Nicotine & Tobacco Research Volume 8, Number 2 (April 2006) 309–313

Nicotine & Tobacco Research Volume 8, Number 2 (April 2006) 309–313

Tobacco-specific nitrosamines in new tobacco
products
Irina Stepanov, Joni Jensen, Dorothy Hatsukami, Stephen S. Hecht
[Received 18 January 2005; accepted 7 July 2005]
New tobacco products, designed to attract consumers who are concerned about the health effects of tobacco, have
been appearing on the market. Objective evaluation of these products requires, as a first step, data on their
potentially toxic constituents. Tobacco-specific nitrosamines (TSNAs) are an important class of carcinogens in
tobacco products, but virtually no data were available on their levels in these products. In the present study, we
analyzed several new products—Ariva, Stonewall, Exalt, Revel, Smokey Mountain, and Quest—for TSNAs and
compared their TSNA levels with those in nicotine replacement products and conventional smokeless tobacco and
cigarette brands. TSNAs were not detected in Smokey Mountain, which is a tobacco-free snuff product. The lowest
levels among the new products containing tobacco were in Ariva and Stonewall (0.26–0.28 mg/g wet weight of
product). The highest levels in the new products were found in Exalt (3.3 mg/g tobacco), whereas Revel and Quest
had intermediate amounts. Only trace amounts were found in nicotine replacement products, and conventional
brands had levels consistent with those reported in the literature. These results demonstrate that TSNA levels in
new tobacco products range from relatively low to comparable with those found in some conventional brands.
Introduction
Tobacco-specific nitrosamines (TSNAs) are widely
considered to be among the most important carcinogens
in smokeless tobacco products and cigarette
smoke (Bartsch & Spiegelhalder, 1996; Hecht, 1998;
Hecht & Hoffmann, 1988; Magee, 1996; Preston-
Martin & Correa, 1989). Two of these tobaccoalkaloid
derived compounds, 4-(methylnitrosamino)-
1-(3-pyridyl)-1-butanone (NNK) and N9-nitrosonornicotine
(NNN), are consistently carcinogenic in
laboratory animals, with NNK showing higher
activity (Hecht, 1998). The other two commonly
measured TSNAs are N9-nitrosoanabasine (NAB)
and N9-nitrosoanatabine (NAT). NAB is a weak
carcinogen and NAT apparently lacks activity (Hecht,
1998). NNK and NNN have recently been evaluated
by the International Agency for Research on Cancer
(2005) as carcinogenic to humans (Group 1).
In recent years, some new types of tobacco products
have been appearing on the market. Ariva is a lozenge
that contains compressed powdered tobacco and is
advertised as ‘‘the alternative for smokers in a smokefree
environment.’’ Stonewall Hard Snuff is another
compressed tobacco lozenge. Both products are made
with tobacco cured by a process that minimizes
nitrosamine formation. General and Exalt are types
of Swedish snus in which the GothiaTek process,
designed to ‘‘continuously reduce or eliminate alleged
harmful components in tobacco,’’ is used. Revel is an
American smokeless tobacco product designed for
‘‘tobacco satisfaction without smoking.’’ Smokey
Mountain is a tobacco-free and nicotine-free herbal
snuff, designed as a snuff substitute. Quest is a
cigarette available in three varieties: 1, low nicotine;
2, extra low nicotine; and 3, nicotine free. Quest
cigarettes are ‘‘the first that gradually step you to
nicotine free smoking.’’
With the exception of one review that includes
data for Ariva, Exalt, and Revel, but without
experimental details, we are aware of no published
studies on TSNA levels in these products (Rodu
& Jansson, 2004). These analyses were carried
out here, in parallel with analyses of TSNAs in
nicotine replacement products, and in conventional
ISSN 1462-2203 print/ISSN 1469-994X online # 2006 Society for Research on Nicotine and Tobacco
DOI: 10.1080/14622200500490151
Irina Stepanov, Ph.D., Stephen S. Hecht, Ph.D., The Cancer Center,
University of Minnesota, Minneapolis, MN; Joni Jensen, M.P.H.,
C.C.R.C., Dorothy Hatsukami, Ph.D., Transdisciplinary Tobacco Use
Research Center, University of Minnesota, Minneapolis, MN.
