lack of reliable data, sampling did neither take into 
account consumption habits and preferences of e-cigarette consumers nor market share 
of certain brands/products.  

1.1.1  Sample preparation and analysis 

E-liquid  samples  were  analysed  following  an  in-house  developed  method.  Briefly, 
samples  were  diluted  in  a  mixture  of  acetonitrile/water  (50:50,  v/v),  filtered  and 
analysed  by  high-performance  liquid  chromatography  coupled  to  tandem  mass 
spectrometry  (HPLC-MS/MS).  Quantification  of  nicotine  (NIC)  and  related  compounds 
was  carried  out  by  internal  standardisation  with  stable  isotope  labelled  analogues. 
Tobacco alkaloids from cigarettes were extracted by ultrasound assisted extraction with a 
mixture  of  methanol/water  (80:20,  v/v).  Extracts  were  then  centrifuged,  filtered  and 
analysed  following  the  same  procedure  as  the  e-liquid  samples.  Table  1  shows  the 

7 

 
 
 
 
 
chemical  structures  and  acronyms  of  the  analysed  compounds.  Details  of  the  analytical 
method are given in Annex A1.1. 

Table 1: Chemical structures and acronyms of the analysed substances 

Compound  Acronym  Structure  Compound 

Acronym  Structure 

Nicotine 

NIC 

4,4'-Dipyridyl 

DIPY 

Nornicotine  NNIC 

N-Nitrosonornicotine  NNN 

Anatabine  ATB 

N-Nitrosoanatabine  NATB 

Anabasine  ABS 

N-Nitrosoanabasine  NABS 

Myosmine  MYO 

Cotinine 

COT 

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

NNAL 

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

NNK 

Norcotinine   NCOT 

Cytisine 

CYS 

8 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1.1.2  Tobacco alkaloid content of liquids used in e-cigarettes 

The measured concentrations of NIC and related compounds in the analysed e-liquids are 
listed in Annex 1 in Table A-2. In agreement with previous literature reports (Farsalinos 
et  al.,  2015;  Goniewicz  et  al.,  2012;  Peace  et  al.,  2016),  measured  NIC  concentrations 
were often significantly lower than the concentrations declared by manufacturers on the 
e-liquid  labels  (Figure  1).  A  slight  variability  of  contents  might  be  unavoidable  in  the 
manufacture  of  e-liquids.  Therefore,  guidance  on  tolerable  deviations  of  the  actual  NIC 
contents  from  the  contents  declared  on  the  product  labels  was  taken  from  the  British 
guide  for  manufacture,  importation,  testing  and  labelling  of  vaping  products,  including 
electronic  cigarettes,  e-liquids,  e-shisha  and  directly-related  products  (BSI,  2015).  This 
guide requires that the actual NIC contents of e-liquid preparations is within ±10% of the 
content  indicated  on  the  label.  More  than  half  of  the  tested  samples  with  declared  NIC 
contents did not comply with the 10% threshold, suggesting a need for improvement of 
the  production  process.  One  sample  exceeded  the  indicated  NIC  content  by  more  than 
10%,  while  the  majority  of  the  samples  contained  less  than  90%  of  the  NIC  content 
indicated on the packaging.  

Four  products  contained  NIC  at  levels  higher  than  20  mg/ml,  which  is  the  upper  limit 
specified  in  Directive  2014/40/EU  (EU,  2014).  However,  it  has  to  be  noted  that  these 
products were produced and acquired before the TPD came into force. 

Figure  1:  Difference  between  the  NIC  concentrations  declared  on  the  label  and  the 
measured concentration in the analysed samples.  

9 

 
 
 
 
 
The  study  highlighted  the  presence  of  measurable  amounts  on  NIC  in  45  out  of  91 
samples labelled as "zero nicotine". For these e-liquids, NIC concentrations ranged from 
0.0005 mg/ml to 13.57 mg/ml. According to the British guide for vaping products, zero-
nicotine-content labelled e-liquids shall not contain more than 0.1 mg NIC per millilitre of 
finished product. Eight of the 91 tested zero-nicotine e-liquids exceeded the threshold of 
0.1 mg NIC per millilitre of finished product. 

These  findings indicate  that  the  information  about  NIC  levels  on  product  packages  may 
be inaccurate, which highlights the importance of both proper manufacturing and quality 
control, and surveillance in order to guaranty the quality of the products on the market.  

Nicotine  for  the  preparation  of  e-liquids  is  usually  derived  from  tobacco.  As  such,  it 
usually  contains  also  other  tobacco  alkaloids.  While  the  TPD  only  requires  using 
ingredients  of  high  purity  for  the  manufacture  of  nicotine-containing  liquids,  the  British 
guide  PAS  54115:2015  is  more  specific  by  setting  the  purity  requirement  to  either 
European  Pharmacopoeia  (Ph.  Eur.)  or  United  States Pharmacopoeia  (USP)  grade.  Both 
Pharmacopoeias  specify  the  purity  of  NIC  to  at  least  99%.  The  level  of  total  impurities 
tolerated  by  the  USP  is  maximum  1.00%,  while  Ph.  Eur.  set  it  to  maximum  0.80%. 
Individual minor tobacco alkaloids (impurities) may not exceed 0.50% (USP) and 0.30% 
(Ph. Eur.) respectively. 

Minor tobacco alkaloids, myosmine and nornicotine were found in all tested e-liquids that 
contained  NIC,  at  concentrations  in  the  ranges  0.112-62.85  µg/mL  and  0.179-26.65 
µg/mL,  respectively  (Annex  1,  Table  A-2).  Cotinine,  anatabine  and  anabasine  were 
present in most of the samples at concentration levels up to 78.62 µg/mL, 49.01 µg/mL 
and 40.34 µg/mL,  respectively. Norcotinine was found in only 10 samples, in the range 
0.010-3.010  µg/mL.  The  average  relative  contribution  of  individual  minor  tobacco 
alkaloids  to  the  measured  total  tobacco  alkaloid  content  (including  NIC)  was  below 
0.1 %. None of the tested samples contained N-nitroso derivatives of tobacco alkaloids. 

Comparative evaluation of tobacco alkaloids in e-liquids and traditional tobacco 

The  analysis  of  traditional  cigarettes  from  several  brands  showed  NIC  as  the  most 
abundant alkaloid with concentrations ranging from 12.21 mg/g to 15.88 mg/g (Annex 1, 
Table  A-3).  All  samples  presented  a  similar  pattern  of  minor  tobacco  alkaloids,  with 
nornicotine  (391.15-584.54  µg/g)  and  anatabine  (304.75-386.60  µg/g)  as  the  most 
abundant  ones.  Lower  concentrations  were  obtained  for  cotinine,  anabasine  and 
myosmine.  Norcotinine  was  also  the  least  abundant  alkaloid  in  traditional  cigarette 
tobacco.  

Although  differences  exist  among  the  different  tobacco  varieties/species,  there  are 
generally  certain  correlations  between  the  contents  of  NIC  and  the  minor  alkaloids 
(Sisson  &  Severson,  1990).  As  displayed  in  Figure  2,  the  profile  of  minor  alkaloids 
extracted from cigarette tobacco was rather stable, despite different brands of cigarettes 
were  analysed.  Therefore,  in  an  attempt  to  identify  the  source  of  NIC  applied  in  the 
production  of  e-liquids,  the  relative  concentrations  of  minor  tobacco  alkaloids  were 
investigated (sum of tobacco alkaloids=100%). 

10 

 
 
 
 
 
Figure 2: Relative concentrations of minor alkaloids in traditional cigarettes. 

Traditional  cigarettes

)

%

(

n
o
i
t
a
r
t
n
e
c
n
o
c

e
v
i
t
a

l

e
R

4.00

3.50

3.00

2.50

2.00

1.50

1.00

0.50

0.00

3.06

NNIC

0.05

MYO

0.64

COT

0.01

NCOT

0.44

ABS

2.15

ATB

The concentrations of these minor tobacco alkaloids are much lower in e-liquids than in 
traditional  cigarette  tobacco.  However,  their  profiles  were  not  as  consistent  as  in 
cigarette  tobacco  extracts,  but  varied  enormously.  Direct  comparison  of  the  profiles 
obtained  for  tobacco-flavoured  e-liquids  and  other  nicotine-containing  flavours  did  not 
show  any  significant  difference  (Figure  3).  Lisko  et  al.  (2015),  who  concluded  that 
concentrations  of  minor  alkaloids  highly  depend  on  the  applied  manufacturing  and 
purification  of  NIC  extracts,  thus  affecting  their  relative  concentration  in  the  final 
e-liquids,  obtained  similar  results.  Poor  manufacturing  practices  might  be  another 
explanation  for  the  poor  correlation  between  NIC  and  minor  alkaloids  in  e-liquids.  In 
addition,  oxidation  processes  may  also  influence  the  profiles  of  minor  alkaloids.  When 
NIC  is  exposed  to  air  during  manufacturing  or  storage,  oxidation  can  occur  resulting  in 
the formation of minor alkaloids (Kisaki et al., 1978).  

Figure 3: Relative concentrations of minor alkaloids in e-liquids. 

e-liquids

)

%

(

n
o

i
t
a
r
t
n
e
c
n
o
c

e
v
i
t
a
l
E
R

0.25

0.20

0.15

0.10

0.05

0.00

-0.05

-0.10

0.04

0.03

0.04

0.04

0.03

0.02

0.00

0.00

0.04

0.02

0.07

0.07

Other flavours

Tobacco flavour

MYO

NNIC

COT

NCOT

ABS

ATB

11 

 
 
 
 
 
 
 
 
 
 
Principal components analysis (PCA), a multivariate statistical technique, was carried out 
to further investigate the obtained results (Figure 4). 
The  blue  ellipse  (only  partially  displayed  in  Figure  4)  indicates  the  95%  confidence 
interval. With high probability, points outside the blue ellipse do not belong to the group 
of  points  (e-liquid  test  samples)  within  the  ellipse.  The  two  points  below  the  ellipse 
represent  a  sample  that  was  wrongly  labelled  (labelled  as  zero  nicotine,  but  containing 
about 13.6 mg NIC per millilitre liquid), and the sample with the highest measured NIC 
content  (46.4  mg/ml)  among  all  tested  samples.  The points  within  the  yellow  rectangle 
represent extracts of cigarette tobacco. The graphical separation of the group of nicotine-
containing  e-liquids  from  tobacco  extracts  allows  concluding  that  it  is  possible  to 
discriminate  e-liquids  and  tobacco  products  based  on  their  tobacco  alkaloid  profile.  In 
turn,  it  demonstrates  also  that  it  is  not  possible  to  define  characterising  elements  of 
e-liquids  solely  via  their  tobacco  alkaloid  profiles.  This  outcome  could  be  expected  as 
most  producers  would  use  NIC  complying  with  USP  or  Eur.  Ph.  specifications  for  the 
preparation of e-liquids. Particularly NIC needs to be of at least 99% purity, whereas the 
relative  NIC  content  of  cigarette  tobacco  in  the  current  study  was  between  92.9%  and 
94.2% of total tobacco alkaloids, which agrees well with the 95% reported by Sisson and 
Severson (1990). The small difference might be explained by both the larger number of 
tobacco  alkaloids  determined in  the  current  study,  which  contributed  around  1%  to  the 
total tobacco alkaloid content, and slight differences in the extraction protocols.  

