Inhalation Toxicology

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Comparison of the content of tobacco alkaloids

and tobacco-specific nitrosamines in ‘heat-not-
burn’ tobacco products before and after aerosol
generation

Won Tae Jeong, Hyun Ki Cho, Hyung Ryeol Lee, Ki Hoon Song & Heung Bin
Lim

To cite this article: Won Tae Jeong, Hyun Ki Cho, Hyung Ryeol Lee, Ki Hoon Song & Heung
Bin Lim (2018) Comparison of the content of tobacco alkaloids and tobacco-specific nitrosamines
in ‘heat-not-burn’ tobacco products before and after aerosol generation, Inhalation Toxicology,
30:13-14, 527-533, DOI: 10.1080/08958378.2019.1572840

To link to this article: https://doi.org/10.1080/08958378.2019.1572840

Published online: 11 Feb 2019.

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INHALATION TOXICOLOGY
2018, VOL. 30, NOS. 13-14, 527–533
https://doi.org/10.1080/08958378.2019.1572840

RESEARCH ARTICLE

Comparison of the content of tobacco alkaloids and tobacco-specific

nitrosamines in ‘heat-not-burn’ tobacco products before and after
aerosol generation
Won Tae Jeong, Hyun Ki Cho, Hyung Ryeol Lee, Ki Hoon Song and Heung Bin Lim
Department of Industrial Plant Science & Technology, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea

ABSTRACT ARTICLE HISTORY

Standardized methods for collecting smoke and for measuring smoke components in heat not burn Received 9 November 2018
tobacco product (HTP) are yet to be established, and there is a lack of consensus as to whether the Revised 14 January 2019
content of HTP cigarettes can be assayed in the same manner as for conventional cigarettes. Since Accepted 17 January 2019
HTPcigarettes do not generate ash when smoked, we compared the levels of tobacco alkaloids (TAs)
KEYWORDS
and tobacco-specific nitrosamines (TSNAs) of HTP cigarettes before and after aerosol generation. HTP Heat not burn product;
cigarettes were smoked according to two international standardization methods. The TAs and TSNAs tobacco alkaloids; tobacco-
contents of the cigarettes were analyzed by UPLC-Q-TOF and UPLC-MSMS, respectively. Smoking was specific nitrosamine
found to significantly decrease the content of nicotine, nornicotine, anatabine, and anabasine by
53
100% in all samples, and the maximum inhalable amounts of these entities were determined to
be 4.24 mg/cig, 103.52 lg/cig, 258.72 lg/cig, and 33.03 lg/cig, respectively. By contrast, smoking sig-
nificantly increased the content of NNK and NAB. we suggested that the reduced nicotine content
minus the nicotine content remaining in the filter is an amount that could potentially be inhaled dur-
ing smoking. The increase of NNK and NAB in HTP cigarette after aerosol generation is expected to be
caused by the precursor, but more specific behavioral studies should be performed.

