Analysis of four tobacco-specific nitrosamines in mainstream cigarette smoke of Virginia cigarettes by LC-MS/MS

Abstract

An improved method was developed for the determination of the four major tobacco-specific nitrosamines(TSNAs) in mainstream
cigarette smoke. The new method offers decreased sample preparation and analysis time as compared to traditional methods.
This method uses isotope dilution liquid chromatography coupled with a tandem mass spectrometer with electrospray ionization
and is significantly more sensitive than traditional methods. It also shows no evidence of artifactual formation of TSNAs.
Sample concentrations were determined for four TSNAs in mainstream smoke using two isotopically labeled TSNAs analogues as
internal standards. Mainstream smoke was collected on an industry standard 44-mm Cambridge filter pad, extracted with 0.1
mol/L ammonium acetate, purified by solid-phase extraction, and analyzed without further sample cleanup. The analytical column
is a 3.9 mm × 150 mm Waters Symmertry Shield RP18 column and volume fraction of the mobile phase is 50% methanol, 50% water containing 0.1% acetic acid. The results show that
the linear range is 0.5–100.0 mg/L except for N-nitrosoanabasine (NAB) from 0.25 to 50.0 mg/L. The limits of detection are
0.1 mg/L for N-nitrosonornicotine (NNN), 0.08 mg/L for 4-(methylnitrosamino)-1-(3-py-ridyl)-1-butanone (NNK), 0.05 mg/L for
N-nitrosoanatabine (NAT) and 0.06 mg/L for NAB. The recoveries of the four TSNAs are from 90.2% to 105.7%.

Full text

J. Cent. South Univ. Technol. (2008) 15: 627−631
DOI: 10.1007/s11771−008−0117−5
Analysis of four tobacco-specific nitrosamines in
mainstream cigarette smoke of Virginia cigarettes by LC- MS/MS
WU Ming-jian(吴名剑)1, 2, DAI Yun-hui(戴云辉)2, 3, TUO Su-xing(庹苏行)2,
HU Nian-nian(胡念念)2, LI Yong(李 勇)1, CHEN Xiao-ming(陈小明)1
(1. School of Chemistry and Chemical Engineering, Xiangtan University, Xiangtan 411105, China;
2. Technical Center of China Tobacco Hunan Industrial Corp. Ltd., Changde 415000, China;
3. School of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China)
Abstract: An improved method was developed for the determination of the four major tobacco-specific nitrosamines(TSNAs) in
mainstream cigarette smoke. The new method offers decreased sample preparation and analysis time as compared to traditional
methods. This method uses isotope dilution liquid chromatography coupled with a tandem mass spectrometer with electrospray
ionization and is significantly more sensitive than traditional methods. It also shows no evidence of artifactual formation of TSNAs.
Sample concentrations were determined for four TSNAs in mainstream smoke using two isotopically labeled TSNAs analogues as
internal standards. Mainstream smoke was collected on an industry standard 44-mm Cambridge filter pad, extracted with 0.1
mol/L ammonium acetate, purified by solid-phase extraction, and analyzed without further sample cleanup. The analytical column is
a 3.9 mm×150 mm Waters Symmertry Shield RP18 column and volume fraction of the mobile phase is 50% methanol, 50% water
containing 0.1% acetic acid. The results show that the linear range is 0.5−100.0 mg/L except for N-nitrosoanabasine (NAB) from
0.25 to 50.0 mg/L. The limits of detection are 0.1 mg/L for N-nitrosonornicotine (NNN), 0.08 mg/L for 4-(methylnitrosamino)-1-
(3-py-ridyl)-1-butanone (NNK), 0.05 mg/L for N-nitrosoanatabine (NAT) and 0.06 mg/L for NAB. The recoveries of the four TSNAs
are from 90.2% to 105.7%.
