1.

Introduction

Electronic cigarettes (e-cigarettes) are designed to transfer mix- tures of

air and vapors into the respiratory system [13]. They use plastic or metal
cylinders that contain electronic vaporization systems, a battery, in some
cases, a charger, electronic controls and, optionally, replaceable cartridges.
Different humectants, e.g. propylene glycol or glycerin, flavorings and
nicotine at various con- centrations are generally contained in the cartridges.
They can be disposable (Type 1 e-cigarette) or rechargeable (Type 2 e-
cigarette). Concern has been raised for the compounds incorporated into
smokers as consequence of e-cigarette vaping.
Exhaled breath, namely the alveolar breath [4], may provide sig-
nificant clues on the compounds that are retained in humans as
consequence of this activity. Studies on VOCs in exhaled breath from
e-cigarette smokers have been developed using solid phase
microextraction inside a breath collection device [5] or exposure
chambers which are subsequently sampled by absorption into solid
phase sorption tubes. These tubes are then analyzed by desorption into
gas chromatography coupled to mass spectrometry (GCMS) [6]. In
other cases, the absorption cartridge has been installed at the outlet of a
smoking machine and the retained compounds are eluted with CS2 and
methanol for subsequent analysis by GCMS [7].
In the present study, we describe a simplified method using a Bio-
VOCs exhaled air sampler developed by the UK Health and Safety
Laboratory (Markes International Ltd, Llantrisant, UK) for the
comparison of the smoke generated by Type 1 and Type 2 e-cigarettes,
tobacco cigarettes and the exhaled breath after vap- ing or smoking. This
device has been used in the analysis of both exhaled alveolar air and
mouth air [815]. Now, we are using BIO-VOCs for a rapid method of
characterization of the volatile organic constituents in tobacco cigarettes
and e-cigarettes. Blend type American tobacco cigarettes with filters
(length 83 mm, length of filter 23 mm, diameter 8 mm) were used as
test examples. Cigarettes with low nicotine content (0.6 mg), low tar (8
mg) and low carbon monoxide (9 mg) were chosen. The compounds ana-
lyzed in the present study were mostly in the gas phase. The results add to
the current knowledge of exposure of smokers to organic compounds that
so far have been mostly characterized in particu- late phase transfer
processes [1623].

1. Experimental

1.1. Sampling cartridges

 Volatile organic compounds were concentrated by sorption into stainless
steel sorbent cartridges (89 mm long 0.64 cm outer diameter) packed with
200 mg of Tenax TA 35/60 mesh (Markes International Ltd, Pontyclun,
UK). The sorbent cartridges were pre- conditioned using helium (5N
grade; 100 ml/min) at 320 C for 2 h and then at 335 C for 30 min. In
later conditioning cycles these car- tridges were reconditioned at 335 C
for 20 min with the same flow carrier gas. Once cleaned, the cartridges
were sealed with brass Swagelock storage endcaps fitted with PTFE
ferrules and stored in solvent-free clean environments.

1.1. Sampling

Exhaled breath was sampled with a Bio-VOC system 30 min after

tobacco cigarette or e-cigarette smoking. To avoid metabolic differences
all volunteers were asked to smoke with the tobacco cigarettes and Type 1
and 2 e-cigarettes considered in this study. People inspired and expired
deeply three times, then retained the breath for 20 s and blew into the Bio-
VOC body through a disposable cardboard mouthpiece at their highest
capacity. The air remaining in the Bio-VOC was transferred into the
sorbent cartridge by push- ing a screw-in plunger through the Bio-VOC
body. This procedure was repeated five times in each smoking test and all
exhaled VOCs were accumulated in the same cartridge. Thus, a total
volume of 750 mL of exhaled breath was collected.
Tobacco cigarette and e-cigarette smoke were sampled by con- necting
the mouth outlets to the Bio-VOC outlet. The screw-in plunger was used
to pull smoke into the Bio-VOC cylinder. Then, the tobacco cigarette or e-
cigarettes were removed and the cartridge was connected to the Bio-VOC
outlet and the screw-in pluger was used to push the smoke present in the
Bio-VOC into the cartridge which sorbed the VOCs from the sample. The
sampled volume with this procedure was 150 mL.
Indoor ambient air was also sampled for comparison using this device.
The procedure was the same as that used for tobacco cigarette and e-
cigarette smoke but without connecting any of those devices to the
sorbent cartridge. In this case the procedure was repeated four times and a
total volume of 600 mL was collected.