Correspondence: Stephen S. Hecht, Ph.D., The Cancer Center,
University of Minnesota, 420 Delaware Street SE – MMC 806,
Minneapolis, MN 55455, USA. Tel: +1 (612) 624-7604; Fax: +1 (612)
626-5135; E-mail: hecht002@umn.edu
Nicotine & Tobacco Research Volume 8, Number 2 (April 2006) 309–313
smokeless tobacco products and cigarette tobacco,
for comparison.
Method
Tobacco samples
The 19 brands collected for analysis represent
smokeless spit-free tobacco products, compressed
tobacco lozenges, tobacco-free herbal snuff, new
cigarettes with reduced nicotine content, nicotine
replacement therapy products, and conventional
smokeless tobacco products and cigarettes. The
products were purchased between June 2003 and
January 2005. General was ordered online from Snus
Worldwide, Sweden. Exalt was purchased from retail
stores in Washington, D.C., and Golden Valley,
Minnesota; it was also ordered online from Snus
Worldwide, Sweden (for comparison). Revel was
ordered online (houseoxford.com) and also purchased
in a convenience store in Fort Worth,
Texas. Ariva was also obtained from different
locations: A pharmacy in Belleville, Illinois; a
pharmacy in Richmond, Idaho; and a Virginia
cigarette factory outlet. Stonewall was purchased
from a tobacco shop in Minneapolis, Minnesota.
Quest cigarettes were ordered online through
smokes-spirits.com and 001Cigarettes.com; cigarettes
purchased in Richmond, Idaho, were analyzed
for comparison. Conventional moist snuff, tobaccofree
herbal snuff (Smokey Mountain), and commercial
premium cigarettes were obtained from retailers
in Minneapolis. Nicotine replacement therapy
products were purchased from a pharmacy in
Minneapolis. For 24 h prior to analysis, the tobacco
was conditioned at room temperature in a chamber
at a relative humidity of 60%.
Apparatus
Tobacco-specific nitrosamines were analyzed on a
model 5890 gas chromatograph (GC; Hewlett
Packard, Palo Alto, California) interfaced with a
model 610 thermal energy analyzer (TEA; Orion
Research, Beverly, Massachusetts). The gas chromatography
conditions were as follows: DB-1301
capillary column (30m60.32mm60.25 mm; 6%
[cyanopropylphenyl]methylpolysiloxane; J&W Scientific,
Folsom, California) and a 2m60.53mm deactivated
fused silica pre-column; flow rate 2.6 mL/min
He; splitless injection port temperature 225uC. The
following oven temperature program was used: 80uC
for 2 min, then 12uC/min to 150uC, then 7 min at
150uC, then 12uC/min to 200uC, then 10min at 200uC.
Reagents
Reference NNN, NNK, NAB, 5-methyl-N9-nitrosonornicotine
(5-McNNN), and 5-(methylnitrosamino)
-1-(3-pyridyl)-l-pentanone (C5-NNK) were synthesized
as described previously (Amin, Desai, Hecht, &
Hoffmann, 1996; Carmella, McIntee, Chen, & Hecht,
2000; Stepanov, Carmella, Hecht, & Duca, 2002).
NAT was purchased from Toronto Research
Chemicals Inc., Toronto, Ontario, Canada.
Tobacco-specific nitrosamine analyses
Moist snuff, cigarette tobacco, and lozenges. We used
a slight modification of a method described previously
(Adams, Brunnemann, & Hoffmann, 1983;
Stepanov, Hecht, Ramakrishnan, & Gupta, 2005).
