1. bookVolume 29 (2020): Issue 2 (August 2020)
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Variations of TSNA Levels in Tobaccos Upon Heating at Moderate Temperatures

Published Online: 25 Sep 2020
Page range: 84 - 96
Received: 19 Mar 2020
Accepted: 28 Jul 2020
Journal Details
License
Format
Journal
First Published
23 Oct 2013
Publication timeframe
4 times per year
Languages
English

Tobacco-specific nitrosamines (TSNAs) including nitrosoanabasine (NAB), nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and nitrosonornicotine (NNN) are naturally present at trace levels in tobacco. During tobacco processing, preparation of expanded tobacco, and when tobacco is used in heat-not-burn type cigarettes, the tobacco is exposed to different degrees of heat. Heating of tobacco has been reported in the literature to increase the level of TSNAs. Since the increase of TSNAs in heated tobacco is still not well understood, the present study evaluated TSNA levels in six types of tobacco as a function of moderate heat exposure. These tobaccos included: flue-cured lower stalk, flue-cured upper stalk (US), Burley lower stalk, Burley upper stalk (US), and two Oriental blends (Turkey, Greece, Bulgaria, Northern Republic Macedonia). Heating was performed in sealed glass tubes at oven temperatures of 100 °C, 150 °C, 200 °C, and 250 °C for time intervals of 2 min and 5 min. The temperatures inside the glass tubes were lower than the targets and were monitored separately as a function of glass tube heating. The study showed no meaningful differences within tobacco type (by stalk position) but showed considerable differences in the levels of TSNAs between different tobaccos, with the Burley tobaccos having the highest levels, and the Orientals the lowest. For all tobacco types, TSNAs increase to some extent when temperature increases. For 2-min heating, the increase in TSNAs is relatively small up to about 200 °C, but the levels almost double when the oven temperature increases to 250 °C. For 5-min heating, the increase in TSNAs starts at about 150 °C with a maximum at 200 °C which can reach more than double the initial TSNA level. Longer heating at 250 °C (5 min) starts to cause TSNAs decomposition and the levels are reduced.

Keywords

INTRODUCTION

Tobacco-specific nitrosamines including nitrosoanabasine (NAB), nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and nitrosonornicotine (NNN) are naturally present at trace levels in tobacco. These levels vary considerably depending on many factors such as the type of tobacco (flue-cured, Burley, Oriental), the location of tobacco production, the leaf position, the leaf processing procedure, the tobacco age, etc. The level of TSNAs in tobacco is of considerable interest since these compounds are listed among the harmful and potentially harmful constituents (HPHC) of tobacco products on which Food & Drug Administration (FDA) requires reporting (1). Also, NNK and NNN have been classified as human carcinogens (Class 1) by the International Agency for Research on Cancer (IARC) (2). Tobacco heating has been reported to increase the level of TSNAs in tobacco (3, 4). In addition to the heating of tobacco behind the burning cone of a common burning cigarette, there are other instances when tobacco is heated, although at lower temperatures. Such cases include processing (e.g., drying), Burley toasting, preparation of expanded tobacco, as well as the use in “heat-not-burn” type cigarettes. Since the increase of TSNAs in heated tobacco is still not well understood, this was investigated in the present study. Tobacco-specific nitrosamines (TSNAs) can be analyzed by various methods that have been extensively reported in the literature (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). These methods were applied on tobacco, cigarette smoke, and several tobacco products such as moist snuff. An adaptation of one of these methods (18) has been used in the present study and was applied to TSNA analysis in tobaccos. The study and the selection of the temperature range in which the tobacco was heated (100, 150, 200, 250 °C) intended to provide information for the process in heat-not-burn type cigarettes and other instances in which tobacco is subject to moderate temperatures.

EXPERIMENTAL
Materials

Several chemicals including ammonium acetate, ammonium formate, formic acid, and acetonitrile were obtained from Sigma/Aldrich (St. Louis, MO, USA). Nicotine, nitrosoanabasine (NAB), nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), nitrosonornicotine (NNN), NAB-d4, NAT-d4, NNK-d4 and NNN-d4 were obtained from Toronto Research Chemicals Inc. (TRC) (North York, ON, Canada). Pure water (18.2 MΩ/cm) was obtained from a Barnstead water purification unit (Thermo Fisher Scientific, Waltham, MA, USA). The GC vials were 2 mL with screw top caps with septa. For filtration 0.45-μm PVDF filters were used (Whatman Autovial, GE Healthcare, Little Chalfort, UK).