1.2. Transfer of the VOC into the GCMS

VOCs trapped in the sorbent cartridges were transferred with helium

(5N grade; no inlet split flow) to a thermal desorption (TD) instrument
equipped with a Unity Series 2 Thermal Desorber and an Ultra 50:50
Multi-tube Auto-sampler (Markes International Ltd). The compounds
were desorbed from the cartridges at 300 C for 5 min (desorption flow 40
mL/min) and re-concentrated in a graphitized carbon sorbent cold trap (U-
T11GPC-2S for General
Purpose; Markes International Ltd) cooled at 20 C. This cold trap was
heated to 300 C over 5 min while passing a helium flow of
7.5 ml/min (split flow 6 ml/min) for VOC transfer to an uncoated and
deactivated fused-silica capillary transfer line of 1 m length (inter- nal and
outer diameters 0.25 and 0.35 mm, respectively) heated at 200 C. Total
split ratio was 5:1.
For the Type 2 e-cigarette analyses, inlet split flow during car- tridge
desorption was 50 mL/min and desorption trap conditions operated at a
carrier helium flow of 28.5 mL/min and an outlet split flow of 27 mL/min.
Total split ratio was 95:1.

1.1. GCMS operational conditions

The transfer line introduced the compounds into a Gas Chro- matograph
7890 (GC; Agilent Technologies Inc., Santa Clara, CA) coupled to a Mass
Spectrometer 5975C Inert XL MSD. The GC was equipped with a DB-
5MS UI capillary column (length 60 m; internal diameter 0.32 mm; film
thickness 1 m; Agilent J&W GC Columns). Helium (5N grade) was the
carrier gas at a flow of 1.5 ml/min (con- stant flow mode). The GC oven
temperature program started at 40 C (holding time 10 min) then it
increased to 150 C at 5 C/min and to 210 C at 15 C/min (final holding
time 10 min).
A transfer line heated to 280 C carried the compounds from the GC to
the MS. The MS source and quadrupol temperatures were 230 C and 150
C, respectively. The MS operated in electron impact mode. The detector
was full scanned between 30 and 380 amu.

1.2. Compound identification and quantification

VOCs were identified based on retention times and library iden-

tification of the mass spectrum from each chromatographic peak
(NIST2009, Mass Spectral Search Program, version 2.0f). Quantifi-
cation was performed by the external standard method.
Calibration curves encompassed nine calibration solutions in methanol
(Merck KGaA, Darmstadt, Germany) at different con- centration in the
range between 0.5 and 200 g/ml. They were prepared from commercial
solutions: UST Modified Gasoline Range Organics (1000 g/ml in
methanol; Supelco, Inc. Bellefonte, PA, USA), FIA Paraffin Standard
(Accustandard Inc., New Haven, CT), and the individual standards: 2-
methylbutane, 1-pentene, cis-2- pentene, trans-2-pentene and 4-methyl-1-
pentene, all grade GC Standard (Sigma-Aldrich Co., St. Louis, Mo).
 A Calibration Solution Loading Ring (CSLRTM, Markes Interna- tional
Ltd., Llantrisant, UK) was used to introduce the calibration solution into
clean sorbent cartridges which allowed controlled vaporization and
purging of the solvent (carrier gas flow at 50 ml/min during 3 min).
The different standard solutions were directly introduced into the
cartridges which were subsequently analyzed in the TD-GCMS. This
allowed the determination of lin- ear concentration ranges and limits of
detection. Recoveries were determined by introduction of standard
solutions into the Bio-VOCs heated at 50 C. Repetitivity was also
determined by sequential analysis of standards introduced into the Bio-
VOCs.
\
2. Results and discussion
2.1. Exhaled breath and air concentrations

The gas chromatograms corresponding to indoor air from a building

of Barcelona and exhaled breath of volunteers present in this indoor
environment without smoking are compared in Fig. 1. Compound
identification is reported in Table 1. Acetone and isoprene were the
main compounds in exhaled breath. These are two endogenous
compounds usually present in this type of sample. Both
chromatograms also had some common peaks such as benzene,
toluene, styrene, benzaldehyde, -limonene, decanal, nonanoic acid, and
a siloxane series. Benzene and toluene may con- stitute trace amounts
of vehicular exhaust in the area. The siloxane series may represent
some background input of the analytical sys- tem. The other
compounds may reflect a relationship between in-door atmospheric
VOCs and exhaled breath of residents in this environment.