Introduction
Heat-not-burn tobacco products (HTP), which generate
aerosols produced by heating the tobacco to relatively low
temperatures without actually burning it, have recently
attracted a significant degree of public attention. In 2014,
Philip Morris International (PMI) first launched IQOS as a
new cigarette tobacco product in Japan and Italy. IQOS is
currently sold in about 30 countries, and other commercial
tobacco companies are launching similar products (Caputi
et al., 2017). Unlike liquid electronic cigarettes, they are
deemed to be similar to traditional cigarettes because of the
use of bona fide cigarette materials, resulting in throat-hit
and comparable tastes (Caputi, 2016). Hence, most smokers
are quite interested in HTP, and by October 2017 these
types of cigarettes had captured more than 13% of the mar-
ket share of all tobacco products in Japan, In Korea, HTP
accounted for more than 10% of the tobacco products sold
as of January 2018. According to Philip Morris International
(PMI) and British American Tobacco (BAT), when compar-
ing the Hoffman list of components of the mainstream
smoke of the aerosols of the HTP system with standard
tobacco (3R4F) smoke, the levels of TPM, tar, and nicotine
were similar to what is found in flammable cigarettes, but
the release of hazardous and potentially harmful substances
(HPHTs) was reduced by more than 90% while the cytotox-
icity and genotoxicity were also reported to be reduced
(Schaller et al., 2016a; Forster et al., 2018). In addition, there
have been reports that emissions of carbonyl compounds,
ammonia, TSNAs, aldehydes, and volatile organic com-
pounds (VOCs) in the tobacco smoke component are
reduced by 80% or more compared to conventional tobacco
products (El-Hellani et al., 2018; Forster et al., 2018; Li X
et al., 2018; Mallock et al., 2018; Pauwels et al., 2018;
Uchiyama et al., 2018) However, since no evaluations on
health effects from long-term use have been conducted, it is
premature to say that HTP cigarettes are less harmful than
flammable cigarettes. In addition, the measurement of a
smoke component of HTP is limited to the “Hoffmann list”,
and direct comparison errors may occur due to the influ-
ence of moisture value (Mallock et al., 2018). Investigations
of other potentially toxic components are still lacking, as
well. Therefore, various evaluations, including measurement
of harmful components in standardized HTP cigarettes
needs to be performed.
When cigarettes are burned, more than 4800 compounds
are produced and released, of which 69 have been reported
to be carcinogens (Hoffmann et al., 2001). In contrast to
other components of smoke, nicotine and nitrosamines are
inherent components of tobacco present both in tobacco