Instrumentation

The instruments used for the analysis consisted of an Agilent 1290 HPLC binary system with a binary pump, an autosampler with cooling capability, and a column thermostatted compartment. The HPLC chromatographic separation was achieved on a Luna® 3 μm C18 150 × 3 mm from Phenomenex (Torrance, CA, USA). The MS/MS system was an API-6500 triple quadrupole mass spectrometer (AB Sciex, Framingham, MA, USA). The LCMS/MS system was controlled using Analyst 1.6.2 software, and the peak integration was performed with MultiQuant 3.0.1 software. The heating was performed in a Thermoline Furnace 62700 (Thermo Fisher, Waltham, MA, USA). Tobacco moisture was measured with a halogen moisture analyzer HE53 (Mettler Toledo, Greifensee, Switzerland).

TSNAs standards preparation

An extraction solution of 100 mM ammonium acetate in water was prepared by dissolving 7.7 g of ammonium acetate into 1 L of purified water. The standard solutions were prepared from a stock solution of 162.65 μg/mL N-nitrosonornicotine (NNN), 164.036 μg/mL N’-nitrosoanatabine (NAT), 41.508 μg/mL N-nitrosoanabasine (NAB) and 161.10 μg/mL nicotine-derived nitrosamine ketone (NNK). Standard 1 was prepared by 1/1000 dilution of the stock solution with the extraction solution and used to prepare Standards 2–7, 1000 μmL each. To each 1 mL standard was added 20 μL of the internal standard solution. The internal standard solution contained 1.6 μg/mL NNN-d4, 1.6 μg/mL NAT-d4, 1.33 μg/mL NAB-d4 and 1.6 μg/mL NNK-d4. The concentration of internal standard (I.S.) in the final solution is indicated in Table 1.

Concentrations of deuterated TSNAs in the samples containing internal standard solution.

NNN-d4 (ng/mL)NAT-d4 (ng/mL)NAB-d4 (ng/mL)NNK-d4 (ng/mL)
40.66341.00910.37740.275

The concentrations of the utilized standards are given in Table 2.

Concentrations of TSNAs in the standard solutions.

StandardNNN (ng/mL)NAT (ng/mL)NAB (ng/mL)NNK (ng/mL)
1162.65164.0441.51161.10
281.3382.0220.7580.55
340.6641.0110.3840.28
420.3320.505.1920.14
510.1710.252.5910.07
65.085.131.305.03
72.542.560.652.52
Sample heat processing

Heating of the tobacco was performed in sealed Pyrex glass tubes. This was done to avoid potential loss of material by evaporation when the tobacco was heated. Glass tube dimensions were 8 mm i.d. and a length of 15 cm with a 1.0-mm wall thickness. About 600 mg tobacco (± 20 mg) were weighed into each tube. The tubes were sealed at one end and were subjected to a mild vacuum prior to sealing the other end. The purpose of applying a vacuum was to prevent the tubes from breaking when exposed to heat due to the expansion of gases in the tube. Figure 1 shows several sealed tubes containing powdered tobacco (Burley lower stalk) before heating.

Figure 1

Sealed tubes containing powdered tobacco (Burley lower stalk) before heating.

Four different temperatures were used for heating the tubes: 100 °C, 150 °C, 200 °C, and 250 °C. Two time intervals were selected for heating: 2 min and 5 min. The actual temperature of the tobacco was not measured during the experiments performed for TSNAs measurement. However, in a published study (3) the heating inside of a tobacco rod was evaluated, when exposed from exterior to 100 °C or 200 °C. The study (3) indicated a delay in the temperature reached by the middle of a tobacco rod as compared to the applied external temperature. In the present study, an additional issue was that upon removing the glass tube with the tobacco from the oven, the cooling of the material was not instantaneous and it took 3–4 min for the tube to reach room temperature. For this reason, the temperature inside a sample (of Burley) was measured separately as a function of heating time. For this purpose a thermocouple was inserted into the sample during the heating of the glass tube containing the sample. At 5 min the heating was stopped by removing the glass tube from the oven and exposing it to room temperature. The temperature variation inside the sample is shown in Figure 2.

Figure 2

Temperature variation inside a tobacco sample placed in a glass tube as a function of heating time of the tube. Heating was stopped at 5 min and the samples were exposed to room temperature.