2.2. Smoke from tobacco cigarettes and e-cigarettes

Representative chromatograms of the VOC in the smoke com- position

of tobacco cigarettes and Type 1 and Type 2 e-cigarettes are shown in Fig.
2. As expected a strong contrast was observed between tobacco cigarette
and e-cigarette smoke. The former con- tained a wealth of compounds
including nicotine and related products such as nicotyrine, 7-methyl-1H-
indole, myosmine, ison- icoteine. The occurrence of myosmine,
isonicoteine and nicotyrine together with nicotine in tobacco cigarette
smoke has been reported in previous studies [24,25]. 2,5-dimethylfuran is
another compound characteristic of tobacco cigarette smoke that has been
proposed as a specific marker [2529]. In the present study, this
compound was present in the chromatogram of the tobacco cigarette
smoke and absent in those of the e-cigarette smoke (Fig. 2; Table 1).
 Besides these specific compounds several aromatic compounds such as
benzene, toluene, xylenes, ethylbenzene and styrene were also found in
the chromatogram of tobacco cigarette smoke (Fig. 2). These compounds
are not specific for tobacco cigarette smoke, as several of them are found
in the BTEX mixtures associated to traffic emissions. However, as
documented elsewhere [2527,30,31], ben- zene, a known carcinogen, is
common in tobacco cigarette smoke
In this respect, the relative proportion of benzene and toluene in the
samples described in this study, 44% and 56%, respectively, is in
agreement with the relative proportion of these compounds measured in
other tobacco smoke cigarettes measured with other sampling methods,
43% and 57%, respectively [31].
Other compounds commonly related with traffic emissions were also
present in the tobacco cigarette smoke chromatogram,
E.g. n-heptane, n-octane, 1-ethyl-2-methylbenzene, 1-ethyl-3-
methylbenzene and naphthalene. The occurrence of these com- pounds in
tobacco cigarette smoke has also been reported [25, 27]. In addition to
these VOCs, many polar compounds were also represented in the tobacco
cigarette smoke chromatogram,
E.g. ethanol, acetone, acetic acid, butane-2, 3-dione, methyl ethyl
ketone, methyl furan, isovaleraldehyde, pyridine, methyl pyridine, Benz
aldehyde, phenol, benzonitrile, acetophenone. These com- pounds have
also been found in tobacco cigarette smoke in previous studies [7, 25, 26,
30, and 32]. Some aldehydes such as croton aldehyde are also identified
with this method. This compound has also been found in tobacco cigarette
smoke in analyses using the donator- phenyl hydrazine method [33].
Chromatographic peaks for several unsaturated compounds were also
found in the tobacco cigarette smoke sample, such as buta-1, 3-diene,
isoprene, hex-1-ene, hep-1-ene and -limonene.

Several of them are known natural products that can also be found in
many plant species. Their presence in tobacco cigarette smoke is
consistent with previous studies [7,25,27,30].
The analytical approach of the present study has been designed for the
identification and quantification of the volatile com- pounds. However,
some compounds found in the present study (Table 1) have also been
identified in the particulate phase in analytical methods specifically
designed for the compounds present in this phase, e.g. acetic acid,
crotonaldehyde, n-heptane, phenol, -limonene, benzoic acid,
hydroquinone, nicotine, 7- methyl-1H-indole, myosmine and nicotine [23].
These compounds are generally polar and formed by pyrolysis or
distillation of the tobacco components under the high temperature
conditions of smoking. Condensation processes lead to their distribution
between the gas and particulate phases.
In contrast, the smoke of the e-cigarettes was mainly com- posed of
propylene glycol and glycerin which is consistent with the product
description of the manufacturers (note that the chro- matographic peaks
are overloaded). In addition the smoke of the e-cigarettes contained
nicotine and related products such as mios- mine and nicotyrine. The
smoke of Type 2 e-cigarette also contained vanillin and ethyl vanillin
which were likely added as a flavor.
1.1. Exhaled breath from tobacco cigarette and e-cigarette users
2. Representative chromatograms of the VOCs in the exhaled breath of
tobacco cigarette and Type 1 and Type 2 e-cigarette users are shown in
Fig. 2. The chromatogram of exhaled breath of a tobacco cigarette
smoker showed a simplified mixture of the com- pounds found in the
previously described smoke of these cigarettes ) indicating that most of
the original smoke components were retained in the lungs. Thus, the
relative intensity of most of the higher molecular weight VOCs, those of
higher chromatographic retention time, decreased significantly. However,
some compounds that are specific of tobacco cigarette smoke such as
nicotine, nico- tyrine and 2,5-dimethylfuran were found in the exhaled
breath. Their occurrence in the VOC composition can be used to indicate
the exposure of the individuals to tobacco smoke compounds. Other
VOCs such as benzene, toluene or -limonene were less specific of
tobacco cigarette smoke but they still were dominant peaks in the
exhaled breath chromatograms of the tobacco cigarette smokers.
Isoprene was the most abundant exhaled breath peak. As men- toned
above, this is an endogenous compound.
3. In the exhaled breath of the e-cigarette smokers the chro- matographic
peaks of propylene glycol and glycerin were absent indicating that they
remained in the respiratory system of the smokers. Comparison of both
original e-cigarette smoke and exhaled breath of the e-cigarette smokers
also showed a strong decrease of the peaks corresponding to nicotine and
related com- pounds. On the other hand, two main peaks in the
chromatograms from exhaled breath were those corresponding to acetone
and isoprene which likely represent endogenous sources. In addition,
benzene, toluene and 2, 5-dimethylfuran were also found. These peaks
were below limit of detection in the e-cigarette smoke vapors. Their
occurrence in exhaled breath could reflect past expo- sures of the
volunteers.
3.1. Figures of merit
Linear concentration ranges over three magnitudes of con- centration were found for most
compounds analyzed (Table 2).
 In some cases, e.g. n-hexane, naphthalene, these ranges were about 200.
The limits of detection ranged between 0.05 and
0.65 ng. The transformation of these limits into concentration Val- use
(g/m3) must be done by reference to the sampled volume that depends on
the number of Bio-VOC replicates (N). Thus, amount detection limit (ng)
is equivalent to concentration detec- tion limit (g/m3) when multiplying
the former by 1000/ (150 N). The number of replicates in the analyses is
indicated in section The highest limits, e.g. toluene (0.65 ng), were due to
back- ground atmospheric levels by use of this compound in nearby labs.
Repeatability (residual standard deviation of ten measurements) ranged
between 5.9 and 23% which is consistent with previous measurements
with Bio-VOCs in other studies [11]. Recover- ies of standards introduced
into the Bio-VOCs and analyzed as described in the experimental section
ranged between 92 and 114% ( table2).