Key words: tobacco-specific nitrosamines; mainstream cigarette smoke; Virginia cigarette; LC-MS/MS
1 Introduction
Tobacco-specific nitrosamines(TSNAs) are known
components of tobacco products and tobacco smoke[1].
Four of the most mentioned TSNAs are N-nitrosonor-
nicotine (NNN), 4-(methylnitrosamino)-1-(3-py-ridyl)-
1-butanone (NNK), N-nitrosoanatabine (NAT) and
N-nitrosoanabasine (NAB)[2].
Because of their carcinogenic potential, it is
necessary to develop a sensitive, specific and simple
method for the determination of TSNAs. Several
methods, including gas chromatography(GC)[3−5], high-
performance liquid chromatography(HPLC)[6], gas
chromatography/mass spectrometry(GC/MS)[7] and gas
chromatography coupled with thermal energy analyzer
(TEA)[8], have been used to analyze TSNAs. Among
these methods, GC coupled with TEA which offers
reasonable sensitivity and is nitroso-specific, is the most
commonly used method. However, this technique still
has several disadvantages, such as complex sample
cleanup procedure involving liquid-liquid extractions and
solid-phase extractions and relative high limit of
detection.
A recent publication presented by WAGNER et al[9]
described the analysis of four TSNAs by LC-MS/MS.
This LC-MS/MS method offers several advantages over
the GC-TEA method. It has greater specificity,
significantly lower limits of detection, and a greater
linear dynamic range. However, this method cannot
quantitatively analyze Virginia cigarettes which are
popularly smoked by Chinese，due to their relative low
concentration of TSNAs and heavy matrix effect.
In this work, a LC-MS/MS method was developed
that overcomes the disadvantages of current methods
meanwhile takes full advantage of the specificity and
sensitivity of tandem mass spectrometry. The sample
preparation involves only extraction of Cambridge filter
pad(CFP) on an orbital shaker followed by a solid phase
extraction(SPE) cleanup. Experiments on Virginia and
Kentucky reference 2R4F research cigarettes with this
method show good results.
2 Experimental
2.1 Apparatus
Surveyor liquid chromatography coupled with a
TSQ quantum discovery max triple quadruple mass
Foundation item: Project(2005JC02) supported by China Tobacco Hunan Industrial Corporation Limited
Received date: 2008−01−29; Accepted date: 2008−03−12
Corresponding author: WU Ming-jian, Senior engineer; Tel: +86−736−7299030; E-mail: wumingjian301@sohu.com

J. Cent. South Univ. Technol. (2008) 15: 627−631
628
spectrometry operated by Xcalibur 1.4 software (Thermo
Electron Corporation, USA) was employed in this study.
SM400 linear smoking machine was manufactured by
Filtrona Instrument & Automation Ltd., UK, and SPE
instruments were manufactured by Waters Corporation
(Milford, MA, USA). Statistic analysis was performed
using statistical analysis system(SAS) software (SAS
Institute, USA).
2.2 Materials and reagents
The Kentucky reference 2R4F research cigarettes
were purchased from the University of Kentucky
(Lexington, Kentucky, USA) and other brand of
cigarettes were purchased from local supermarkets.
Cambridge filter pads(CFP) were purchased from
Borgwaldt Tecknik (Hamburg, Germany); Waters Oasis
MCX 30-mg SPE cartridges and Waters Symmetry
Shield RP18 column (3.9 mm×150 mm i.d. ×5 µm)
were purchased from Waters Corporation (Milford, MA,
USA). HPLC-grade methanol was obtained from
MERCK Chemicals (Darmstadt, Germany). NNN, NNK,
NAT and NAB were purchased from Toronto Research
Chemicals Inc. (Toronto, Canada). The deuterated
standards, d4-NNN and d4-NNK were purchased from
C/D/N Isotopes (Quebec, Canada). Acetic acid was
purchased from Dima Technology (Richmond Hill,
USA). Ammonium acetate was analytical grade and
MIlli-Q quality water was used to prepare the mobile
phase.