Figure  4:  Principle  component  analysis  –  Score  plot  of  PC1  and  PC2  for  nicotine 
containing e-liquids and cigarette tobacco extracts  

(a, mislabelled sample; b, sample with the highest measured NIC content) 

a 

b 

12 

 
 
 
 
 
 
 
The  presence  of  minor  tobacco  alkaloids  in  e-liquids  is  not  sufficient  to  indicate  the 
source  of  NIC  (natural  or  synthetic),  and  therefore  identify  the  product  as  a  tobacco 
product,  as  specified  in  the  TPD  (EU,  2014).  Synthetic  NIC,  which  is  offered  on  the 
market specifically for the preparation of e-liquids, might, despite high purity claims, also 
contain  minor  tobacco  alkaloids,  most  likely  intermediates  of  the  synthesis  ("TFN® 
Nicotine,"  2016).  A  possibility  to  distinguish  NIC  derived  from  Nicotiana  tabaccum  from 
synthesized  NIC  might  be  offered  by  chiral  chromatography,  as  natural  NIC  occurs 
primarily  as  (-)-(S)-stereoisomer,  and  chemically  synthesized  NIC  is  likely  to  be  a 
racemic mixture of (-)-(S)- and (-)-(R)-forms. This option would need, however, further 
investigations. 

Volatile  profiles  of  tobacco  and  non-tobacco  flavoured 

e-liquids 

The aim of the evaluation of the flavour profiles of e-liquids was to identify possibilities to 
discriminate  tobacco-derived  flavours  from  non-tobacco  flavours.  For  that  purpose, 
different  e-liquids  were  analysed  by  gas  chromatography  -  high-resolution  mass 
spectrometry for their volatile and semi-volatile constituents.  

The  NIST  mass  spectral  library  was  used  for  peak  identification.  Additionally,  Linear 
Retention Indices (LRIs) were calculated for each peak, and compared to literature data 
retrieved  from  several  online  databases.  Finally,  literature  was  searched  in  order  to 
evaluate  whether  the  particular  substance  was  already  identified  as  genuine  tobacco 
constituent  (Rodgman  &  Perfetti,  2013).  This  resulted  in  a  set  of  90  substances,  which 
corresponded to about 90 % of all chemical compounds found in the different e-liquids. 
Details of the analysed substances are provided in Annex 2 in Table A-5. The number of 
substances is rather small compared to natural tobacco, in which more than 5000 volatile 
substances  were  identified  so  far  (Perfetti  &  Rodgman,  2011).  However,  the  difference 
can be explained by the influence of the type of tobacco used for the preparation of the 
flavour  extract,  the  extraction  procedure  and  solvent  used,  and  a  potential  further 
fractionation of the obtained extract. The low concentration of flavourings in the e-liquid, 
which is  about  1  %  of  the  total  volume,  additionally  impedes  the  detectability  of  minor 
flavour constituents.  

13 

 
 
 
 
 
 
1.2  Samples 

E-liquid  samples  were  categorized  based  on  the  declared  flavour  into  the  following 
categories:  tobacco,  coffee,  vanilla,  menthol,  fruit,  and  other  flavours.  In  total,  108  e-
liquids  were  analysed.  About  40 %  of  the  analysed  samples  contained  tobacco  flavours 
according  to  the  label  declaration  (Table  2).  Details  of  the  individual  samples  analysed 
are presented in Annex 2 in Table A-4. 

Table 2: Number of test samples per flavour category 

Flavour 

Tobacco 

Coffee 

Vanilla 

Mint 

Fruit 

Other 

Number of samples 

43 

8 

8 

9 

29 

11 

1.2.1  Sample preparation and analysis 

The  sample  preparation  was  designed  with  a  view  to  minimise  potential  sample 
discrimination during sample preparation. Aliquots of the test samples were only diluted 
in methanol, fortified by the stable isotope labelled internal standard, and introduced into 
the  gas  chromatograph  hyphenated  to  a  QTOF  high  resolution  mass  spectrometer. 
Sample  injection  occurred  via  a  thermal  desorption  inlet  system.  Chromatograms  were 
recorded  in  total  ion  scan  mode  over  the  mass-to-charge  range  of  45  to  450.  The 
internal standard was used to monitor fluctuations of the instrument sensitivity. As they 
were rather low, absolute signal abundances (peak areas) were used for data evaluation. 
Annex 2 provides the list of identified substances, the selection and classification of test 
samples as well as details of the applied analytical method. 

1.3  Data Analysis 

Data analysis was based on a data matrix with about 9700 entries. 
The first step concerned the identification of volatiles with high discriminative power with 
the aim to reduce the number of analytes for model building. The second step concerned 
the  development  of  a  model  for  the  flavour  based  discrimination  of  tobacco-flavoured 
e-liquids from non-tobacco-flavoured e-liquids. 

14 

 
 
 
 
 
  
 
 
1.3.1  Classification of samples 

The  volatile  composition  of  e-liquids  is  complex  and  not  uniform  at  all.  They  contain 
typically a large number of different substances at varying concentration levels. Several 
volatile  substances  occurring  in  tobacco  extracts  are  also  constituents  of  e-liquids 
belonging  to  other  non-tobacco  flavoured  categories.  Figure  5  displays  a  map  of  the 
relative  prevalence  of  the  measured  substances  in  the  differently  flavoured  e-liquids. 
Green  colour  indicates  the  presence  of  the  particular  substance  in  all tested  samples  of 
this  e-liquid  category,  whereas  red  colour  means  the  opposite.  Pale  colours  point  to 
relative prevalence in-between the two extremes.  

Figure 5: Prevalence of measured substances in different e-liquid categories 

Tobacco

Coffee

Fruit

Mint

Vanilla

Others

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Principle component analysis of the whole data set was employed for elucidating whether 
there is a significant difference in the composition of e-liquids. Figure 6 shows a graph of 
the  first  two  principle  components.  Only  menthol  flavoured  e-liquids  and  some  vanilla 
flavoured e-liquids could be distinguished from the bulk of samples.  

Figure 6: PCA score plot of e-liquid volatiles:  

]
2
[
t

15 

 
 
 
 
 
 
 
The  prediction  of  whether  a  particular  e-liquid contains  a  tobacco  extract  is  not  simple, 
as there is not a single identifier for tobacco extracts, and many substances measured in 
tobacco  extracts  also  occur  in  other  plant  extracts.  Hence,  the  overlap  of  the  chemical 
profiles of different plant based flavourings, together with their variability in composition 
prevents  the  application  of  simple  mathematical  models.  A  number  of  multivariate 
statistical  methods/algorithms,  i.e.  partial  least  square  discrimination  analysis  (PLS-DA) 
and decision tree (DT) prediction, were employed for class prediction. 

1.3.1.1  Partial least square discrimination 

PLS-DA  aims  to  find  relations  between  the  measured  signals  (raw  data  matrix)  and  the 
classification matrix (flavour category). Two different levels of classification were tested. 
One  comprised  five  flavour  classes  (tobacco,  mint,  vanilla,  fruit,  and  others)  while  the 
other distinguished only between tobacco and  non-tobacco flavour.  Each model was set 
up  with  the  same  79  trainings  samples,  and  only  those  volatile  substances  with  high 
discrimination  power  were  used  for  fitting.  The  developed  models  were  validated  both 
against a subset of the training samples (cross-validation) as well as against independent 
test samples.  

For  cross-validation  of  the  developed  statistical  models  the  79  samples  were  split  in 
three groups. Two of the three groups were used to build the PLS-DA model. The model 
is then used to classify the samples in the third group, and results are evaluated for their 
agreement  with  the  known  class  membership  of  samples.  This  procedure  is  ten  times 
repeated,  each  time  with  random  allocation  of  the  samples  to  different  groups.  Table  3 
presents the outcome of the cross-validation of the PLS-DA models. 

Table 3: Cross-validation of the PLS-DA model based on five flavour groups 

The  accuracy  (percentage  of  correct  classifications)  was  92 %  for  tobacco-flavoured 
samples about, whereas the overall accuracy reached about 85 %.  

The  samples  highlighted  in  Table  4  in  green  represent  tobacco-flavoured  e-liquids, 
whereas  all  others  correspond  to  different  flavours.  Of  the  21  independent  validation 
samples,  three  out  of  the  ten  tobacco  flavoured  samples  and  two  samples  with  other 
flavours  were  misclassified  (red  ellipses).  Challenges  for  correct  classification  are 
provided  by  samples  that  contain  next  to  tobacco  another  characteristic  flavour,  e.g. 
sample  F03,  which  was  labelled  by  the  producer  as  "shisha  coffee",  contains  besides 
tobacco also a coffee flavouring. The same was true for sample C02, which was labelled 
as  "shisha  vanilla".  Despite  these  two  class-overlapping  samples,  the  accuracy  of 
classification agrees well with the outcome of cross validation. 

16 

 
 
 
 
 
 
Table  4:  Individual  results  from  validation  of  the  PLS-DA  model  based  on  five  flavour 
groups with independent samples 

Flavour identifier: A: tobacco; B: mint; C: vanilla; D and K: fruit; E and F: others. green highlighted: contains 
tobacco flavour; red ellipses indicate wrong classifications 

Reducing the number of classes to tobacco-flavoured and non-tobacco flavoured (binary 
classification), and restricting the model only to parameters of high significance (p>0.05) 
provided similar overall accuracy of classification, but improved accuracy in identification 
of  tobacco-flavoured  e-liquids.  Table  5  contains  accuracy  data  obtained  by  cross 
validation of the model according to the method described earlier.  

Table 5: Cross-validation of the PLS-DA model based on two flavour groups 

Validation  of  the  developed  model  with  independent  samples  led  to  very  promising 
results. All thirty samples were correctly classified (Table 6).  

17 

 
 
 
 
 
 
 
 
 
Table  6:  Individual  results  from  validation  of  the  PLS-DA  model  based  on  two  flavour 
groups with independent samples 

Green highlighted cell: samples containing tobacco flavour 

1.3.1.2  Decision tree 

Decision  tree  classification  is  an  analysis  method  used  in  data  mining  of  large  data 
collections.  It  builds  classification  models  in  the  form  of  a  tree  structure,  thereby 
breaking  down  the  input  data  set  into  smaller  subsets.  Cross  validation  was  performed 
using  the  method  specified  earlier.  The  overall  accuracy  was  less  favourable  than  with 
PLS-DA (Table 7). Especially the correct classification rate of tobacco containing e-liquids 
was in cross-validation lower compared to PLS-DA. 

Table 7: Accuracy table for cross validation of decision tree model 

However,  the  validation  with  independent  samples  revealed  a  high  level  of  correct 
classifications  (Table  8).  Two  out  of  16  tobacco-flavoured  samples  were  wrongly 
classified  (red  ellipses).  However,  sample  A30  contains  besides  tobacco  flavour 
significant amounts of menthol, which might cause the misclassification.  

18 

 
 
 
 
 
 
Table 8: Validation of decision tree model with independent samples 

Green highlighted cell: samples containing tobacco flavour, red ellipses indicate misclassifications 

19 

 
 
 
 
 
 
Conclusion 

In  agreement  with  previous  literature  reports,  the  measured  NIC  concentrations  of 
e-liquids were often significantly lower than the concentrations indicated on the package. 
The  data  showed  that  the  profiles  of  minor  alkaloids  vary  considerably  among  the 
different  samples  tested.  As  most  of  the  NIC  currently  used  to  produce  e-liquids  is 
obtained  from  natural  tobacco,  the  minor  alkaloid  profiles  are  generally  affected  by  the 
quality  of  the  purification  processes.  Based  on  the  obtained  results,  the  analysis  of 
tobacco  alkaloids  does  not  seem  to  be  suitable  for  tracing  back  the  origin  of  NIC 
contained in e-liquids to a natural source or made by chemical synthesis. The presence of 
minor  tobacco  alkaloids  might  be  an indicator  for  the  natural  origin  of  NIC.  However,  it 
cannot  be  excluded  that  chemically  synthesized  NIC  contains  similar  impurities. 
Additionally, modern purification processes might lead for both natural and synthetic NIC 
to qualities that do not introduce in the e-liquid detectable levels of other minor tobacco 
alkaloids.  