CONTACT Heung Bin Lim heungbin@chungbuk.ac.kr Department of Industrial Plant Science & Technology, Chungbuk National University, Cheongju,
Chungbuk 28644, Republic of Korea.
Supplemental data for this article can be accessed at https://doi.org/10.1080/08958378.2019.1572840.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
528 W. T. JEONG ET AL.
products and in the smoke and they are of clear relevance
to human health as they are considered to be harmful sub-
stances (Hecht, 1998; Mayer, 2014). Nicotine is a tobacco
alkaloid (TA) and related compounds such as nornicotine,
anatabine, anabasine, cotinine, and myosmine are typically
also present and they are highly addictive to humans
(Kataoka et al., 2009). Nitrosamines were first reported in
the 1970s, and 35 different entities have been reported so
far, with more than 8 entities present in tobacco and the
smoke (Nestor et al., 2014). Among them, nitrosamines pro-
duced by nitrosation of nicotine, nornicotine, anatavine, and
anabasine in tobacco are referred to as TSNAs, and N0 -
nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-
bipyridyl)-1-butanone (NNK), N0 -nitrosoanatabine (NAT),
and N0 -nitrosoanabasine (NAB) are common in addition to
being highly carcinogenic (Humans, 2007). Of note, it has
been reported that TSNAs are not present in tobacco leaves
when they are harvested in the field but are produced dur-
ing the curing process (Fisher et al., 2012), and they are
released into the smoke by distillation, pyrorelease, and
pyrosynthesis when the tobacco product is smoked
(Moldoveanu et al., 2008; Jaccard et al., 2018). As these enti-
ties are of clear relevance to the health of smokers, they are
included as toxic substances for priority step-by-step moni-
toring as specified by the World Health Organization
Framework Convention on Tobacco Control (WHO FCTC)
(Hoffmann et al., 2001).
Likewise, the presence of nicotine and TSNAs in fillers
and mainstream smoke in HTP cigarettes have been identi-
fied and reported (Schaller et al., 2016b; Forster et al.,
2018). Unlike conventional cigarettes, the filler of HTP also
exists after aerosol generation. Hence, we were interested in
whether it was possible to predict the amount of inhalation
by analyzing the content of TAs (nicotine, nornicotine, ana-
tabine, anabasine, cotinine, and myosmine) and TSNA con-
tained in electronic cigarettes before and after aerosol
generation. Therefore, in this study, we measured the nico-
tine and TSNAs content of HTP fillers both before and after
use, and discussed whether the amount inhaled could be
estimated by differences in the results. To the best of our
knowledge, this is the first attempt.
Methods
Chemicals and materials
Ammonium formate, 26
30% ammonia solution
(NH4OH), and acetic acid were purchased from Sigma-
Aldrich. Methanol, acetonitrile, and water (all of these sol-
vents were HPLC grade) were purchased from Merck
(Darmstadt, Germany). NNN, NNK, NAT, NAB, NNN-d4,
NNK-d4, NAT-d4, NAB-d4 nicotine, nicotine-d4, nornico-
tine, anabasine, anatabine, cotinine, and myosmine were
purchased from Toronto Research Chemicals (Toronto,
Canada). University of Kentucky research cigarette 3R4F
and CORESTA Approved Monitor No. 7 (CM7) were used
as conventional cigarettes, and the HNB products were
regular cigarettes (and their corresponding heating devices)
as sold by three companies in Korea. 3R4F and CM7 were
purchased from Kentucky Tobacco Research and
Development Center (Lexington, USA) and Borgwaldt
(Heini GmbH, Germany), respectively. All of the HNB
products were purchased at local outlets in Korea. All of the
samples were conditioned for two days at 22  C and 60%
humidity according to International Organization for
Standardization (ISO) 3402 prior to smoking and extraction.
Smoking machine condition
HTP cigarette smoking was carried out with a Heinr
Borgwaldt (Heini GmbH, Germany) LX20 linear smoking
device according to ISO (International Organization for
Standardization) 3308:2000 and Health Canada Intense
(HCI) T-115 smoking method. After the smoking cigarette
was inserted into the heating device, the heating button was
activated and the smoking started when the lamp was fully
lit. Under ISO regime, the HTP devices were smoked with a
35 mL puff volume every 60 s, a puff duration of 2 s, with
no blocking of filter ventilation. Under HCI regime, the
HTP devices were smoked with a 55 mL puff volume every
30 s, a 2-s puff duration, with 100% blocking of filter venti-
lation. The puff number of the mainstream smoke of manu-
facturing goods I (MG I), manufacturing goods II (MG II),
and manufacturing goods III (MG III) were set as 12 times,
10 times, and 8 times, respectively.
Standard and sample preparation procedure
TAs and nicotine-d4 were dissolved in methanol and diluted
to prepare an intermediate stock solution (nicotine 1000 lg/
mL, the other TAs 100 lg/mL). The calibration curves of
TAs were prepared in the range of 1–50 lg/mL for nicotine
and 0.01–5 lg/mL for nornicotine, anatabine, anabasine,
cotinine, and myosmine. TSNAs and their internal stand-
ards were dissolved in acetonitrile and mixed solutions con-
sisting of 50 lg/mL NNN, NNK, NAT, and 10 lg/mL NAB
were prepared. The calibration curves of TSNAs were pre-
pared from 0.5–80 ng/mL for NNN, NNK, and NAT, and
from 0.25–20 ng/mL for NAB. The extraction of TAs and
TSNAs from samples was performed with reference to
CORESTA Recommended Method No. 72 and Li et al.
(2015). Briefly, the powdered sample (0.3 g) was accurately
dispensed and transferred to an amber triangular flask.
Ammonium acetate (12 mL of a 100 mM solution), 60 lL of
nicotine-d4 (1000 lg/mL), and 12 lL of TSNAs deuterated
standard mixture solution (50 lg/mL for NNN-d4, NNK-d4,
NAT-d4, and 10 lg/mL for NAB) as internal standards were
added and the sample was shaken at 200 rpm for one hour.
The extracts were filtered through a membrane filter with a
0.22 lm pore size and 100 lL of extract was transferred to a
2 mL vial for TA analysis and diluted 10 times with metha-
nol. The extracts were purified using an Oasis HLB 60 mg
cartridge (Waters) for the analysis of TSANs. The cartridge
was first conditioned with 4 mL of methanol and 4 mL of
distilled water, and then 4 mL of extraction solution was
loaded (after being adjusted to pH 9.0–9.5 with ammonia
solution). The cartridge was washed with 4 mL of washing