2.3 Smoke particulate collection
Before smoking, each CFP and cigarette were
conditioned at 22 ℃ and 60% relative humidity for at
least 48 h. Individual pad was then placed inside the
filter holders to collect mainstream smoke. Cigarette was
smoked using SM400 linear smoking machine. Each
cigarette was smoked at 1 puff/min, 2 s puff during, and
35 mL puff volume. All samples were smoked to a butt
mark of 3 mm past the tipping paper overwrap.
2.4 Sample preparation
Waters Oasis MCX 30-mg SPE cartridge was firstly
washed with 1 mL of methanol, and then with 1 mL of
water. Each pad with trapped smoke particulate was
spiked with 50 µL (5 mg/L) of internal standard mixtures
and extracted with 10 mL of ammonium acetate (0.1
mol/L) for 30 min using a wrist action shaker operated at
175 r/min. 1.0 mL of extraction solution was loaded onto
the SPE column. After the sample was loaded, the
cartridge was washed by 1 mL of water. Analytes and
their corresponding internal standards were eluted with
1.0 mL of methanol solution with 50 % water. The eluant
was transferred into an autosampler vial.
2.5 Sample analysis
All samples were analyzed using a Surveyor liquid
chromatography coupled with a TSQ quantum discovery
max triple quadruple mass spectrometry. Separation of
TSNAs from the smoke extract was performed using
reversed phase HPLC with a Waters Symmetry Shield
RP18 column (3.9 mm×150 mm i.d. ×5 µm) at 65 ℃.
The mobile phase was 50% methanol, 50% water
containing 0.1% acetic acid and the flow rate was 0.25
mL/min. The labeled NNN was used as internal standard
for the quantification of NNN and NAT. The labeled
NNK was used as internal standard for the quantification
of NNK and NAB.
Ionization of the TSNAs was achieved using the
electrospray ionization(ESI) source operated in positive
ion mode. The typical operation conditions of tandem
mass spectrometry were as follows: spray voltage 5 kV;
sheath gas pressure 172 Pa, aux gas pressure 34 Pa,
capillary temperature 350 ℃, collision gas pressure 0.2
Pa, collision energy 10 V, scanning time 0.2 s.
Mass spectral data on precursor and product ions
were collected in selected reaction monitoring(SRM)
mode. The precursor and product ions are shown in
Fig.1.
Fig.1 Precursor/product ions of TSNAs: (a) NNN; (b) NAT; (c) NAB; (d) NNK

J. Cent. South Univ. Technol. (2008) 15: 627−631
629
Standard stock solutions were prepared by weighing
the appropriate commercially available analyte and
dissolving with acetonitrile. Calibration standards were
prepared in the range of 0.5−100.0 mg/L for NNN, NNK
and NAT, 0.25−50.0 mg/L for NAB.
2.6 Artifact formation
A separate smoking run was conducted to determine
the extent, if any, of artifact formation of the four TSNAs
during sample collection. Five CFPs were treated with 40
mg of ascorbic acid dissolved in 2 mL of methanol. The
treated CFPs were allowed to dry and then conditioned at
22 ℃ for 24 h and a humidity of 60% before use. Ten
smoke samples were collected on five treated and five
untreated CFPs during the same collection run. Samples
were extracted and analyzed as described in the
experimental section.
3 Results and discussion
3.1 Extraction of TSNAs from CFP
WAGNER et al[9] suggested that aqueous solution
(0.1 mol/L ammonium acetate) can make an adequate
and selective extraction of TSNAs from the CFP, which
may simplify the sample preparation. To examine this
possibility, several solutions for extraction of TSNAs in
smoke samples were initially evaluated. In general, it
was found that methylene chloride, ethyl acetate,
methanol and acidic aqueous solutions all adequately
extracted the TSNAs from the CFP within 30 min.
Methylene chloride, ethyl acetate and methanol extracted
nearly all the particulate from the CFP, which made the
sample purifying procedure become very complex.