The  flavour  profiles  of  the  investigated  test  samples  are  complex.  The  distinction 
between  tobacco-flavoured  e-liquids  and  non-tobacco-flavoured  e-liquids  is  challenging. 
About  90  substances  were  identified  in  tobacco-containing  e-liquids.  They  were 
systematically  analysed  in  about  110  test  samples,  of  which  70 %  were  applied  for 
setting-up  statistical  classification  models  and  about  30 %  were  used  to  validate  the 
developed models. Two different multivariate classification methods were applied to the 
test  samples,  in  particular  partial  least  square  discrimination  and  decision  tree 
classification.  Both  methodologies  provided  suitable  results,  allowing  the  discrimination 
of  tobacco-flavoured  and  non-tobacco-flavoured  e-liquids  with  a  high  level  of  accuracy. 
However,  partial  least  square  discrimination  performed  slightly  better.  Samples 
containing both tobacco flavours and other characteristic flavours challenge both models. 
Including new training samples in the models of e.g. mixed flavours and, if applicable, of 
new flavours should be envisaged in order to maintain their discrimination power. 

20 

 
 
 
 
ANNEX  1  –  Analytical  method  for  the  determination  of 

tobacco alkaloids in e-liquids, test samples, and results 

A1.1. Analysis of tobacco alkaloids by HPLC-MS/MS in HILIC mode 

1. Standards and reagents 

Following reference substances were acquired from Sigma-Aldrich, Diegem, Belgium. Information on 
purity, concentration and solvent are given within brackets. 

in 

(1) 
(2) 
(3) 
(4) 
(5) 

(6) 
(7) 
(8) 
(9) 
(10) 
(11) 
(12) 
(13) 
(14) 
(15) 
(16) 
(17) 
(18) 

(1.000  ±  0.005  mg/mL 

(+)-anabasine hydrochloride (1.000 ± 0.005 mg/mL in methanol)  
(–)-cotinine (1.000 ± 0.005 mg/mL in methanol) 
Cytisine (≥99%) 
4,4′-dipyridyl (98%) 
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol 
methanol) 
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (≥98%) 
Myosmine (≥98%) 
(–)-nicotine (1.00 ± 0.05 mg/mL in methanol) 
N-nitrosoanabasine (≥99%) 
N-nitrosoanatabine (≥97%) 
(±)-N-nitrosonornicotine (1.000 ± 0.005 mg/mL in methanol) 
(R,S)-norcotinine (1.000 ± 0.005 mg/mL in methanol) 
(±)-nornicotine (1.000 ± 0.005 mg/mL in methanol) 
(±)-cotinine-D3 (1.0 mg/mL in methanol) 
(±)-nicotine-D4 (100 μg/mL in acetonitrile) 
(±)-nornicotine-D4 (100 μg/mL in methanol)  
Ammonium acetate (NH4Ac) LCMS grade from Sigma-Aldrich 
Acetonitrile LCMS grade 

(R,S)-anatabine (≥95%) was purchased at Cayman Chemical, Ann Arbor, Michigan, USA. 

Mixed standard solution of stable isotope labelled internal standards (IS):  

Mix IS 
NIC-d4 
NNIC-d4 
COT-d3 

Conc. (µg/mL) 
5.0622 
0.8943 
0.3130 

Calibration solutions for tobacco alkaloids: 
NIC: 20 ng/mL- 2000 ng/mL 
Minor tobacco alkaloids: 10 ng/mL-1000 ng/mL 

21 

 
 
 
 
 
 
 
 
 
 
2. Methods 

2.1. Sample preparation 

The relative proportions of nicotine content compared to the contents of minor tobacco 
alkaloids required two separated assays per test sample. Two dilutions were prepared of 
each tested e-liquid, in order to bring the analyte concentrations into the linear range of the 
HPLC-MS/MS instrument. Cigarette tobacco was initially extracted with aqueous methanol. 
The extract was further diluted according to the scheme of dilutions of e-liquids. 

A) Determination of minor tobacco alkaloids: low dilution (20-fold dilution) of e-liquid: 

(1) 

(2) 

(3) 

Add 100 µL e-liquid sample to 1.900 mL ACN/water (1:1) in a 2 mL Eppendorf 
vial and vortex  
Pipette 0.5 mL dilution into a HPLC vial, add 100 µL IS solution and homogenate 
on a Vortex mixer. 
Filtrate through 0.20 µm regenerated cellulose filter syringe filter 

B) Determination of nicotine: high dilution (20.000- fold dilution): 

1.  Add 100 µL e-liquid sample to 1.900 mL ACN/water (1:1) in a 2 mL Eppendorf vial and vortex  
2.  Pipette 10 µL dilution into 10 mL ACN/water (1:1) in a 15 mL centrifuge plastic tube 
3.  Pipette 0.5 mL dilution into a HPLC vial, add 100 µL IS solution and vortex 
4.  Filtrate through 0.20 µm regenerated cellulose filter syringe filter 

C) Cigarettes 

5.  Add 10 mL MeOH/water (80:20, v/v) to the tobacco content of 1 cigarette (aprox. 500 mg) in a 

15 mL centrifuge plastic tube 
6.  Ultrasound extraction for 30 min 
7.  Centrifuge at 3500 RPM 5 min and collect supernatant 
8.  Follow same dilution scheme as for the e-liquids 

22 

 
 
 
 
 
 
 
 
 
 
 
 
 
2.2. HPLC-MS/MS analysis  

HPLC Column: 
Ascentis Express F5 (10 cm x 2.1 mm, 2.7 µm) from Supelco 
Guard column Discovery Ascentis Express F5 (0.5 cm x 2.1 mm, 2.7 µm) 

Mobile Phase A: 5 mM aqueous NH4Ac 
Mobile Phase B: acetonitrile 

Mobile phase flow: 0.500 ml/min 
Column temperature: 45 °C 
Injection volume 0.5 µL 

Gradient elution program: 

Time (min) 

% B 

0.50 

3.00 

6.00 

9.50 

9.51 

14.50 

95 

80 

70 

70 

95 

95 

MS/MS method 
Instrument: AB SCIEX™ QTRAP® 6500 

Instrument parameters: 
CUR: 
CAD: 
IS: Positive 
TEM  
GS1  
GS2 
DP 
EP 
ST1 

30 
Medium 
4500 
500 
70 
50 
20 
10 
-20 

23 

 
 
 
 
 
 
 
 
 
 
 
Acquisition in two time segments/periods: 
Period 1 (weakly retained compounds) 

Q1 Mass (m/z)  Q3 Mass (m/z) 

Dwell time 
(msec) 

CE (ev) 

CXP (V) 

ID 

163.1 
163.1 
177.1 
177.1 
180.1 
180.1 
178.1 
178.1 
208.1 
208.1 
147.0 
147.0 
157.0 
157.0 
190.1 
190.1 
192.1 
192.1 
210.1 
210.1 

146.1 
135.1 
80.0 
98.0 
80.0 
101.0 
148.1 
120.1 
122.0 
106.0 
105.0 
129.9 
77.0 
130.1 
160.1 
79.1 
162.1 
133.1 
93.0 
149.0 

5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 
5 

15 
30 
30 
28 
30 
28 
15 
25 
15 
29 
31 
29 
45 
35 
13 
37 
15 
29 
31 
19 

12 
12 
12 
12 
12 
12 
4 
10 
14 
14 
12 
14 
12 
8 
12 
12 
12 
8 
18 
16 

NCOT_1 
NCOT_2 
COT_1 
COT_2 
COT-d3_1 
COT-d3_2 
NNN_1 
NNN_2 
NNK_1 
NNK_2 
MYO_1 
MYO_2 
DIPY_1 
DIPY_2 
NATB_1 
NATB_2 
NABS_1 
NABS_2 
NNAL_1 
NNAL_2 

Period 2 (stronger retained compounds) 

Q1 Mass (m/z)  Q3 Mass (m/z) 

191.1 
191.1 
149.1 
149.1 
153.1 
153.1 
163.1 
163.1 
167.1 
167.1 
161.0 
161.0 
163.1 
163.1 

147.9 
133 
132.1 
80.0 
84.0 
110.1 
132.1 
106.1 
136.0 
84.0 
144.0 
80.0 
94.1 
146 

Dwell time 
(msec) 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 

CE (ev) 

CXP (V) 

ID 

16 
16 
6 
10 
10 
10 
12 
12 
12 
12 
12 
10 
12 
12 

CYS_1 
CYS_2 
NNIC_1 
NNIC_2 
NNIC-d4_1 
NNIC-d4_2 
NIC_1 
NIC_2 
NIC-d4_1 
NIC-d4_2 
ATB_1 
ATB_2 
ABS_1 
ABS_2 

27 
43 
17 
27 
27 
23 
20 
22 
20 
25 
28 
32 
25 
20 

24 

 
 
 
 
 
 
Figure 1. MRM chromatograms for a calibration standard containing all target compounds 

XIC from 20160215.wiff (sample 10) - Cal6_01, Period 1, +MRM (20 transitions): NCOT_1 (163.1 /  146.1)
XIC from 20160215.wiff (sample 10) - Cal6_01, Period 1, +MRM (20 transitions): NCOT_1 (163.1 /  146.1)
XIC from 20160215.wiff (sample 10) - Cal6_01, Period 1, +MRM (20 transitions): NCOT_1 (163.1 /  146.1)
XIC from 20160215.wiff (sample 10) - Cal6_01, Period 1, +MRM (20 transitions): NCOT_1 (163.1 /  146.1)

y
t
i
s
n
e
t
n
I

8.5e7

8.0e7

7.5e7

7.0e7

6.5e7

6.0e7

5.5e7

5.0e7

4.5e7

4.0e7

3.5e7

3.0e7

2.5e7

2.0e7

1.5e7

1.0e7

5.0e6

0.0e0

COT

NNN

MYO

NABS

DIPY

NNAL
NATB

NNN

NCOT

NIC

CYS

ATB

0.711

ABS

NNIC

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

4.4

4.6

4.8
Time, min

5.0

5.2

5.4

5.6

5.8

6.0

6.2

6.4

6.6

6.8

7.0

7.2

7.4

7.6

7.8

8.0

8.2

8.4

8.6

8.8

9.0

9.2

9.4

Legend 

NIC 

NNIC 

ATB 

ABS 

MYO 

COT 

Cotinine 

NCOT 

Norcotinine 

Nicotine 

DIPY 

4,4'-Dipyridyl 

Nornicotine 

NNN 

N-Nitrosonornicotine 

Anatabine 

NATB 

N-Nitrosoanatabine 

Anabasine 

NABS 

N-Nitrosoanabasine 

Myosmine 

NNAL 

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

NNK 

CYS 

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

Cytisine 

25 

 
 
 
 
 
 
A1.2. Test samples 

Table A- 1: E-liquid samples analysed for tobacco alkaloid contents 

Code 
001 
002 

003 
004 
005 
006 
007 
008 

009 
010 
011 
012 
013 
014 

015 
016 
017 
018 
019 
020 

021 
022 
023 
024 
025 

Description 
Tobacco 
Smooth 

Menthol 
Blueberry 
Smooth 
Rainbow 
Apple 
Virginia Tobacco 

Blueberry 
Tobacco 
Strawberry 
Cherry 
Cherry Limeade 
Calamity Jane Tobacco 

Ol' River Tobacco 
Rocket Blend 
Calamity Jane Tobacco 
Cherry Limeade 
Ol' River Tobacco 
Cherry Limeade 