solution (95% water with 5% methanol and 0.5% ammonia
solution) and then washed with 4 mL of 0.01% formic acid
in water. Analytes and their corresponding internal stand-
ards were eluted with 4 mL of methanol containing 30%
water and 0.1% acetic acid. The eluent was transferred into
a 2 mL vial. The content of the vials was subjected to
UPLC-Q-TOF and UPLC-MS/MS, respectively.
Analytical condition for TAs and TSNAs
TAs and TSNAs were analyzed using a Waters ACQUITY
ultra performance liquid chromatography system coupled
with a Waters Xevo G2 Q-TOF and MS/MS. Separation of
TAs was carried out using a Waters BEH C18 column
(Waters Corporation, Milford, MA, USA) (2.1  100 mm,
1.7 lm) and the mobile phase was eluted with 20 mM
ammonium formate (A) pH 8.7 adjusted with ammonia
solution, and with acetonitrile (B). The temperature of the
column was set to 40  C, while the flow rate was set at
0.3 mL/min, and the sample injection amount was set to
1 lL. The contents of the mobile phase were varied as fol-
lows: 10–15% B from 0 to 3 min; 15–22% B from 3 to
5 min; 22–40% B from 5 to 10 min; 40–50% B from 10 to
13 min; 50–70% B 13 to 15 min; run at 10% B for 3 min.
Separation of TSNAs was carried out using a Waters BEH
C18 column (2.1  50 mm, 1.7 lm) and the mobile phase
was composed of water containing 0.1% acetic acid (A) and
methanol containing 0.1% acetic acid (B). The temperature
of the column was set to 40  C, while the flow rate was set
at 0.4 mL/min, and the sample injection amount was set to
5 lL. The contents of the mobile phase were varied as fol-
lows: 2–98% B from 0 to 4.5 min; after maintaining this for
0.5 min, the B mobile phase returned to its initial condition.
For the mass detector, the capillary voltage and the cone
voltage were set at 2.8 kV and 18 V, respectively, in the posi-
tive mode. The cone gas and the desolvation gas were set to
50 L/h and 550 L/h, respectively, and the source and desolva-
tion temperatures were set at 150  C and 500  C, respect-
ively. TAs were performed in the MSe mode, the low
collision energy was set at 6 eV and the high collision
energy was set at 30–50 eV. TSNAs and their internal stand-
ards were monitored for quantitative analysis in multiple
reaction monitoring (MRM).
Statistics
The results are expressed as means ± the standard deviation
(SD) and were subjected to analysis of variance using SPSS
Table 1. The content of six TAs in general and HNB cigarettes before aerosol generation by UPLC-Q-TOF.
Parameters 3R4F CM7
Cigarette weight (g) 0.79 0.81
Nicotine 23.76 ± 1.02 23.72 ± 0.32
Nornicotine 654.27 ± 47.61 664.33 ± 24.03
Anatabine 869.60 ± 28.07 971.17 ± 13.58
Anabasine 133.52 ± 7.16 150.26 ± 4.37
Cotine 87.96 ± 4.96 11.28 ± 2.53
Myosmine 26.69 ± 0.34 N.D
The unit of nicotine is mg/g, and the other TAs are lg/g.
INHALATION TOXICOLOGY 529
12.0 software (SPSS Inc., Chicago, IL, USA). Differences
between sample means were determined to be statistically
significant at p < .05 using the Duncan’s multiple compari-
son test. The TA and TSNA analyses were performed in
triplicate and as six replicates, respectively.
Results
TA content of tobacco in conventional and
HTP cigarette
Table 1 shows the content of TAs measured in two types of
conventional cigarettes and three types of HTP cigarettes
(manufacturing goods I, II and III). Nicotine, nornicotine,
anatabine, and anabasine were detected in both the conven-
tional and the HTP cigarettes, but cotinine was detected
only in the conventional cigarettes while myosmine was not
detected in the CM7 cigarettes only. The four most abun-
dant alkaloids in all of the samples were nicotine, followed
by anatabine, nornicotine, and anabasine. When comparing
the contents calculated by weight of each alkaloid of con-
ventional versus HTP cigarettes, the content of myosmine in
the HTP cigarettes was much higher than in 3R4F cigarettes.
Moreover, for the HTP cigarettes, the contents of nicotine,
nornicotine, anatabine, and anabasine were lowest in MG
III, and contained 58%, 68%, 35%, and 68% less, respectively
than 3R4F cigarettes. Even though the contents of the four
alkaloids in MG I and II versus conventional cigarettes did
not differ significantly, the amount that can be inhaled is
presumably small since the weight per cigarette is low.
Comparison of the TA content in HTP cigarettes before
and after aerosol generation
The amounts of TAs in the three types of HTP cigarettes
after aerosol generation under the ISO and HCI regimes are
shown in Table 2. The content of nicotine, nornicotine, ana-
tabine, and anabasine in MG I was significantly lowered to
a maximum of 17.34 mg/g, 431.33 lg/g, 908.51 lg/g, and
115.13 lg/g, respectively, after aerosol generation. The con-
tent of nicotine, nornicotine, anatavine, and anabasin in
MG II was significantly lowered to a maximum of 19.28 mg/
g, 430.97 lg/g, 1176.02 lg/g, and 150.12 lg/g, respectively,
after aerosol generation. Especially, alkaloids were further
reduced under HCI than ISO regime. In the case of MG III,
the nicotine content pattern was similar to that of MG I
and II, but the other alkaloids were all reduced irrespective
of the smoking conditions. Overall, the content of TAs in
the HTP cigarettes after aerosol generation was clearly
Manufacture Goods I Manufacture Goods II Manufacture Goods III
0.31 0.30 0.27
22.31 ± 0.33 25.97 ± 1.18 10.10 ± 0.26
468.39 ± 10.13 503.40 ± 30.98 212.73 ± 1.95
1012.60 ± 27.15 1378.05 ± 70.96 572.10 ± 4.21
129.78 ± 5.45 184.24 ± 18.25 42.76 ± 0.36
N.D N.D N.D
404.71 ± 5.76 400.88 ± 4.38 370.73 ± 2.89
 530 W. T. JEONG ET AL.