However, ammonium acetate solution only extracted
TSNAs and some water miscible matrixes and resulted in
a relatively simple sample cleanup. On account of this,
ammonium acetate was chosen as extractant of TSNAs
from CFP.
3.2 Sample cleanup
To clean up smoke extraction solution, a solid-phase
extraction was performed. The solution used for washing
and eluting of Oasis MCX SPE was investigated. It was
found that water can selectively wash down some sample
matrix and 1׃1 (volume ratio) water-methanol can elute
analytes and some other matrix. So water was selected to
wash sample matrix and 1׃1 (volume ratio) water-
methanol was selected to elute analytes from the
cartridge.
3.3 Optimization of chromatographic conditions
Several types of HPLC columns, including Thermo
Hypersil GOLD, Waters Xterra C18 and Waters
Symmetry RP18 columns, were experimented to achieve
the maximum separation and optimize peak shape of
TSNAs and internal standard. In a preliminary study, the
results showed that good separation of TSNAs and
internal standard were achieved on a Symmetry RP18
column (3.9 mm×150 mm i.d.×5 µm) by using an
isocratic elution and the mobile phase consisting of water,
methanol and acetic acid. Then, a rapid, simple, and
specific isotope dilution LC condition for the determina-
tion of tobacco- specific nitrosamines in mainstream
cigarette smoke was presented. Because both TSNAs and
internal standard were easily protonized in a stronger
acidic mobile phase before entering the ionization
ambient (chamber), acetic acid was added into methanol-
water to adjust pH and function as modifier. Separation
conditions such as percentages of water, methanol and
acetic acid were optimized. The optimized chromato-
graphic conditions were listed in Section 2.4. Fig.2
shows the SRM traces of chromatograms for the internal
standards and TSNAs obtained from the mainstream
smoke particulate of a Virginia cigarette sample.
JANSSON et al[10] reported that the E and Z isomers
of NNN can result in the shoulder effect and peak splitt-
ing on HPLC separation. Similar results under our HPLC
separation conditions were observed. NNN and NNK
exhibit some degree of shoulder effects or split peaks.
However, this behavior is temperature dependent. Split
peaks of NNN and NNK can be observed at 20 ℃, but
good peak shapes of all TSNAs were achieved at 65 ℃.
3.4 Calibration curves and limits of detection
Calibration curves were calculated by plotting peak
area ratio (y) of analyte and internal standard against
concentrations (x, mg/L) of analyte. The calibration
curves ranged from 0.5−100.0 mg/L for all TSNAs and
0.25−50.0 mg/L for NAB. Excellent linearity was
obtained with typical values for the correlation
coefficient(R2) between 0.999 1 and 0.999 9. The calibra-
tion range could be extended to higher and lower
amounts. But this is not routinely done since this
working calibration range is sufficiently wide to measure
smoke TSNAs from current cigarette samples.
Standard deviations calculated from multiple
measurements of standard concentrations were used to
estimate the limits of detection(LOD), set equal to 3S0,
where S0 is the standard deviation of the analyte
concentration at zero concentration. The estimated LODs
for NNN, NNK, NAT, and NAB were 0.10, 0.08, 0.05
and 0.06 mg/L, respectively. In all cases, the LODs were
significantly below the levels determined in the test
samples.
3.5 Accuracy and precision
Method accuracy was evaluated by spiking known
amount of the TSNAs on CFP containing smoke samples

J. Cent. South Univ. Technol. (2008) 15: 627−631
630
Fig.2 Chromatograms of TSNAs in Virginia cigarette sample
from 2R4F and a brand of Virginia cigarette. Analytes
were added at two concentrations: one at half the amount
in 2R4F or in the Virginia cigarette smoke and the other
at double. The accuracy was calculated as the
experimentally determined concentration from replicate
analysis divided by the nominal concentration. These
data are listed in Table 1. Recoveries were excellent with
all values range from 90.2% to 105.7%. These results
indicate a lack of any significant matrix effect for the
determination of the selected TSNAs.