Calamity Jane Tobacco 
Coffee 
Coffee 
Coffee 
Tobacco 

Country acquisition  Country production 

UK 
UK 

UK 
UK 
UK 
UK 
UK 
UK 

UK 
UK 
UK 
UK 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 

PRC 
PRC 

PRC 
PRC 
PRC 
PRC 
PRC 
PRC 

PRC 
PRC 
PRC 
PRC 
USA 
USA 

USA 
USA 
USA 
USA 
USA 
USA 

USA 
ES 
ES 
ES 
DE 

Flavour classification 
Tobacco 
Tobacco 

Nicotine content declared (mg/mL) 
0 
0 

Fresh 
Fruity 
Tobacco 
Tobacco 
Fruity 
Tobacco 

Fruity 
Tobacco 
Fruity 
Fruity 
Fruity 
Tobacco 

Tobacco 
Sweet 
Tobacco 
Fruity 
Tobacco 
Fruity 

Tobacco 
Non-alcoholic drink 
Non-alcoholic drink 
Non-alcoholic drink 
Tobacco 

26 

0 
0 
6 
12 
18 
18 

18 
18 
18 
24 
0 
0 

0 
0 
12 
12 
18 
18 

18 
0 
6 
16 
0 

 
 
Code 
026 

027 
028 
029 
030 
031 
032 

033 
034 
035 
036 
037 
038 

039 
040 
041 
042 
043 
044 
045 

046 
047 
048 
049 
050 
051 

052 
053 
054 

Description 
Black Mamba 

Black Fire 
Pfeife 
Kentucky 
American Blend 
Maxx Blend 
Dessert 

7 leaves 
RY4 
Toffee 
Karamell Sahne 
After Nine 
Marzipan 

Ice Bombon 
Gummi-barchen 
Kase-kuchen 
Apfel-kuchen 
Honig 
Cookie 
Zitrone fresh 

Kirsch Amarena fresh 
Erdbeer fresh 
Walderdbeer fresh 
Apfel fresh 
Brombeer fresh 
Kiba fresh 

Pfirsich Maracuja fresh 
Himbeer fresh 
Wassermelone fresh 

Country acquisition  Country production 

DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 

DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 

Flavour classification 
Tobacco 

Nicotine content declared (mg/mL) 
0 

Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 

Tobacco 
Tobacco 
Sweet 
Sweet 
Sweet 
Sweet 

Sweet 
Sweet 
Sweet 
Sweet 
Sweet 
Sweet 
Fresh 

Fresh 
Fresh 
Fresh 
Fresh 
Fresh 
Fresh 

Fresh 
Fresh 
Fresh 

27 

0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
0 
0 

0 
0 
0 

 
 
Code 
055 

056 
057 
058 
059 
060 
061 

062 
063 
064 
065 
066 
067 

068 
069 
070 
071 
072 
073 
074 

075 
076 
077 
078 
079 
080 

081 
082 
083 

Description 
Pfirsich Maracuja 

Zitrone 
Honigmelone 
Kiba 
Himbeer 
Frucht-mix Tropic 
Erdbeer 

Cocos 
Apfel 
Amarena kirsch 
Tobacco 
Black Mamba 
Black Fire 

Pfeife 
Kentucky 
American Blend 
Maxx Blend 
Dessert 
7 leaves 
RY4 

Toffee 
Karamell Sahne 
After Nine 
Marzipan 
Ice Bombon 
Gummi-barchen 

Kase-kuchen 
Apfel-kuchen 
Honig 

Country acquisition  Country production 

DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 

DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 

Flavour classification 
Fruity 

Nicotine content declared (mg/mL) 
10 

Fruity 
Fruity 
Fruity 
Fruity 
Fruity 
Fruity 

Fruity 
Fruity 
Fruity 
Tobacco 
Tobacco 
Tobacco 

Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 

Sweet 
Sweet 
Sweet 
Sweet 
Sweet 
Sweet 

Sweet 
Sweet 
Sweet 

28 

10 
10 
10 
10 
10 
10 

10 
10 
10 
10 
10 
10 

10 
10 
10 
10 
10 
10 
10 

20 
20 
20 
20 
20 
20 

20 
20 
20 

 
 
Code 
084 

Description 
Cookie 

085 
086 
087 
088 
089 
090 

091 
092 
093 
094 
095 
096 

097 
098 
099 
100 
101 
102 
103 

104 
105 
106 
107 
108 
109 

110 
111 
112 

Zitrone fresh 
Kirsch Amarena fresh 
Erdbeer fresh 
Walderdbeer fresh 
Apfel fresh 
Brombeer fresh 

Kiba fresh 
Pfirsich Maracuja fresh 
Himbeer fresh 
Wassermelone fresh 
Pfirsich Maracuja 
Zitrone 

Honigmelone 
Kiba 
Himbeer 
Frucht-mix Tropic 
Erdbeer 
Cocos 
Apfel 

Amarena kirsch 
Tobacco 
Black Mamba 
Black Fire 
Pfeife 
Kentucky 

American Blend 
Maxx Blend 
Dessert 

Country acquisition  Country production 

DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 

DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 
DE 
DE 
DE 

DE 
DE 
DE 

Flavour classification 
Sweet 

Nicotine content declared (mg/mL) 
20 

Fresh 
Fresh 
Fresh 
Fresh 
Fresh 
Fresh 

Fresh 
Fresh 
Fresh 
Fresh 
Fruity 
Fruity 

Fruity 
Fruity 
Fruity 
Fruity 
Fruity 
Fruity 
Fruity 

Fruity 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 

Tobacco 
Tobacco 
Tobacco 

29 

20 
20 
20 
20 
20 
20 

20 
20 
20 
20 
20 
20 

20 
20 
20 
20 
20 
20 
20 

20 
20 
20 
20 
20 
20 

20 
20 
20 

 
 
Code 
113 

114 
115 
116 
117 
119 
120 

121 
122 
123 
124 
125 
126 

Description 
7 leaves 

RY4 
Et Spiritum 
Unknown 
Crisp Mint 
Unknown 
Unknown 

Unknown 
Unknown 
Arabic tobacco 
USA Mix 
Turkish blend 
Southeast County 

Cuba Cigar 

127 
128  Honey Toasted Tobacco 
129 
130 
131 
132 
133 

Old Dry Tabacco 
The Palm 
Coco & Havana 
Coco & Havana 
Coco & Havana 

134 
135 
136 
137 
138 
139 

140 
141 
142 

Coco & Havana 
Tobacco Mint 
Menthol 
Arctic Mint 
Mint 
Ice Mint 

Ice Mint 
Ice Mint 
Ice Mint 

Country acquisition  Country production 

DE 

DE 
FR 
- 
UK 
UK 
UK 

UK 
UK 
NL 
NL 
NL 
NL 

NL 
NL 
IT 
NL 
NL 
NL 
NL 

NL 
NL 
NL 
NL 
NL 
NL 

NL 
NL 
NL 

DE 

DE 
FR 
- 
- 
- 
- 

- 
- 
NL 
NL 
NL 
NL 

NL 
NL 
IT 
NL 
NL 
NL 
NL 

NL 
NL 
NL 
NL 
NL 
NL 

NL 
NL 
NL 

Flavour classification 
Tobacco 

Nicotine content declared (mg/mL) 
20 

Tobacco 
Exotic 
- 
Fresh 
- 
- 

- 
- 
Tobacco  
Tobacco  
Tobacco  
Tobacco  

Tobacco  
Tobacco  
Tobacco  
Tobacco  
Tobacco  
Tobacco  
Tobacco  

Tobacco  
Fresh 
Fresh 
Fresh 
Fresh 
Fresh 

Fresh 
Fresh 
Fresh 

30 

20 
11 
- 
6 
- 
- 

- 
- 
0 
0 
0 
0 
0 

0 
0 
0 
0 
6 
12 

18 
0 
0 
0 
0 
0 

6 
12 
18 

 
 
Code 
143 

144 
145 
146 
147 
148 
149 

150 
151 
152 
153 
154 
155 

156 
157 
158 
159 
160 
161 
162 

163 
164 
165 
166 
167 
168 

169 
170 
171 

Description 
Vanilla 

Shisha Vanille 
Vanille 
Ice Vanille 
Vanilla Orange 
Vanilla Orange 
Vanilla Orange 

Vanilla Orange 
Passion Fruit 
Mandarin 
Coconut 
Pear 
Strawberry 

Strawberry 
Strawberry 
Strawberry 
Cotton Candy 
White Chocolate 
Banana Chocolate 
Pumpkin Pie 

Biscuit 
Banana Split 
Banana Split 
Banana Split 
Banana Split 
Cola 

Shisha Coffee 
Cappuccino 
Herbal Tea 

Country acquisition  Country production 

NL 

NL 
FR 
NL 
NL 
NL 
NL 

NL 
NL 
NL 
NL 
NL 
NL 

NL 
NL 
NL 
NL 
NL 
NL 
NL 

IT 
NL 
NL 
NL 
NL 
NL 

NL 
NL 
NL 

NL 

NL 
FR 
NL 
NL 
NL 
NL 

NL 
NL 
NL 
NL 
NL 
NL 

NL 
NL 
NL 
NL 
NL 
NL 
NL 

IT 
NL 
NL 
NL 
NL 
NL 

NL 
NL 
NL 

Flavour classification 
Sweet 

Sweet 
Sweet 
Sweet 
Sweet 
Sweet 
Sweet 

Sweet 
Fruity 
Fruity 
Fruity 
Fruity 
Fruity 

Fruity 
Fruity 
Fruity 
Sweet 
Sweet 
Sweet 
Sweet 

Sweet 
Sweet 
Sweet 
Sweet 
Sweet 
Non-alcoholic drink 

Non-alcoholic drink 
Non-alcoholic drink 
Non-alcoholic drink 

31 

Nicotine content declared (mg/mL) 
0 
0 

0 
0 
0 
6 
12 
18 

0 
0 
0 
0 
0 
6 

12 
18 
0 
0 
0 
0 

0 
0 
6 
12 
18 
0 

0 
0 
6 

 
 
Code 
172 

Description 
Herbal Tea 

173 
174 
175 
176 
177 
178 

179 
180 
181 
182 
183 
184 

185 
186 
187 
188 
189 
190 
191 

192 
193 
194 
195 
196 
197 

198 
199 
200 

Caramel Machiato 
Caramel Machiato 
Caramel Machiato 
Beer 
Red Wine 
Kriek 

Champagne 
Tequila 
Tequila 
Tequila 
Tequila 
Exotic 

Enjoy 
Supreme 
Relax 
Relax 
Jet Fresh 
Jet Fresh 
Jet Fresh 

Thyme 
Sage 
Rosemary 
Arctic Mint 
Tobacco zero 
Nevada tobacco 

Mint 
Tiramisu 
Cheese cake 

Country acquisition  Country production 

NL 

NL 
NL 
NL 
NL 
NL 
FR 

NL 
NL 
NL 
NL 
NL 
FR 

FR 
FR 
FR 
FR 
FR 
FR 
FR 

NL 
NL 
NL 
BE 
DE 
DE 

FI 
FI 
CR 

NL 

NL 
NL 
NL 
NL 
NL 
FR 

NL 
NL 
NL 
NL 
NL 
FR 

FR 
FR 
FR 
FR 
FR 
FR 
FR 

CZ 
CZ 
CZ 
- 
- 
- 

- 
DE 
CR 

Flavour classification 
Non-alcoholic drink 

Non-alcoholic drink 
Non-alcoholic drink 
Non-alcoholic drink 
Alcoholic drink 
Alcoholic drink 
Alcoholic drink 