Humidity-conditioned tobacco (200–500 mg) and
10mL of citrate-phosphate buffer (pH54.5) containing
ascorbic acid were added to a 30mL Nalgene
centrifuge tube (Nalge Nunc International,
Rochester, New York) to which 200 ng each of
5-MeNNN and C5-NNK internal standards
were added. The samples were homogenized for
30 s with a Polytron tissue homogenizer (Brinkmann
Instruments, Westbury, New York) and sonicated
for 1 hr. The buffer extracts were separated from the
particles of tobacco by high-speed centrifugation
(15,000 rpm, 10 min). The extracts were filtered into
50-mL glass screw-top centrifuge tubes (Kimble,
Vineland, New Jersey), and the pH was adjusted to 7
by adding 100 mL of 10N NaOH. Each sample was
applied to a 20-mL ChemElut cartridge (Varian,
Harbor City, California), eluted with 3620mL
CH2Cl2, and the eluants were combined and
concentrated to dryness with a model SVT200H
Speedvac concentrator (Thermo Savant,
Farmingdale, New York). Residues were dissolved
in 0.5mL of CH2Cl2 and further purified by solidphase
extraction using Sep-Pak Plus silica cartridges
(Waters Corp., Milford, Massachusetts), preequilibrated
with CH2Cl2. The cartridges were
washed with 5mL of CH2Cl2/ethyl acetate: 50/50,
and the TSNAs were eluted with 10mL of ethyl
acetate. The ethyl acetate eluants were concentrated
to dryness (Speedvac). The dry residues were
transferred into gas chromatography microvials with
3650 mL methanol, concentrated to dryness, and
redissolved in 100 mL of acetonitrile. Then 3 mL of the
prepared sample was injected into the GC-TEA.
Patch and gum. The patches were cut in three pieces
and, after removal of the backing, rolled into 30mL
Nalgene centrifuge tubes containing 10mL of citratephosphate
buffer (pH54.5). The gum was cut into
small pieces, added to Nalgene tubes containing
10mL of citrate phosphate buffer, and homogenized
for 30 s with a Polytron tissue homogenizer. Then
100 ng each of 5-MeNNN and C5-NNK internal
standards were added to the samples. The samples
were incubated overnight at 37uC and the next day
310 TOBACCO-SPECIFIC NITROSAMINES IN NEW TOBACCO PRODUCTS
were sonicated for 1 hr. The buffer extracts were
separated from the particles of patch or gum and
transferred into 50-mL glass screw-top centrifuge
tubes. The pH was adjusted to 7 by adding 100 mL of
10N NaOH, and each sample was extracted three
times with equal volumes of ethyl acetate. The
extracts were combined, dried with approximately
10 g of Na2SO4 and concentrated to dryness with a
Speedvac concentrator. Residues were dissolved in
0.5mL of CH2Cl2 and further purified by solid-phase
extraction and analyzed as described for tobacco
products.
Results
Table 1 summarizes levels of TSNAs in the products.
TSNAs were not detected in Smokey Mountain, a
tobacco-free snuff product. The lowest TSNA levels
among the new products that contain tobacco were
found in the compressed tobacco lozenges Ariva and
Stonewall, which had totals of 0.19 and 0.28 mg/g wet
weight of product, most of which was due to NAT.
The highest nitrosamine levels among the new
tobacco products were found in Exalt, and these
were independent of the place of purchase. TSNA
levels were relatively low in Quest cigarette tobacco,
as compared with Marlboro, Camel, Winston, and
Newport. The amounts of NNN were similar in
Quest 1, 2, and 3; the levels of the other TSNAs were
similar in Quest 1 and 2 and lower in the nicotine-free
Quest 3.
Nicotine replacement therapy products did not
contain detectable levels of NAT or NAB. Traces of
NNN were found in three out of six gum pieces taken
for analysis (0.002 mg/piece), and traces of NNK were
found in six patches analyzed (0.008 mg/patch). The
levels of TSNAs in the conventional products
represent means for two to six analyses, carried out
at different times.