reduced and it decreased further when smoked under the

2880.86
F-value

4.20NS
HCI regimen compared to the ISO. We estimated that the

–
–
–
–
reduced amount of alkaloids could be converted
into aerosols.

366.95 ± 1.03bc
0.26 ± 0.01c
0.21

N.D
N.D
N.D
N.D
HCI

The TSNA content of tobacco in conventional and

After smoking

HTP cigarettes
Manufacture good III

376.81 ± 6.57ab Table 3 shows the content of TSNAs measured in two types
0.75 ± 0.17b

of conventional cigarettes and three types of HTP cigarettes.

0.21

N.D
N.D
N.D
N.D
ISO

Although the TSNAs NNN, NNK, NAT, and NAB, were

detected in all of the samples, the content of TSNAs in
3R4F was higher than that of all of the HTP cigarettes.
Before smoking

10.10 ± 0.26a

370.73 ± 2.89a
212.73 ± 1.95
572.10 ± 4.21
42.76 ± 0.36

Except for MG I cigarettes, in all of the samples were the

0.27

most abundant TSNA was NAT, followed by NNN, NNK,

N.D

and NAB. By contrast, MG I had the highest content of

NNN and was higher than CM7 in this regard.
Interestingly, despite the similar levels of TAs in conven-
267.43
379.98
394.83
112.10
F-value