As indicated in Figure 2, the temperature to which the glass tube with sample is exposed is different from the actual temperature of the sample. Assuming a linear idealized temperature profile to which the tobacco is exposed, the temperature variation in the tobacco can be described by the following formula: T(t)=Tinit.+b1t(0<t<tmax)T(t)=Tinit.+b1tmaxb2t(tmax<t<tfinal)\begin{array}{*{20}{c}} {T\left( t \right) = {T_{{\text{init}}.}} + {{\text{b}}_1}\,\,t\left( {0 < t < {t_{{\text{max}}}}} \right)} \\ {T\left( t \right) = {T_{{\text{init}}.}} + {{\text{b}}_1}\,\,\,\,{t_{{\text{max}}}} - {{\text{b}}_2}\,\,\,\,t\left( {{t_{{\text{max}}}} < t < {t_{{\text{final}}}}} \right)} \end{array} where T(t) is the temperature of the sample as a function of time t, Tinit. is the initial temperature (about 22 °C), tmax is the length of time the sample is heated, tfinal is the time of cooling to room temperature, and b1 and b2 are heating and cooling rates, respectively. The fixed oven temperatures 100 °C, 150 °C, 200 °C, and 250 °C for which the results are indicated in this study, are in fact described correctly by sample temperature variations as shown in Figure 2 and approximated by equation [1]. However, the linear approximation holds only for the first minute of heating. Afterwards the higher temperatures showed an endothermic leveling when passing 100 °C and the rates for all temperatures decreased as the difference in temperature between the tobacco and the outside of the tube decreased. The initial slopes (°C/s) were proportional to the oven temperature and were equal to [0.0076 × oven temperature – 0.494] with an R2 = 0.999. The initial rate of cooling was also linear and equal to [− 0.0062 × oven temperature + 0.433] with an R2 = 0.939. The cooling rate was slightly lower than the heating rate because the initial temperature difference was lower between the inside and the outside of the tube. The fixed temperatures of the oven, to which the glass tubes were exposed, were precisely controlled (within 1–2 °C) while the true temperature of the samples was subject to variations depending on the exact mass of the glass tube, the mass of the tobacco sample, and on the distribution of the tobacco inside the glass tube during heating, all of which affected parameters b1 and b2. The use of sealed tubes as containers for the heated tobacco avoided potential loss of material by evaporation but at the same time did not allow a precise measurement of the temperature in the tobacco sample. This temperature can be inferred only by comparing the temperature of the heating of the glass tube with that inside the sample shown in the graph of Figure 2.

TSNA analysis

Prior to the analysis, the moisture of the samples was measured using a halogen moisture analyzer. For the analysis, samples of 250 ± 1.0 mg of tobacco (as is) were weighed directly into 20-mL scintillation vials. To each vial 10 mL of the extraction solution were added, then the vial was capped and placed into a shaker for 30 min. The solution was then filtered through a syringe filter into a test tube. 1000 μL of the filtered solution was transferred to a 2-mL GC vial and 20 μL of the TSNA internal standard was added to the sample.

The HPLC separation of TSNAs was performed using a gradient program. The mobile phase A was 100 mM ammonium formate pH 4.9 aqueous solution with 5% acetonitrile and mobile phase B was 0.2% formic acid in acetonitrile. The timetable for the gradient is shown in Table 3.

Solvent gradient employed for the HPLC-MS/MS analysis of TSNAs.

Time% A% BFlow rate (mL/min)
0.0100.00.00.6
1.7100.00.00.6
3.570.030.00.6
4.560.040.00.6
8.050.050.00.6
10.0100.00.00.6

The parameters for the MS/MS detection included: curtain gas CUR = 20 mL/min, collision gas CAD = 4 mL/min, ion spray voltage IS = 4500 V, temperature TEM = 500 °C, ion source gas 1 GS1 = 40 mL/min, ion source gas 2 GS2 = 50 mL/min, entrance potential EP = 10 V, target scan time 0.11 s, scheduled MRM detection window 40 s. Other parameters including the analyzed ions are shown in Table 4.

Other parameters for the MS/MS detection.

CompoundIon for Q1Ion for Q3Retention time (min)DP1(V)CE2 (V)CXP3 (V)
NNN178.1148.14.7330159
NNN-d4182.1152.14.7030169
NAT190.1160.15.352815.510
NAT-d4194.1164.15.31281610
NAB192.1162.15.40261811
NAB-d4196.1166.15.37261710
NNK208.1122.15.163017.613
NNK-d4212.1126.15.15301612

DP = Declustering potential;

CE = Collision energy;

CXP = Collision cell exit potential

Extracted ion chromatograms for the eight compounds measured in the study are shown in Figure 3 for a flue-cured tobacco (upper stalk) heated at 200 °C for 5 min. Both NNN and NNN-d4 show two peaks in the previously described separation conditions. Only the higher of the two peaks was used for the quantitation. Peak splitting for TSNAs can be attributed to the presence of a syn-anti isomers of the nitrosamine group.