We observed substantial variation in nitrosamine
content measured in the same brand at different
times (see Table 2 for some examples). The Swedish
snus General, produced in 2003, was found to
contain lower levels of TSNAs compared with that
produced in 2002. The tobacco of Marlboro and
Camel had variable levels of individual TSNAs, with
the total amount in Marlboro cigarettes generally
lower in 2004 than in 2001 (Table 2).
Discussion
Tobacco companies are trying to create alternative
smokeless tobacco products and safer cigarettes,
which would potentially reduce the risk of tobaccoattributable
cancers. A number of new brands are
being test marketed in the United States. These
products are targeted to smokers and smokeless
tobacco users who wish to reduce or quit tobacco use
or who want to use ‘‘safer’’ products. Manufacturers’
claims include statements of reduced toxin content
and implied reduced risk, but it may take years
before the real health effects of these new tobacco
products are known. TSNAs are among the most
important carcinogens in tobacco, and it is imperative
that objective data on levels of these compounds
be available.
The lowest TSNA levels in the tobacco-containing
products we analyzed were found in the compressed
tobacco lozenges Ariva and Stonewall. Levels of the
strongly carcinogenic NNN and NNK were only 56–
99 ng/g, with most of the TSNA content comprised of
NAT, which is apparently noncarcinogenic. These
products use Star Scientific specially cured tobacco
known to be low in TSNAs. The emergence of these
new products with relatively low levels of carcinogenic
TSNAs is an encouraging sign.
The Swedish snus General, which is manufactured
using the GothiaTek process and quality standard
designed to minimize nitrosamine contamination,
contained relatively low levels of TSNAs, compared
with conventional smokeless tobacco products. The
variation in TSNA content observed in General in
2002 and 2003 (Table 2) is consistent with a study by
the Swedish National Food Administration
(O¨ sterdahl, Jansson, & Paccou, 2004) that demonstrated
a noticeable decrease in TSNA content in
moist snuff on the Swedish market. However, TSNA
levels in Exalt, which is supposedly produced by the
same technology, were comparable with those in
some conventional commercial brands of smokeless
tobacco such as Copenhagen and Kodiak, which
have had relatively high amounts of these compounds
for many years (Hecht & Hoffman, 1988;
Hoffmann et al., 1995; Rodu et al., 2004). Lower
levels were found in Revel; however, these levels were
still considerably higher than nitrosamine levels in
other products such as food and beer (Bartsch &
Spiegelhalder, 1996). Based on comprehensive dietary
surveys, exposure to carcinogenic volatile nitrosamines
such as N-nitrosodimethylamine and Nnitrosopyrrolidine
is estimated to be about 1 mg/day
(Bartsch & Spiegelhalder, 1996). The same exposure
to carcinogenic TSNAs would be reached by using
just 1–2 g of Revel. One packet of Revel contains
approximately 27 g tobacco.
Quest cigarette tobacco contained substantial
amounts of NNN, which did not decrease with
reduced nicotine content. Levels of NNN and other
TSNAs in tobacco are a major determinant of smoke
levels (Fischer, Spiegelhalder, Eisenbarth, &
Preussmann, 1990). Therefore, cigarettes lacking
nicotine will still expose smokers to significant
amounts of known carcinogens. However, NNN
NICOTINE & TOBACCO RESEARCH 311
Table 1. Tobacco-specific nitrosamine levels.