Quantitative differences

The concentrations of some representative VOCs found in the tobacco

cigarette and e-cigarette smoke and in the exhaled breath of the smokers
are shown in Table 3. Concentrations of the same compounds in ambient
indoor air and in volunteers breathing this air without smoking are shown
for comparison. Tobacco cigarette smoke provided the samples containing
high- est concentrations of all compounds analyzed. Besides nicotine
(1300 g/m3) it contained benzene, toluene, xylenes, ethylben- zene and
naphthalene in high abundance (1100, 1400, 1500, 660 and 240 g/m3,
respectively) as well as other compounds such as isoprene (2700 g/m3),
pent-1-ene (700 g/m3), n-pentane (1200 g/m3), n-hexane (975 g/m3), n-
heptane (1400 g/m3) and others. This composition was in strong contrast
with that of smoke from the e-cigarettes in which all these compounds
were virtu- ally absent except nicotine (710720 g/m3). Propylene glycol
and glycerin were not found in the indoor air sample.
In principle, the compositions of exhaled breath reflected the
differences of the cigarette smoke compositions (Table 3). Thus, tobacco
cigarette smoke was the one with highest nicotine con- centration and the
highest content of this compound was found in the exhaled breath after
tobacco cigarette smoking. In the cases shown in the present study, the
differences in nicotine concentra- tion between smoke and exhaled breath
were highest for tobacco cigarettes, indicating that this was the smoking
system with the highest nicotine transfer.
Isoprene is an endogenous compound and similar concentra- tions
should be expected in all exhaled breath samples. However, it was found
between 47 and 87 g/m3 in the e-cigarette smok- ers and 670 g/m3 in the
tobacco cigarette smokers (Table 3). The high concentration of this
compound in this volunteer may respond to a combination of non-
absorbed compound from the tobacco cigarette smoke and generation of
high yield of this com- pound after tobacco cigarette smoking. In fact, the
exhaled breath of the tobacco cigarette smoker shows higher
concentrations of all above mentioned compounds, including benzene,
toluene, xylenes, ethylbenzene and naphthalene, than in the other exhaled
breath samples
Conclusions

The analysis of VOCs in smoke from tobacco cigarette and e- cigarettes

and in the exhaled breath of users of these smoking systems can be
performed by collection with Bio-VOC, absorp- tion in Tenax cartridges
and analysis by TD-GCMS. This method provides consistent results
when comparing the composition of VOCs in cigarette smoke and exhaled
breath of the smokers. It also allows the discrimination between
endogenous and exogenous compounds and compounds reflecting past
exposures to pollut- ants or tobacco smoke. Comparison of the
concentrations between smoke and equivalent exhaled breath of the
smokers illustrated the incorporation of higher burdens of VOCs in the
tobacco cigarette smokers than in the e-cigarette smokers.