The precision of the method was evaluated by
calculating the relative standard deviations(RSD) of the
replicate measurements. Five replicates of 2R4F and a
brand of Virginia cigarettes smoke extract on three days
were analyzed to evaluate both intra- and inter-assay
precisions. The precision for intra-assay samples was
determined by analyzing the same sample five times on
three separate days (n=15). The precision for the
inter-assay samples was determined by the analysis of 15
Virginia samples, 5 of which were collected on three
separate days. Data for 2R4F and Virginia cigarettes are
listed in Table 2.
3.6 Artifact formation investigation
Several groups have investigated the artifact
formation of TSNAs during sample collection and
treatment[11−12]. It has been reported that the formation of
artifactual nitrosamines can be reduced with the addition
of ascorbic acid to the sample collection system.
However, some other investigators did not detect any
Table 1 Accuracy of measurement of TSNAs in mainstream
cigarette smoke
Sample Compound Spiked/
(mg·L−1)
Calculateda)/
(mg·L−1)
Accuracyb)/
%
45.24 45.02 99.5
NNN 135.72 134.53 99.1
45.51 43.86 96.4
NNK 136.53 133.27 97.6
45.10 44.20 98.0
NAT 135.33 135.41 100.1
10.52 10.06 95.6
2R4F
NAB 31.56 30.93 98.0
7.59 7.62 100.4
NNN 22.77 20.32 93.6
7.59 7.08 93.3
NNK 22.77 19.56 94.7
7.52 6.78 90.2
NAT 22.56 22.20 98.4
1.75 1.85 105.7
A brand of
Virginia
cigarette
NAB 5.25 5.13 97.7
Note: a) means data were obtained from the line regression standard curve;
b) means data were calculated by: (calculated concentration)/(spiked
concentration)×100%.
artifact formation when fresh mainstream smoke was
collected directly on untreated CFPs[13−14]. With our
assay, difference in the four selected TSNAs with or
without pretreatment of the CFP with ascorbic acid was
not detected (Table 3).

J. Cent. South Univ. Technol. (2008) 15: 627−631
631
Table 2 Method precision of measurement of TSNAs in
mainstream cigarette smoke
Compound Sample Intra-assaya) Inter-assayb)
2R4F 4.3 7.3
NNN Virginia
cigarette 4.7 6.1
2R4F 4.6 6.8
NNK Virginia
cigarette 3.7 8.7
2R4F 5.3 7.4
NAT Virginia
cigarette 3.9 7.9
2R4F 3.7 8.9
NAB Virginia
cigarette 4.2 9.6
Note: a) means the replicate analysis of the same sample, 5 times a day for 3
d (n=15), expressed as % RSD; b) means replicate analysis of separate
samples, five times a day for 3 d (n=15), expressed as % RSD.
Table 3 Investigation artifact formation of TSNAs from
Virginia cigarette sample (n=5) Concentration/(ng/cigarette)
Sample NNN NNK NAT NAB
Untreated 25.5 22.0 18.6 5.5
2R4F Treated 25.3 22.1 17.5 5.2
Untreated 3.2 5.1 2.8 2.3
Virginia Treated 4.1 4.1 3.1 2.8
4 Conclusions
1) A rapid and reliable LC-MS/MS method for the
quantitative determination of TSNAs in Virginia
cigarette mainstream smoke is developed and validated.
2) This LC-MS/MS method offers decreased sample
preparation and analysis time as compared to traditional
methods. The sample preparation time for 20 samples is
about 1 h and both Virginia and 2R4F cigarettes show
good analysis results.
3) The method can be used for identification and
determination of TSNAs in Virginia and Kentucky
cigarette mainstream smoke. The results show that
LC-MS/MS method is a powerful technique for the
analysis of TSNAs.
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(Edited by YANG Hua)