Alcoholic drink 
Alcoholic drink 
Alcoholic drink 
Alcoholic drink 
Alcoholic drink 
Exotic 

Exotic 
Exotic 
Exotic 
Exotic 
Exotic 
Exotic 
Exotic 

Fresh 
Fresh 
Fresh 
Fresh 
Tobacco 
Tobacco 

- 
Sweet 
Sweet 

32 

Nicotine content declared (mg/mL) 
12 
0 

12 
18 
0 
0 
0 
6 

0 
6 
12 
18 
0 
0 

6 
0 
18 
0 
6 
12 

0 
0 
0 

16 
0 
18 

0 
0 
0 

 
 
Code 
201 

Description 
Tobacco natural 

202 
203 
204 
205 
206 
207 

208 
209 
210 
211 
212 
213 

214 
215 
216 

Cappuccino 
Marbi 
Real tobacco 
LOP 
Venere 
Natural flavor violet 

Energetinio gerimo 
Bubblegum 
Desert ship 
L&M 
Apple mint 
Coffee 

Traditional tobacco 
Vanilka 
Energy mix 

Country acquisition  Country production 

CR 

CR 
HU 
HU 
IT 
IT 
FR 

LT 
NL 
NL 
NL 
PL 
PL 

RO 
CZ 
CZ 

CR 

CR 
HU 
HU 
IT 
IT 
LT 

LT 
- 
FR 
- 
PRC 
PRC 

CZ 
CZ 
CZ 

Flavour classification 
Tobacco 

Nicotine content declared (mg/mL) 
48 

Non-alcoholic drink 
Tobacco 
Tobacco 
Sweet 
Exotic 
Fresh 

Non-alcoholic drink 
Sweet 
Tobacco 
Tobacco 
Fresh 
Non-alcoholic drink 

Tobacco 
Sweet 
Non-alcoholic drink 

18 
12 
0 
0 
4 
12 

12 
6 
12 
11 
0 
6 

12 
8 
8 

33 

 
 
 
 
 
A1.3. Contents of tobacco alkaloids determined in e-liquid test samples 

Table A- 2: Results for tobacco alkaloids in tested e-liquids, and relative deviation of nicotine content from declared content 

(red  highlighted  cells:  zero-nicotine  labelled  products  containing  nicotine  at  levels  above  0.1  mg/ml;  orange  highlighted  cells:  nicotine 
content deviates more than 10% from declared nicotine content) 

Sample 
code 

NIC 
declared 
(mg/mL) 

NIC 
measured 
(mg/mL) 

MYO 
(µg/mL) 

NNIC 
(µg/mL) 

COT 
(µg/mL) 

NCOT 
(µg/mL) 

ABS 
(µg/mL) 

ATB 
(µg/mL) 

∆ declared 
(%) 

001 
002 
003 
004 
005 
006 
007 
008 
009 
010 
011 
012 
013 
014 
015 
016 
017 
018 
019 
020 

0 
0 
0 
0 
6 
12 
18 
18 
18 
18 
18 
24 
0 
0 
0 
0 
12 
12 
18 
18 

0.000 
0.000 
0.000 
0.000 
5.997 
11.224 
17.971 
16.854 
18.721 
17.803 
17.886 
22.965 
0.000 
0.001 
0.000 
0.000 
9.741 
8.687 
15.287 
13.449 

0.000 
0.000 
0.000 
0.000 
2.320 
3.657 
10.475 
8.366 
8.366 
5.553 
11.539 
3.625 
0.000 
0.000 
0.000 
0.000 
4.084 
0.288 
7.502 
0.985 

0.000 
0.000 
0.000 
0.000 
4.680 
5.843 
15.472 
17.367 
22.728 
10.362 
13.164 
19.444 
0.000 
0.000 
0.000 
0.000 
3.502 
3.692 
4.097 
7.403 

0.000 
0.000 
0.000 
0.000 
1.498 
0.893 
3.995 
2.807 
3.084 
3.009 
2.886 
2.505 
0.000 
0.000 
0.000 
0.000 
5.663 
0.527 
8.484 
6.096 

0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.196 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 

0.000 
0.000 
0.000 
0.000 
5.832 
6.727 
10.813 
45.529 
49.014 
22.820 
46.239 
11.127 
0.000 
0.000 
0.000 
0.000 
1.248 
0.000 
1.927 
1.815 

0.000 
0.000 
0.000 
0.000 
7.306 
10.120 
8.276 
61.309 
64.397 
30.757 
63.746 
14.079 
0.000 
0.000 
0.000 
0.000 
0.365 
0.000 
0.633 
0.661 

0.0 
-6.5 
-0.2 
-6.4 
4.0 
-1.1 
-0.6 
-4.3 

-18.8 
-27.6 
-15.1 
-25.3 

34 

 
 
  
  
  
  
  
  
  
  
Sample 
code 

NIC 
declared 
(mg/mL) 

NIC 
measured 
(mg/mL) 

MYO 
(µg/mL) 

NNIC 
(µg/mL) 

COT 
(µg/mL) 

NCOT 
(µg/mL) 

ABS 
(µg/mL) 

ATB 
(µg/mL) 

∆ declared 
(%) 

-2.5 

-14.3 
0.0 

021 
022 
023 
024 
025 
026 
027 
028 
029 
030 
031 
032 
033 
034 
035 
036 
037 
038 
039 
040 
041 
042 
043 
044 
045 

18 
0 
6 
16 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

17.550 
0.000 
5.142 
16.001 
0.003 
0.001 
0.000 
0.000 
0.000 
0.004 
0.000 
0.000 
0.001 
0.038 
0.001 
0.002 
0.038 
0.003 
0.009 
0.004 
0.132 
0.174 
0.002 
0.014 
0.001 

1.128 
0.000 
1.828 
1.327 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.112 
0.160 
0.000 
0.000 
0.000 

4.597 
0.000 
5.681 
6.913 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
眃.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.179 
0.388 
0.000 
0.000 
0.000 

0.762 
0.000 
0.373 
1.107 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 

0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 

35 

0.000 
0.000 
8.731 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 

0.000 
0.000 
15.370 
0.257 
0.141 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
0.000 

 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Sample 
code 

NIC 
declared 
(mg/mL) 

NIC 
measured 
(mg/mL) 

MYO 
(µg/mL) 

NNIC 
(µg/mL) 

COT 
(µg/mL) 

NCOT 
(µg/mL) 

ABS 
(µg/mL) 

ATB 
(µg/mL) 

∆ declared 
(%) 

046 
047 
048 
049 
050 
051 
052 
053 
054 
055 
056 
057 
058 
059 
060 
061 
062 
063 
064 
065 
066 
067 
068 
069 
070 

0 
0 
0 
0 
0 
0 
0 
0 
0 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 

0.002 
0.000 
0.000 
0.002 
0.001 
0.003 
0.001 
0.001 
ND 
8.320 
8.117 
8.633 
8.374 
8.472 
8.668 
8.241 
8.360 
8.649 
9.339 
8.698 
8.723 
8.361 
9.005 
8.587 
8.773 

0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
ND 
ND 
ND 
1.773 
2.525 
5.101 
1.803 
5.508 
5.582 
3.811 
2.863 
2.651 
1.961 
1.213 
1.285 
1.237 
0.987 
0.969 
1.323 

0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
0.827 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 

0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
0.505 
ND 
ND 
ND 
0.217 
ND 
ND 
ND 
ND 
ND 

0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
ND 
ND 
ND 
4.297 
4.108 
4.262 
4.287 
4.702 
4.723 
7.794 
4.182 
4.260 
4.845 
5.171 
3.316 
3.251 
4.382 
4.282 
3.828 

0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
ND 
ND 
ND 
1.773 
2.228 
3.888 
1.823 
5.844 
5.952 
1.721 
3.730 
2.416 
2.102 
0.966 
0.922 
0.839 
0.999 
0.990 
1.018 

0.000 
0.000 
0.000 
0.000 
0.000 
0.000 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 

36 

-16.8 
-18.8 
-13.7 
-16.3 
-15.3 
-13.3 
-17.6 
-16.4 
-13.5 
-6.6 
-13.0 
-12.8 
-16.4 
-9.9 
-14.1 
-12.3 

 
 
  
  
  
  
  
  
  
  
  
Sample 
code 

NIC 
declared 
(mg/mL) 

NIC 
measured 
(mg/mL) 

MYO 
(µg/mL) 

NNIC 
(µg/mL) 

COT 
(µg/mL) 

NCOT 
(µg/mL) 

ABS 
(µg/mL) 

ATB 
(µg/mL) 

∆ declared 
(%) 

071 
072 
073 
074 
075 
076 
077 
078 
079 
080 
081 
082 
083 
084 
085 
086 
087 
088 
089 
090 
091 
092 
093 
094 
095 

10 
10 
10 
10 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 

8.639 
8.498 
8.700 
9.020 
19.169 
17.674 
17.710 
18.391 
17.727 
17.182 
16.895 
14.503 
17.672 
17.450 
16.561 
17.247 
17.144 
18.442 
17.911 
15.009 
17.828 
18.339 
18.155 
19.352 
17.940 

0.935 
1.140 
1.052 
1.183 
1.328 
0.834 
1.199 
1.266 
1.192 
1.402 
2.952 
5.512 
1.195 
1.638 
4.144 
1.249 
6.323 
1.512 
6.079 
5.245 
1.439 
1.478 
1.241 
1.223 
1.468 

4.127 
3.846 
3.414 
3.190 
8.286 
9.785 
7.108 
10.417 
8.247 
7.593 
9.138 
7.730 
8.087 
8.108 
7.648 
8.270 
11.532 
7.006 
5.626 
17.228 
6.224 
5.741 
5.113 
5.774 
5.735 

0.898 
0.880 
0.944 
0.821 
1.675 
1.601 
1.610 
1.551 
1.787 
1.630 
2.125 
3.257 
1.918 
1.883 
3.087 
1.737 
2.113 
1.938 
1.529 
1.715 
1.568 
1.957 
1.842 
1.867 
2.193 

ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 

ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
1.520 
ND 
ND 
ND 
ND 
1.750 
ND 
1.568 
2.061 
ND 
ND 
ND 
ND 
ND 

ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
1.124 
ND 
ND 
ND 
ND 
0.706 
ND 
0.562 
0.876 
ND 
ND 
ND 
ND 
ND 

-13.6 
-15.0 
-13.0 
-9.8 
-4.2 
-11.6 
-11.4 
-8.0 
-11.4 
-14.1 
-15.5 
-27.5 
-11.6 
-12.7 
-17.2 
-13.8 
-14.3 
-7.8 
-10.4 
-25.0 
-10.9 
-8.3 
-9.2 
-3.2 
-10.3 

37 

 
 
Sample 
code 

NIC 
declared 
(mg/mL) 

NIC 
measured 
(mg/mL) 

MYO 
(µg/mL) 

NNIC 
(µg/mL) 

COT 
(µg/mL) 

NCOT 
(µg/mL) 

ABS 
(µg/mL) 

ATB 
(µg/mL) 

∆ declared 
(%) 

096 
097 
098 
099 
100 
101 
102 
103 
104 
105 
106 
107 
108 
109 
110 
111 
112 
113 
114 
115 
116 
117 
119 
120 
121 

20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
20 
11 
- 
6 
- 
- 
- 

17.037 
16.997 
18.552 
17.984 
17.188 
18.460 
17.768 
16.990 
18.321 
17.757 
16.982 
17.938 
17.722 
19.086 
17.257 
17.061 
17.910 
17.384 
16.684 
9.941 
17.749 
4.983 
22.139 
22.416 
16.027 