11.08

tional and HNB cigarettes, especially for 3R4F, for MG I

–

and MG II, there was a clear difference between the levels

Table 2. The content and parameters of six TAs in three HNB cigarettes before and after aerosol generation (under ISO and HCI) by UPLC-Q-TOF.

of TSNAs. The results indicate that the content of TAs in

411.94 ± 14.09ac
202.03 ± 40.42c
6.69 ± 0.09c
72.43 ± 2.16c

34.12 ± 3.27c

HTP cigarettes before smoking was not proportional to the

0.23

N.D
HCI

content of TSNAs.
After smoking
Manufacture good II

Comparison of the TSNA content in HTP cigarettes

128.88 ± 18.31b
338.26 ± 52.57b
72.60 ± 12.01b

446.47 ± 15.53b
11.74 ± 1.40b

before and after aerosol generation

0.23

N.D
ISO

The levels of TSNAs of the three types of HNB cigarettes

after aerosol generation under the ISO and HCI regimes are
shown in Table 4. When NNN, NNK, NAT, and NAB were
503.40 ± 30.98a
1378.05 ± 70.96a
184.24 ± 18.25a
Before smoking

compared before and after aerosol generation of three dif-

25.97 ± 1.18a

400.88 ± 4.38a

ferent HTP cigarettes, the content of NNN was found to be

0.30

N.D

81.02 ng/g in MG III cigarettes smoked according to HCI

regime, which was significantly lower than before smoking.
The content of NAT of MG II smoked according to ISO
147.00
450.39
438.86
115.90

5.87NS
F-value

and HCI regimes increased by 682.27 ng/g and 437.43 ng/g,

–

respectively, compared to before smoking, and by contrast,

the NAT content of MG III smoked according to ISO and
104.095 ± 11.18c

406.34 ± 9.77ac

HCI regimes was significantly lower, being reduced by

4.97 ± 0.52c
37.063 ± 8.24c

14.654 ± 4.32c

Different letter in the same row (a–c) is significantly different at p < .05.

221.72 ng/g and 267.40 ng/g, respectively.

0.24

N.D
HCI

The levels of NNK and NAB in all of the HTP cigarette

After smoking

types were significantly increased after aerosol generation

Significant at p < .05
p < .01 and p < .001, respectively.

compared to before smoking, and the content of the smoked

Manufacture good I

The unit of nicotine is mg/g, and the other TAs are ng/g.
93.099 ± 30.47b
232.67 ± 63.99b
40.782 ± 15.32b

430.12 ± 13.48b

cigarettes was significantly higher under the ISO regime

10.58 ± 2.10b

than under the HCI regime. The difference in the content

0.24

N.D
ISO

of NNK between ISO and HCI was 38.42 ng/g (9.22 ng/cig)
in MG I, 6.99 ng/g (1.60 ng/cig) in MG II, and 78.18 ng/g
(16.42 ng/cig) in MG III, while for the content of NAB it
468.39 ± 10.13a
1012.60 ± 27.15a
Before smoking

22.31 ± 0.33a

129.78 ± 5.45a

404.71 ± 5.76a

was 34.78 ng/g (8.34 ng/cig) in MG I, 25.41 ng/g (5.59 ng/

cig) in MG II, and 48.70 ng/g (10.23 ng/cig) in MG III.
0.31

N.D

These results indicated that the production of TSNAs from

TAs by heat increased and that the amount of TSNAs pro-
Values are mean ± SD.

duced was different according to the smoking conditions.

Cigarette Weight (g)

NS: not significant.