Product
Tobacco-specific nitrosamine level (mg/g product wet weight)
NNN NNK NAT NAB Total
New tobacco products
Hard snuff
Ariva 0.019 0.037 0.12 0.008 0.19a
Stonewall 0.056 0.043 0.17 0.007 0.28b
Swedish snus
General 0.98 0.18 0.79 0.06 2.0c
Spit-free tobacco packets
Exalt
Purchased in Sweden 2.3 0.27 0.98 0.13 3.7d
Purchased in the United States 2.1 0.24 0.68 0.05 3.1b
Revel
Mint flavored 0.62 0.033 0.32 0.018 0.99b
Wintergreen flavored 0.64 0.032 0.31 0.017 1.0b
Tobacco-free snuff
Smokey Mountain nd nd nd nd ndb
Nicotine-reduced cigarettes
Quest 1 (low nicotine) 0.93 0.17 0.31 0.013 1.4d
Quest 2 (extra-low nicotine) 0.82 0.19 0.19 0.01 1.2d
Quest 3 (nicotine free) 0.83 0.054 0.045 0.003 0.93d
Nicotine replacement therapy products
NicoDerm CQ (patch, 4-mg nicotine)f nd 0.008 nd nd 0.008b
Nicorette (gum, 4-mg nicotine)f 0.002 nd nd nd 0.002b
Commit (lozenge, 2-mg nicotine)f nd nd nd nd ndb
Conventional tobacco products
Smokeless tobacco
Copenhagen
Snuff 2.2 0.75 1.8 0.12 4.8b
Long cut 3.9 1.6 1.9 0.13 7.5b
Skoal
Long cut straight 4.5 0.47 4.1 0.22 9.2b
Bandits 0.9 0.17 0.24 0.014 1.3b
Kodiak
Ice 2.0 0.29 0.72 0.063 3.1b
Wintergreen 2.2 0.41 1.8 0.15 4.5b
Cigarette tobacco
Marlboro
Full flavor 2.9 0.96 2.3 0.1 6.3e
Light 2.8 0.68 1.1 0.051 4.6b
Ultra-light 2.9 0.75 1.1 0.058 4.8b
Camel
Full flavor 2.5 0.90 1.7 0.091 5.2e
Light 2.7 0.55 1.3 0.061 4.6b
Ultra-light 2.8 0.77 1.2 0.055 4.8b
Winston (full flavor) 2.2 0.58 0.56 0.025 3.4b
Newport (full flavor) 1.1 0.83 1.9 0.055 3.9b
Note. NAB, N9-nitrosoanabasine; NAT, N9-nitrosoanatabine; nd, not detected; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone;
NNN, N9-nitrosonornicotine. aMean of five analyses, each performed in duplicate. bSingle analysis performed in duplicate. cMean of two
analyses, each performed in duplicate. dMean of three analyses, each performed in duplicate. eMean of four analyses, each performed
in duplicate. fValues are expressed per piece.
Table 2. Variation of tobacco-specific nitrosamine levels in some products.
Product
Date of
purchase
Number of
samples
Tobacco-specific nitrosamine level (mg/g tobacco)
NNN NNK NAT NAB Total
General 2002 2 1.2 0.28 0.93 0.076 2.5
2003 2 0.78 0.075 0.65 0.049 1.6
Marlboro (full flavor) July 2001 2 4.3 1.8 2.7 0.14 8.9
Oct. 2003 2 3.0 1.2 4.9 0.19 9.3
Oct. 2004 2 2.0 0.37 0.71 0.03 3.1
Jan. 2005 2 2.5 0.49 1.0 0.046 4.0
Camel (full flavor) July 2001 2 3.1 1.4 1.6 0.11 6.2
Oct. 2003 2 1.9 1.2 2.8 0.15 6.1
Oct. 2004 2 2.1 0.40 0.75 0.032 3.3
Jan. 2005 2 3.0 0.56 1.5 0.074 5.2
Note. NAB, N9-nitrosoanabasine; NAT, N9-nitrosoanatabine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN, N9-nitrosonornicotine.
312 TOBACCO-SPECIFIC NITROSAMINES IN NEW TOBACCO PRODUCTS
and NNK levels in Quest were substantially lower
than in currently available conventional brands.
Overall, the results of the present study demonstrate
that TSNA levels in new tobacco products
range from relatively low to comparable with those
found in some conventional brands.
Acknowledgments
This study was supported by National Institutes of Health grants CA-
81301 and DA-13333.
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