3.538 
3.478 
1.364 
1.385 
1.860 
2.524 
1.507 
2.562 
1.285 
2.457 
3.003 
2.903 
2.373 
2.553 
3.230 
2.365 
2.541 
2.736 
2.869 
2.653 
11.932 
5.986 
5.606 
17.663 
4.850 

5.377 
5.901 
5.576 
4.720 
5.381 
7.268 
5.452 
4.750 
5.582 
6.354 
4.295 
3.991 
5.327 
5.532 
4.452 
4.975 
5.203 
4.485 
4.197 
4.683 
15.736 
3.632 
221.744 
16.016 
11.178 

3.335 
2.345 
2.130 
2.453 
2.602 
3.226 
2.377 
2.884 
2.632 
3.107 
4.155 
3.664 
4.777 
5.402 
4.451 
4.315 
4.424 
5.129 
3.905 
2.807 
12.427 
12.337 
81.624 
41.067 
7.142 

ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
0.271 
0.193 
1.608 
0.621 
ND 

ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
1.012 
20.904 
0.290 
6.164 
1.773 
1.390 

ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
0.190 
76.210 
0.259 
0.690 
1.271 
0.951 

-14.8 
-15.0 
-7.2 
-10.1 
-14.1 
-7.7 
-11.2 
-15.1 
-8.4 
-11.2 
-15.1 
-10.3 
-11.4 
-4.6 
-13.7 
-14.7 
-10.4 
-13.1 
-16.6 
-9.6 

-17.0 

38 

 
 
  
  
  
  
Sample 
code 

NIC 
declared 
(mg/mL) 

NIC 
measured 
(mg/mL) 

MYO 
(µg/mL) 

NNIC 
(µg/mL) 

COT 
(µg/mL) 

NCOT 
(µg/mL) 

ABS 
(µg/mL) 

ATB 
(µg/mL) 

∆ declared 
(%) 

122 
123 
124 
125 
126 
127 
128 
129 
130 
131 
132 
133 
134 
135 
136 
137 
138 
139 
140 
141 
142 
143 
144 
145 
146 

- 
0 
0 
0 
0 
0 
0 
0 
0 
0 
6 
12 
18 
0 
0 
0 
0 
0 
6 
12 
18 
0 
0 
0 
0 

14.728 
ND 
ND 
0.390 
0.159 
0.001 
0.004 
0.028 
ND 
0.009 
5.719 
11.639 
17.560 
0.001 
ND 
ND 
ND 
ND 
5.636 
10.927 
16.848 
0.001 
ND 
ND 
ND 

3.990 
0.143 
0.175 
0.372 
0.179 
0.126 
0.125 
0.134 
0.128 
0.131 
0.640 
1.345 
1.941 
0.143 
0.132 
0.117 
0.143 
0.127 
0.769 
2.440 
2.191 
0.125 
0.154 
0.118 
0.134 

ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 

1.181 
0.068 
0.062 
0.199 
0.096 
0.065 
0.076 
0.113 
0.065 
0.090 
4.200 
8.187 
11.983 
0.071 
0.061 
0.067 
0.075 
0.064 
0.338 
1.590 
1.150 
0.067 
0.063 
0.067 
0.066 

0.798 
0.024 
0.003 
0.263 
0.120 
0.058 
0.428 
0.058 
ND 
0.635 
22.499 
47.750 
72.391 
0.022 
0.001 
0.015 
0.007 
ND 
11.097 
78.625 
40.499 
ND 
ND 
0.006 
0.001 

7.926 
0.155 
0.155 
0.314 
0.206 
0.156 
0.179 
0.171 
0.158 
0.258 
0.786 
1.385 
1.931 
0.158 
0.157 
0.160 
0.170 
0.158 
0.672 
3.412 
2.093 
0.161 
0.158 
0.158 
0.162 

4.421 
ND 
ND 
0.295 
0.103 
ND 
ND 
ND 
ND 
ND 
ND 
0.031 
0.070 
ND 
ND 
ND 
ND 
ND 
0.084 
0.229 
0.143 
ND 
ND 
ND 
ND 

39 

-4.7 
-3.0 
-2.4 

-6.1 
-8.9 
-6.4 

 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Sample 
code 

NIC 
declared 
(mg/mL) 

NIC 
measured 
(mg/mL) 

MYO 
(µg/mL) 

NNIC 
(µg/mL) 

COT 
(µg/mL) 

NCOT 
(µg/mL) 

ABS 
(µg/mL) 

ATB 
(µg/mL) 

∆ declared 
(%) 

147 
148 
149 
150 
151 
152 
153 
154 
155 
156 
157 
158 
159 
160 
161 
162 
163 
164 
165 
166 
167 
168 
169 
170 
171 

0 
6 
12 
18 
0 
0 
0 
0 
0 
6 
12 
18 
0 
0 
0 
0 
0 
0 
6 
12 
18 
0 
0 
0 
6 

ND 
6.158 
11.983 
18.390 
0.001 
ND 
ND 
ND 
0.955 
6.027 
11.426 
17.560 
5.719 
ND 
ND 
ND 
ND 
ND 
5.624 
12.339 
18.034 
0.001 
ND 
ND 
0.580 

0.127 
0.967 
1.659 
2.733 
0.121 
0.116 
0.138 
0.134 
0.684 
0.357 
2.093 
0.882 
1.844 
0.170 
0.115 
0.150 
0.140 
0.120 
0.633 
1.204 
1.540 
0.118 
0.188 
0.123 
0.251 

0.061 
0.472 
0.998 
1.827 
0.067 
0.066 
0.064 
0.068 
0.617 
0.497 
0.833 
0.712 
2.112 
0.062 
0.069 
0.066 
0.060 
0.062 
0.318 
0.358 
0.825 
0.065 
0.064 
0.066 
0.335 

0.002 
14.733 
33.977 
52.317 
ND 
ND 
0.006 
0.007 
1.042 
0.499 
61.001 
1.030 
0.566 
ND 
0.002 
0.074 
0.067 
0.091 
3.926 
7.870 
13.258 
0.057 
0.057 
0.034 
0.161 

ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 

0.157 
0.266 
0.360 
0.512 
0.164 
0.163 
0.156 
0.158 
0.606 
0.264 
1.385 
0.439 
0.348 
0.157 
0.164 
0.158 
0.158 
0.156 
0.197 
0.237 
0.255 
0.159 
0.160 
0.156 
0.197 

ND 
ND 
0.035 
0.071 
ND 
ND 
ND 
ND 
0.525 
0.046 
0.195 
0.412 
0.959 
ND 
ND 
ND 
ND 
ND 
0.025 
0.081 
0.128 
ND 
ND 
ND 
0.045 

40 

2.6 
-0.1 
2.2 

0.5 
-4.8 
-2.4 

-6.3 
2.8 
0.2 

-90.3 

 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Sample 
code 

NIC 
declared 
(mg/mL) 

NIC 
measured 
(mg/mL) 

MYO 
(µg/mL) 

NNIC 
(µg/mL) 

COT 
(µg/mL) 

NCOT 
(µg/mL) 

ABS 
(µg/mL) 

ATB 
(µg/mL) 

∆ declared 
(%) 

172 
173 
174 
175 
176 
177 
178 
179 
180 
181 
182 
183 
184 
185 
186 
187 
188 
189 
190 
191 
192 
193 
194 
195 
196 

12 
0 
12 
18 
0 
0 
0 
6 
0 
6 
12 
18 
0 
0 
6 
0 
18 
0 
6 
12 
0 
0 
0 
16 
0 

11.485 
ND 
10.809 
16.136 
0.001 
ND 
ND 
0.578 
0.001 
5.042 
9.919 
15.305 
ND 
ND 
0.602 
ND 
18.153 
0.001 
6.122 
12.221 
ND 
ND 
ND 
14.842 
13.569 

0.402 
0.117 
0.851 
0.698 
0.196 
0.108 
0.121 
0.175 
0.131 
0.733 
1.497 
2.440 
0.116 
0.183 
0.313 
0.131 
1.367 
0.179 
0.687 
1.038 
0.140 
0.108 
0.123 
6.784 
27.610 

0.224 
0.156 
0.210 
0.232 
0.160 
0.157 
0.158 
0.184 
0.159 
1.205 
1.061 
1.505 
0.155 
0.158 
0.267 
0.162 
0.315 
0.160 
0.181 
0.210 
0.162 
0.155 
0.166 
5.994 
26.057 

0.089 
ND 
0.780 
0.723 
0.073 
ND 
ND 
0.032 
ND 
0.097 
0.190 
0.307 
ND 
ND 
0.099 
ND 
1.139 
ND 
0.337 
0.671 
ND 
ND 
ND 
1.582 
11.809 

ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
ND 
0.092 
3.010 

41 

0.731 
0.066 
0.704 
1.162 
0.063 
0.061 
0.061 
0.470 
0.069 
0.612 
0.711 
1.141 
0.061 
0.060 
0.438 
0.068 
1.471 
0.062 
0.441 
0.718 
0.066 
0.063 
0.066 
ND 
9.977 

0.144 
0.063 
0.082 
0.164 
0.035 
0.033 
0.039 
0.058 
0.046 
22.745 
39.548 
62.429 
0.044 
0.047 
0.058 
0.049 
0.210 
0.036 
0.200 
0.308 
0.039 
0.052 
0.041 
5.505 
1.561 

-4.3 

-9.9 
-10.4 

-90.4 

-16.0 
-17.3 
-15.0 

-90.0 

0.8 

2.0 
1.8 

-7.2 

 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
Sample 
code 

NIC 
declared 
(mg/mL) 

NIC 
measured 
(mg/mL) 

MYO 
(µg/mL) 

NNIC 
(µg/mL) 

COT 
(µg/mL) 

NCOT 
(µg/mL) 

ABS 
(µg/mL) 

ATB 
(µg/mL) 

∆ declared 
(%) 

197 
198 
199 
200 
201 
202 
203 
204 
205 
206 
207 
208 
209 
210 
211 
212 
213 
214 
215 
216 

18 
0 
0 
0 
48 
18 
12 
0 
0 
4 
12 
12 
6 
12 
11 
0 
6 
12 
8 
8 

0.011 
0.017 
0.001 
0.001 
46.422 
10.970 
10.029 
1.617 
0.004 
4.595 
11.868 
9.653 
5.898 
10.993 
10.711 
0.003 
6.133 
11.444 
5.945 
5.841 

ND 
ND 
ND 
ND 
62.851 
18.459 
17.231 
1.658 
ND 
2.177 
9.232 
4.066 
4.126 
6.082 
7.266 
ND 
4.166 
3.317 
1.589 
3.936 

0.320 
ND 
ND 
ND 
26.653 
1.745 
2.141 
0.886 
ND 
0.858 
6.783 
0.788 
0.575 
1.162 
0.964 
ND 
5.244 
0.734 
0.570 
0.545 

0.320 
0.311 
0.308 
0.309 
40.342 
5.181 
5.237 
0.537 
0.325 
1.697 
2.051 
1.029 
3.967 
1.389 
6.364 
0.316 
0.776 
3.099 
0.643 
1.318 

ND 
ND 
ND 
ND 
0.468 
0.213 
0.137 
ND 
ND 
ND 
ND 
0.718 
0.010 
ND 
0.091 
ND 
ND 
ND 
ND 
ND 

ND 
ND 
ND 
ND 
ND 
27.977 
23.884 
ND 
ND 
ND 
ND 
ND 
ND 
30.552 
ND 
ND 
25.055 
ND 
ND 
ND 

ND 
ND 
ND 
ND 
7.396 
14.758 
18.739 
0.448 
ND 
0.363 
1.624 
0.513 
0.317 
34.663 
0.721 
ND 
38.378 
0.850 
1.055 
4.771 

-99.9 

-3.3 
-39.1 
-16.4 

14.9 
-1.1 
-19.6 
-1.7 
-8.4 
-2.6 

2.2 
-4.6 
-25.7 
-27.0 

42 

 
 
  
  
  
  
  
  
 
 
 
 
Table A- 3: Results for tobacco alkaloids in cigarette tobacco extracts 

Sample code 

NIC 
(mg/mL) 

MYO 
(µg/mL) 

NNIC 
(µg/mL) 

COT 
(µg/mL) 

NCOT 
(µg/mL) 

ABS 
(µg/mL) 

ATB 
(µg/mL) 

Cigarette 1 

Cigarette 2 

Cigarette 3 

Cigarette 4 (average of two 
determinations) 
Cigarette 3R4F (average of two 
determinations) 

15.759 

15.878 

14.096 

6.815  484.033 

82.742 

7.729  488.820  112.834 

9.595  584.537  112.081 

1.445 

2.180 

2.072 

64.010 

343.027 

86.295 

386.597 

66.357 

304.754 

12.211 

6.399  391.155 

88.880 

1.608 

60.226 

328.569 

15.032 

6.307  413.240 

94.479 

1.912 

82.133 

344.703 

43 

 
 
 
 
ANNEX  2  –  Analytical  method  for  the  evaluation  of  flavour 

profiles 

A2.1. 