Discussion
Nornicotine
Parameters

Anabasine

Myosmine
Anatabine
Nicotine

Cotine

Nicotine is generally the most abundant alkaloid in tobacco

leaves, but tobacco alkaloids such as nornicotine, anatabine,

Table 3. The content of four TSNAs in general and HNB cigarettes before aerosol generation by UPLC-MSMS.
Parameters 3R4F CM7
NNN 2238.06 ± 722.22 358.34 ± 93.76
NNK 663.67 ± 115.82 201.41 ± 19.83
NAT 2478.13 ± 155.87 394.96 ± 58.81
NAB 122.39 ± 35.49 66.09 ± 5.81
The unit of all compounds is ng/g.
anabasine, cotinine, and myosmine coexist in trace amounts
(Matsushima et al., 2014; Tassew & Chandravanshi, 2015).
In our study result, four alkaloids (nicotine, nornicotine,
anatabine, and anabasine) in all of HTP cigarettes were sig-
nificantly decreased after aerosol generation. We regarded
first this reduced amount as a potential amount of inhal-
ation that could be inhaled by smokers. Nicotine and
other alkaloids are released from tobacco heated above
140  C (Rowell & Tarran, 2015). Because the HTP heating
range is between 200  C and 350  C, alkaloids in the sample
can be seen as emissions due to the conversion of aerosols
(Forster et al., 2018). Myosmine, in particular, has been
reported to undergo less conversion to aerosols than other
alkaloids (Liu et al., 2017). As a difference in the two
regimes (ISO and HCI), four alkaloids showed more reduc-
tion in samples smoked under the HCI regime. HCI regime
can detect more chemical components of aerosols in con-
ventional tobacco and HTP because the parameters of the
smoking machine, such as puff volume, puff frequency, and
puff duration, are higher than ISO (Purkis et al., 2012).
These results show that smoking according to an HCI
regime was associated with a larger decrease in TAs in
our results.
Recently, Mallock et al. (2018) and Bekki et al. (2017)
reported that the nicotine content was measured at 1.1 mg/
cig in the aerosol of HTP collected under the HCI regime.
According to Li et al. (2018), the nicotine content of IQOS
aerosols smoked under the ISO and HCI regimes was meas-
ured as 0.5 mg/cig and 1.35 mg/cig, respectively. The PMI
and BAT were published in a peer-reviewed document,
where the nicotine content of HTP aerosols was 1.35 mg/cig
and 0.3 mg/cig, respectively (Schaller et al., 2016b; Forster
et al., 2018). When these results were compared to reduced
nicotine content after aerosol generation in our study, our
calculated values was approximately three times higher. In
order to find the cause of these concentration differences,
we analyzed the mouthpiece filter, which has a high possi-
bility of containing residual alkaloids and investigated the
residual amount. As a result (Table S1), the nicotine meas-
ured by the ISO regulation was 1.21–1.52 mg/cig, HCI was
1.01–1.85 mg/cig, and the other alkaloids were present in
trace amounts. Despite the exclusion of nicotine which
remains in the filter, our figures are still high. However, it is
necessary to consider such factors as the minute smoke loss
and the remaining concentration of the heating device, and
if the behavior is understood, it will be possible to calculate
the intake amount more accurately.
TSNAs in tobacco leaves are generally produced by nitro-
sation of TAs and the curing process, and they may exhibit
differences in terms of the content depending on the
tobacco variety and cultivation environment (e.g., the nitrate
INHALATION TOXICOLOGY 531
Manufacture Goods I Manufacture Goods II Manufacture Goods III
573.77 ± 52.08 200.84 ± 50.37 234.07 ± 31.10
191.94 ± 9.40 67.15 ± 14.65 110.02 ± 7.59
525.39 ± 45.33 325.39 ± 66.83 431.04 ± 50.54
48.40 ± 11.04 19.14 ± 8.88 28.14 ± 9.30
concentration) (Fisher et al., 2012). Recently, the selection
of tobacco cultivars with low nornicotine and nicotine con-
tents, which are precursors of NNN and NNK in tobacco
leaves, and a change in the curing process have resulted in a
lowering of the level of TSNAs (Hecht & Hoffmann, 1988;
Appleton et al., 2013). Like nicotine, they can also be
released by heat during smoking (Jaccard et al., 2018).
Unlike our expectations, NNK and NAB were significantly
increased after aerosol generation, but NNN and NAT
showed no significant pattern or dependence on smoking
conditions. Although estimation of the inhaled amount
according to the content of these components was unclear,
the understanding of the reduction and increase of TSNAs
after aerosol generation has been obtained, in part, from
various literature, including those published by
tobacco companies.
Moldoveanu et al. has intentionally added 2H4-nicotine
and 2H4-noricotine to the cigarette, and then analyzed the
2
H4-TSNAs formed in the smoke by LC-MS/MS, suggesting
that only NNK can be partially converted (Moldoveanu
et al., 2008). Kim and Shin suggested that TSNAs com-
pounds could also be formed from the heating of E-ciga-
rettes (Kim & Shin, 2013). Thus, the increase in NNK and
NAB in HTP after aerosol emission may be a contribution
of the reduced their precursor (Nicotine and anabasine).
Similarly, amine compounds in foods can form N-nitrosa-
mine compounds with sodium nitrite by thermal chemical
reactions (Herrmann et al., 2015). However, the literature
on the chemical composition of HTP and the thermal
behavior of NAB has not been published yet, and further
research is needed.
According to Lipowicz and Seeman (Lipowicz & Seeman,
2017), the transfer of NNN from cigarettes to smoke was
suggested to occur by distillation, and this could explain our
results where only NNN of MG III smoked under the HCI
regime was significantly reduced. If evidently conveyed by
distillation without any other effect, the delivery will depend
on the type of heating device and cigarette. MG I’s heating
system uses a method in which cigarettes are inserted into
the apparatus and heated simultaneously by heating blades
(Smith et al., 2016). On the other hand, the heating system
of MG III does not have a heating blade, and instead, the
whole apparatus is heated (Murphy et al., 2018). In addition,
MG III cigarettes are thinner than MG I cigarettes and have
a structure that is advantageous for distillation because they
have a large contact area with the heating device. Hence,
the decrease of NNN in MG III was thought to be affected
by the type of heating device and tobacco. Unfortunately,
the study of the behavior of NAT due to smoking is lacking,
and no published article was available to provide informa-
tion on the device of the MG II. In conclusion, the study of
 532 W. T. JEONG ET AL.

behavior in TSNAs during smoking should be studied more

383.70
75.99
62.56
5.67NS
F-value
accurately by using a substance such as standard exchanged
deuterium materials.

153.05 ± 37.19bc

163.64 ± 33.83b
54.28 ± 10.09c
208.79 ± 7.31c Conclusions
HCI
After smoking

In our study, we provided the possibility to estimate the

expected amount of inhalation by comparing nicotine con-
Manufacture good III

tent before and after aerosol generation without capturing

203.17 ± 45.56ab
286.97 ± 14.01b
209.32 ± 18.43b
102.98 ± 12.55b

HTP smoke. Although the content of TSNAs is difficult to

ISO

predict in the same way as nicotine due to its tendency to

increase, it suggests that TSNAs can be increased through
the reduction of precursors. Of course, further study on the
exact behavior of TAs and TSNAs is needed. Nevertheless,
Before smoking
234.07 ± 31.10a

431.04 ± 50.54a
110.02 ± 7.59a

28.14 ± 9.30a

our attempts would be able to respond to the system charac-

teristics of different heating devices by tobacco companies
and evaluate individual smoking patterns when smoking
HTP product.
106.51
451.39
95.50
Table 4. The content and parameters of four TSNAs in three HNB cigarettes before and after aerosol generation (under ISO and HCI) by UPLC-MSMS.

0.48NS
F-value

Acknowledgments
We are grateful to Ministry of Food and Drug Safety in Korea for pro-
233.61 ± 56.83a

762.82 ± 41.82c
103.31 ± 11.49c
164.50 ± 9.62bc

viding tobacco sample.

HCI
After smoking

Disclosure statement
Manufacture good II

171.48 ± 12.25b
1007.66 ± 45.02b
128.72 ± 16.81b

The authors declare there is no conflict of interest regarding the publi-

211.10 ± 80.95a

cation of this paper.

ISO

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