Analysis of volatile constituents of e-liquids by thermal 
desorption  high-resolution  gas  chromatography  QTOF  mass 
spectrometry 

1) Standards and reagents 

Isotopically  labelled  standard  2-ethylphenol-D10  was  purchased  from  CDN  isotopes 
(Quebec, Canada). Spiking solution of labelled standard of about 30 µg/mL was prepared 
gravimetrically in methanol. Methanol of LC-MS grade was obtained from VWR (Leuven, 
Belgium).  

2) Sample preparation 

For the analysis of volatile compounds, 30 µL of homogenized sample was taken to the 
vial and dissolved in 900 µL methanol. Labelled internal standard 2-ethylphenol-D10 was 
added (100 µL, 30 µg/mL), the vial was vigorously shaken and placed on the GC/Q-TOF-
MS autosampler for the analysis.  

3) Thermal desorption GC-QTOF analysis 

Operating conditions 

Six  µL  of  the  sample  was  injected  by  MPS  sampler  into  the  liner  of  the  thermal 
desorption  unit  (TDU)  containing  a  micro-vial  insert.  The  TDU  was  programmed  from 
20 °C  held  for  0.1  min  to  100 °C  at  30 °C/min  held  for  15  min  with  purge  flow  100 
mL/min. Volatile compounds were trapped in the PTV inlet on Tenax TA packed liner at 
15 °C. Trapped compounds were injected onto the HP-5MS GC column (30 m x 250 µm 
x  0.25  µm,  Agilent  Technologies)  with  split  ratio  15:1  while  programing  the  PTV  inlet 
from  15 °C  held  for  0.1  min  to  270 °C  at  12 °C/s  held  for  20  min.  The  GC  oven  was 
programmed  from  45 °C  (held  for  2  min)  to  210 °C  at  4°C/min  further  to  300 °C  at 
10 °C/min  (held  for  10  min).  Helium  was  used  as  a  carrier  gas  at  1  mL/min  flow  rate. 
The transfer line temperature was set at 300 °C. The Q-TOF-MS was operated at a mass 
range  of  m/z  45-450.  The  data  acquisition  rate  was  5  Hz  in  2  GHz  extended  dynamic 
range (EDR) mode. 

Instrument configuration 

The TDU-GC/Q-TOF-MS system consisted of: A gas chromatograph (GC) 7890A (Agilent 
Technologies, Santa Clara, CA, USA) was equipped with a cooled injection system CIS, a 
programmable  temperature  vaporizing  (PTV)  inlet  (Gerstel,  Mülheim  an  der  Ruhr, 
Germany)  and  TDU  thermal  desorption  unit  (Gerstel)  operating  automatically  in 
conjunction with MurtiPurpose Sampler MPS (Gerstel). The GC was coupled to an Agilent 
7200 Accurate Mass Q-TOF MS (Agilent Technologies). Operation of the instrument was 

44 

 
 
 
 
 
 
controlled  by  MassHunter  GC/MS  Acquisition  version  B.07.03.2129 
Technologies) and Gerstel Maestro 1 version 1.4.31.10/3.5 (Gerstel). 

(Agilent 

4) Data Analysis 

Mass  Hunter  Qualitative  Analysis  version  B.07.00  (Agilent  Technologies),  Mass  Hunter 
Unknowns  Analysis  version  B.07.01  (Agilent  Technologies),  Mass  Hunter  Quantitative 
Analysis  for  QTOF  version  B.07.01  (Agilent  Technologies),  Mass  Profiler  Professional 
version 12.5 (Agilent Technologies), XCMSTM version v3.5.1 (Scripps Research Institute, 
La  Jolla,  CA,  USA)  and  Mass  MountaineerTM  version  2.00.3.1  (RBC  software,  Reabody, 
MA,  USA)  were  used  for  the  data  analysis.  Multivariate  statistics  were  applied  using 
SIMCA version 14.1.0.2047 (MKS Umetrics, Malmo, Sweeden) and Statgraphics, version 
15.2.06 (StatPoint Technologies, Warrenton, VA, USA). 

5) Analytical precision 

Repeatability expressed as a relative standard deviation (RSD) of the absolute responses 
of  labelled  standard  spiked  in  12  different  e-liquids  was  2%.  Intermediate  precision 
calculated  as  RSD  of  labelled  standard  response  analysed  in  the  period  of  two  months 
was 13%. 

45 

 
 
 
 
 
 
 
 
A2.2. 

Test samples 

In  total  108  samples  of  e-liquids  comprising  different  producers  and  flavour 
characteristics  (tobacco,  mint,  vanilla,  fruits  and  others)  were  selected  for  the  volatile 
analysis (Table 1). 

Table A- 4:  Samples analysed for profile of volatiles 

Sample 
code 

Description 

Produced in 

F04a 
F04b 
F04c 
F05 
F06a 
F08 
F09 
D01 
D02 
D03a 
D03b 
D03c 
D03d 
D04 
D05 
D06 
D07a 
D07b 
D08a 
D08a 
E11 
E12 
E13 
E14 
K01 
K02 
K03 
K04 
K05 
K06 
K07 
K08 
K09 
K10 
K11 

Caramel Machiato 
Caramel Machiato 
Caramel Machiato 
Cappuccino 
Coffee 
Cappuccino 
Coffee 
Passion Fruit 
Mandarin 
Strawberry 
Strawberry 
Strawberry 
Strawberry 
Coconut 
Pear 
Strawberry 
Erdbeer fresh 
Erdbeer fresh 
Erdbeer 
Erdbeer 
Honigmelone 
Himbeer 
Frucht-mix Tropic 
Amarena Kirsch 
Apple 
Cherry 
Apfel-kuchen 
Kirsch Amarena fresh 
Zitrone fresh 
Apfel fresh 
Brombeer fresh 
Pfirsich Maracuja fresh 
Wassermelone fresh 
Himbeer fresh 
Walderdbeer fresh 

Netherlands 
Netherlands 
Netherlands 
Netherlands 
Spain 
Croatia 
China 
Netherlands 
Netherlands 
Netherlands 
Netherlands 
Netherlands 
Netherlands 
Netherlands 
Netherlands 
China 
Germany 
Germany 
Germany 
Germany 
Germany 
Germany 
Germany 
Germany 
China 
China 
Germany 
Germany 
Germany 
Germany 
Germany 
Germany 
Germany 
Germany 
Germany 

46 

NIC declared 
(mg/mL) 
0 
12 
18 
0 
0 
18 
6 
0 
0 
0 
6 
12 
18 
0 
0 
18 
0 
20 
10 
20 
10 
10 
10 
10 
18 
24 
0 
0 
0 
0 
0 
0 
0 
0 
0 

Flavour 

Coffee 
Coffee 
Coffee 
Coffee 
Coffee 
Coffee 
Coffee 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 
Fruit 

 
 
Sample 
code 

K12 
B02 
B03 
B04a 
B04b 
B04c 
B04d 
B05 
B06 
E01a 
E02 
E03 
E04 
E05 
E06 
E07 
E08 
E09 
E10 
F07 
A01 
A02 
A03 
A04 
A05 
A06a 
A06b 
A06c 
A06d 
A07 
A09 
A10 
A11 
A12 
A13 
A14 
A15 
A16 
A17 
A18 
A19a 

Description 

Produced in 

USA 
Cherry Limeade 
Netherlands 
Menthol 
Netherlands 
Arctic Mint 
Netherlands 
Ice Mint 
Netherlands 
Ice Mint 
Netherlands 
Ice Mint 
Netherlands 
Ice Mint 
Netherlands 
Mint 
China 
Menthol 
Netherlands 
Banana Split 
Netherlands 
Cotton Candy 
Netherlands 
White Chocolate 
Netherlands 
Banana Chocolate 
Netherlands 
Pumpkin Pie 
Italy 
Biscuit 
Germany 
Toffee 
Germany 
Marzipan 
Germany 
Honig 
Germany 
Cookie 
Germany 
Karamell Sahne 
Netherlands 
Arabic tobacco 
Netherlands 
USA Mix 
Netherlands 
Turkish blend 
Netherlands 
Southeast County 
Netherlands 
Cuba Cigar 
Netherlands 
Coco & Havana 
Netherlands 
Coco & Havana 
Netherlands 
Coco & Havana 
Coco & Havana 
Netherlands 
Honey Toasted Tobacco  Netherlands 
Old Dry Tabacco 
The Palm 
Tobacco Natural 
Desert ship 
Marbi 
Real tobacco 
Traditional tobacco 
Tobacco 
Virginia Tobacco 
Tobacco 
Calamity Jane Tobacco 

Italy 
Netherlands 
Croatia 
France 
EU 
EU 
Czech Republic 
China 
China 
China 
USA 

47 

NIC declared 
(mg/mL) 
0 
0 
0 
0 
6 
12 
18 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
6 
2 
18 
0 
0 
0 
48 
12 
no Info 
no Info 
12 
0 
18 
18 
0 

Flavour 

Fruit 
Mint 
Mint 
Mint 
Mint 
Mint 
Mint 
Mint 
Mint 
Other 
Other 
Other 
Other 
Other 
Other 
Other 
Other 
Other 
Other 
Other 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 

 
 
Sample 
code 

Description 

Produced in 

A19b 
A19c 
A20a 
A20b 
A21 
A22 
A23 
A24 
A25 
A26 
A27 
A30 
A31 
A32 
A33 
A34 
A35 
A36 
A37 
B01 
B07 
C02 
E15 
F03 
F10 
C01 
C03a 
C03b 
C03c 
C03d 
C04 
C05 

Calamity Jane Tobacco 
Calamity Jane Tobacco 
Ol' River Tobacco 
Ol' River Tobacco 
Rocket Blend 
Tobacco 
Black Mamba 
Black Fire 
Pfeife 
Kentucky 
American Blend 
Tabac Fresh 
Tabac Regular 
Tabac USA 
Tabac British 
Strong Blondy 
Goldy 
Le Gitan 
Tabac Des Iles 
Tobacco Mint 
Tabac Mint 
Shisha Vanille 
Tabac Caramel 
Shisha Coffee 
Tabac Café 
Vanilla 
Vanilla Orange 
Vanilla Orange 
Vanilla Orange 
Vanilla Orange 
Vanille 
Ice Vanille 

USA 
USA 
USA 
USA 
USA 
Germany 
Germany 
Germany 
Germany 
Germany 
Germany 
France 
France 
France 
France 
France 
France 
France 
France 
Netherlands 
France 
Netherlands 
France 
Netherlands 
France 
Netherlands 
Netherlands 
Netherlands 
Netherlands 
Netherlands 
France 
Netherlands 

NIC declared 
(mg/mL) 
12 
18 
0 
18 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
6 
12 
18 
0 
0 

Flavour 

Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Tobacco 
Vanilla 
Vanilla 
Vanilla 
Vanilla 
Vanilla 
Vanilla 
Vanilla 

48 

 
 
 
 
 
 
A2.3. 

Compound identification 

Approximately  100  components  were  found  in  each  e-liquid  sample  with  Mass  Hunter 
search  using  high  resolution.  Identification  of  the  compounds  was  performed  with  the 
NIST  library  mass  spectra  search  and  based  on  compounds  linear  retention  indexes 
(LRI).  In  total  90  compounds  were  tentatively  identified  in  the  tobacco  flavoured  e-
liquids (table 2). Majority of these compounds (more then 70) was also reported in the 
tobacco either as a naturally presented or added as an ingredient for flavour formulation 
(Rodgman & Perfetti, 2013).  

Table A- 5  Compounds identified in e-liquid products declared as a tobacco flavoured 

(RT: retention time, CAS No: Chemical Abstracts Service number, LRI: linear retention index,  
LRI (lit): linear retention index retrieved from literature) 

No.  RT [min] 

Compound 

CAS No. 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

4.149  Methylbutyrate 

5.086  methyl 2-methylbutyrate 

5.539  Ethylbutyrate 

623-42-7 

868-57-5 

105-54-4 

5.679  dihydro-2-methyl-3(2H)-furanone 

3188-00-9 

5.850  ethyl lactate 

6.064  2-methylpyrazine 

6.864  2-furanmethanol 

7.881  o-xylene 

8.138  3-methoxy-1,2-propanediol 

8.443  2,5-dimethylpyrazine 

97-64-3 

109-08-0 

98-00-0 

95-47-6 

623-39-2 

123-32-0 

8.643  2,3-dimethylpyrazine 

5910-89-4 

9.101  2,4-dimethyl-1,3-dioxolane-2-methanol 

53951-43-2 

9.329  2,2-dimethyl-1,3-dioxolane-4-methanol 

100-79-8 

9.400  2-butyl-4-methyl-1,3-dioxolane 

74094-60-3 

9.678  Ethyl-3-oxobutyrate 

10.000  Benzaldehyde 

10.125  5-methylfurfural 

11.393  Trimethylpyrazine 

12.064  Acetylpyrazine 

12.196  1,1-oxybis-2-propanol 

141-97-9 

100-52-7 

620-02-0 

14667-55-1 

22047-25-2 

110-98-5 

12.290  2-hydroxy-3-methyl-2-cyclpentene-1-one 

80-71-7 

12.562  Benzylalcohol 

13.005  1-methyl-2-pyrrolidine 

100-51-6 

872-50-4 

49 

LRI 

721 

779 

805 

810 

816 

824 

855 

895 

903 

912 

918 

932 

939 

941 

949 

960 

963 

1001 

1020 

1024 

1027 

1034 

1047 

LRI (lit)* 

723 

780 

805 

810 

813 

825 

862 

891 

900 

911 

919 

- 

1016 

939 

943 

961 

962 

1000 

1023 

1014 

1034 

1032 

1045 

 
 
 
No.  RT [min] 

Compound 

CAS No. 

LRI 

LRI (lit)* 

24 

25 

26 

27 

28 

29 

30 

31 

32 

33 

34 

35 

36 

37 

13.243  γ-caprolactone 

13.493  2-acetylpyrrole 

13.813  3-methyldecane 

13.914 

cis-linalool oxide 

14.255  allyl hexanoate 

14.396  Tetramethylpyrazine 

14.474 

trans-linalool oxide 

14.656  1-monoacetin 

14.868  Undecane 

15.047  Nonanal 

15.341  Maltol 

15.380  2-phenylethanol 

695-06-7 

1072-83-9 

13151-34-3 

5989-33-3  

123-68-2 

1124-11-4 

34995-77-2 

106-61-6 

1120-21-4 

124-19-6 

118-71-8 

60-12-8 

15.634  3-ethyl-2-hydroxy-2-cyclopenten-1-one 

21835-01-8 

16.443  2,6,6,-trimethyl-2-cyclohexene-1,4-dione 

1125-21-9 

1054 

1061 

1070 

1073 

1082 

1085 

1089 

1093 

1100 

1105 

1113 

1114 

1121 

1143 

1056 

1060 

1069 

1072 

1080 

1085 

1089 

1091 

1100 

1104 

1108 

1116 

1100 

1140 

38 

16.577 

2-hydroxy-3,5,5-trimethyl-cyclohex-2-
enone 

4883-60-7 

1147 

1150 

39 

40 

41 

42 

43 

44 

45 

46 

47 

48 

49 

50 

51 

52 

53 

54 

55 

17.403  Ethylbenzoate 

17.484  Menthol 

17.814  diethylester succinic acid 

17.906  α-monopropionin 

18.073  2-(2-butoxyethoxy)-ethanol 

18.357  Ethylmaltol 

19.122  2-phenoxyethanol 

19.240  1,2-benzisothiazole 

19.422  3,7-dimethyl-6-octen-1-ol 

20.005  Ethylphenylacetate 

20.305  4-methoxybenzaldehyde 

20.449  γ-octalactone 

93-89-0 

15356-70-4 

123-25-1 

624-47-5 

112-34-5 

4940-11-8 

122-99-6 

272-16-2 

106-22-9 

101-97-3 

123-11-5 

104-50-7 

20.760  4-methyl-2-phenyl-1,3-dioxolane 

2568-25-4 

20.847 

trans-cinnamaldehyde 

21.015  Perillaldehyde 

21.268  p-anisyl alcohol 

14371-10-9 

2111-75-3 

105-13-5 

21.381  1-methoxy-4-(1-propenyl)-benzene 

104-46-1 

50 

1170 

1173 

1182 

1185 

1190 

1197 

1220 

1223 

1228 

1245 

1254 

1259 

1268 

1270 

1275 

1283 

1285 

1170 

1171 

1181 

1190 

1192 

1197 

1221 

1221 

1228 

1244 

1251 

1262 

1272 

1266 

1272 

1279 

1289 

 
 
No.  RT [min] 

Compound 

CAS No. 

LRI 

LRI (lit)* 

56 

57 

58 

59 

60 

61 

62 

63 

64 

65 

66 

67 

68 

69 

70 

71 

72 

73 

74 

75 

76 

77 

78 

79 

80 

81 

82 

83 

84 

85 

21.921  Carvacrol 

22.962  Piperonal 

23.430  Nicotine 

499-75-2 

120-57-0 

54-11-5 

1-hydroxy-2,4,4-trimethylpentan-3-yl  2-
methylpropanoate 

23.583 

74367-33-2 

23.602  4-acetylanisole 

23.634  Triacetin 

23.764  p-eugenol 

23.936  γ-nonalactone 

3-hydroxy-2,4,4-trimethypentyl 
methylpropanoate 

24.280 

2-

24.661 

trans-β-damascenone 

24.942 

trans-α-damascone 

24.987  2-phenylethylisobutyrate 

25.075  Vanillin 

25.287  Methyleugenol 

1-(2,6,6,-trimethyl-1-cycloxen-1-yl)-2-
buten-1-one 

25.609 

25.771  p-anisyl acetate 

25.823  Myosmine 

26.257  Coumarin 

26.771 

trans-geranylacetone 

26.875  ethyl vanillin 

27.238  γ-decalactone 

27.654  Anabasine 

27.762  Nicotyrine 

27.822  β-ionone 

28.564  2,4-ditertbutylphenol 

28.577  Anatabine 

29.144  2,3-dipyridyl 

29.904  6-methylcoumarin 

31.021  Diethylphthalate 

35.647  Benzylbenzoate 

100-06-1 

102-76-1 

97-53-0 

104-61-0 

74367-34-3 

23726-93-4 

24720-09-0 

103-48-0 

121-33-5 

93-15-2 

35044-68-9 

104-21-2 

532-12-7 

91-64-5 

3796-70-1 

121-32-4 

706-14-9 

494-52-0 

487-19-4 

79-77-6 

96-76-4 

2743-90-0 

581-50-0 

92-48-8 

84-66-2 

120-51-4 

51 

1301 

1331 

1348 

1352 

1353 

1354 

1358 

1363 

1374 

1386 

1394 

1396 

1399 

1408 

1415 

1420 

1421 

1437 

1451 

1454 

1469 

1483 

1486 

1487 

1512 

1512 

1532 

1558 

1596 

1765 

1298 

1329 

1341 

1351 

1345 

1356 

1356 

1366 

1376 

1391 

1395 

1396 

1391 

1405 

1412 

1419 

1427 

1439 

1453 

1452 

1472 

1486 

1488 

1485 

1513 

1510 

1536 

1549 

1594 

1761 

 
 
No.  RT [min] 

Compound 

CAS No. 

LRI 

LRI (lit)* 

86 

87 

88 

89 

90 

37.565  Neophytadiene 

38.573  1-hexadecanol 

40.570  n-hexadecanoic acid 

43.430  1-octadecanol 

43.670  Benzylcinnamate 

LRI  were 

* 
http://www.pherobase.com/, 
https://pubchem.ncbi.nlm.nih.gov/.  

searched 

in 

databases 

504-961 

36653-82-4 

57-10-3 

112-92-5 

103-41-3 

1839 

1879 

1961 

2083 

2094 

1840 

1880 

1963 

2084 

2096 

such 

as  http://www.flavornet.org/, 
http://webbook.nist.gov/chemistry/, 

52 

 
 
 
 
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Cheng,  T.  (2014).  Chemical  evaluation  of  electronic  cigarettes.  Tobacco  Control, 

23(SUPPL. 2), ii11-ii17. doi: 10.1136/tobaccocontrol-2013-051482 

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composition  and  evaluation  of  nicotine,  tobacco  alkaloids,  pH,  and  selected 
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53 

 
 
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54 

 
 
 
 
 
 
List of figures 

Figure  1:  Difference  between  the  NIC  concentrations  declared  on  the  label  and  the 
measured concentration in the analysed samples. ................................................. 9 

Figure 2: Relative concentrations of minor alkaloids in traditional cigarettes. .............. 11 

Figure 3: Relative concentrations of minor alkaloids in e-liquids. ............................... 11 

Figure  4:  Principle  component  analysis  –  Score  plot  of  PC1  and  PC2  for  nicotine 
containing e-liquids and cigarette tobacco extracts .............................................. 12 

Figure 5: Prevalence of measured substances in different e-liquid categories .............. 15 

Figure 6: PCA score plot of e-liquid volatiles: .......................................................... 15 

55 

 
 
 
 
 
 
 
List of tables 

Table 1: Chemical structures and acronyms of the analysed substances ....................... 8 

Table 2: Number of test samples per flavour category ............................................. 14 

Table 3: Cross-validation of the PLS-DA model based on five flavour groups ............... 16 

Table  4:  Individual  results  from  validation  of  the  PLS-DA  model  based  on  five  flavour 
groups with independent samples...................................................................... 17 

Table 5: Cross-validation of the PLS-DA model based on two flavour groups ............... 17 

Table  6:  Individual  results  from  validation  of  the  PLS-DA  model  based  on  two  flavour 
groups with independent samples...................................................................... 18 

Table 7: Accuracy table for cross validation of decision tree model ............................ 18 

Table 8: Validation of decision tree model with independent samples ......................... 19 

56 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
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