Figure 3

Extracted ion chromatograms for NNN, NAT, NAB and NNK and their d4-labeled compounds measured for a flue-cured tobacco (upper stalk) heated at 200 °C for 5 min.

For the quantitation of TSNAs, calibration curves were constructed by plotting the concentrations of the standard solutions as a function of the peak areas normalized to the area of the corresponding deuterated internal standards. All calibrations were linear and they were expressed using the formula: Y(conc.)=a(Std.Area/I.S.Area)/bY\left( {{\text{conc}}.} \right) = a\left( {{\text{Std}}.\,{\text{Area}}/{\text{I}.\text{S}}.\,{\text{Area}}} \right)/b

Parameters a and b as well as correlation coefficient R2 for the calibration lines are shown in Table 5.

Parameters a, b and R 2 for the MS/MS quantitation.

AnalyteabR2
NNN33.51510.17980.9997
NAT40.85070.04790.9998
NAB35.15280.07680.9992
NNK35.76550.08890.9996

The overall validation of the analytical method for the analysis of TSNAs has been previously reported (18) and no further validation has been performed for the method.

RESULTS
Sample description

The samples analyzed in this study are listed in Table 6, together with the measured moisture % total alkaloid level as well as the time intervals and temperatures to which they were exposed.

Tobacco samples, moisture (%), alkaloid level (%), and the conditions to which the samples were exposed.

Sample acronymTobacco descriptionMoisture (%)Total alkaloids (%)Temperatures (°C)Time of exposure (min)
FC-LoLower stalk flue-cured6.442.0710015020025025
FC-UpUpper stalk flue-cured6.712.8510015020025025
Bu-LoLower stalk Burley6.362.5110015020025025
Bu-UpUpper stalk Burley7.113.4610015020025025
Or (1)Oriental blend (1)6.641.1310015020025025
Or (2)Oriental blend (2)6.231.0210015020025025

Some samples were processed in duplicate or even triplicate, and other samples were analyzed only once. The samples were randomly analyzed, and some duplicate or triplicate evaluations were made to verify the repeatability of the results. Once the sample was heated and extracted, the LC/MS/MS measurement was performed in duplicate.

Average TSNA levels for the starting tobacco

The results of average TSNA levels in ng/g for the starting tobaccos (analyzed before starting the heating experiments) are shown in Table 7. All results are reported for the tobacco “as is,” a conversion to “dry basis” being possible using the moisture data from Table 6.

Initial TSNA levels in the tobacco samples in ng/g (tobacco as is).

Sample acronymNNN (ng/g)NAT (ng/g)NAB (ng/g)NNK (ng/g)
FC-Lo365.2566.533.2283.1
FC-Up354.1458.332.6227.4
Bu-Lo4688.34616.8199.7861.0
Bu-Up4780.34662.1183.2773.3
Or (1)156.654.86.1443.0
Or (2)126.433.54.7932.6

As indicated in Table 7, the levels of TSNAs did not differ meaningfully within tobacco type (by stalk position), but among the various tobacco types they differed considerably. Burley tobaccos have a very high level of TSNAs even before heating. Significantly lower levels compared to the Burleys are present in flue-cured tobaccos. The lowest TSNA levels are present in the Oriental tobaccos.

TSNA levels in the heated tobaccos

The experimental results for levels of TSNAs in the six evaluated tobaccos for the two heating intervals (2 min and 5 min) are indicated in Tables 8 to 10. In these tables, the listed and repeated temperatures (for a specific TSNA in each tobacco) indicate results from repeated whole experiments (including heating of the tobacco and LC/MS/MS measurement).

Variation of TSNAs in flue-cured tobaccos upon heating (in ng/g).

Temperature (°C)Flue-cured lower stalk, FC-LoFlue-cured upper stalk, FC-Up
2 min5 min2 min5 min
NNN (ng/g)
23372.5 ± 2.1372.5 ± 2.1358.3 ± 3.9358.3 ± 3.9
23357.9 ± 0.5357.9 ± 0.5342.9 ± 1.7342.9 ± 1.7
23361.0 ± 2.9361.0 ± 2.9
100397.9 ± 6.9400.8 ± 0.2378.9 ± 9.1389.5 ± 5.3
100388.8 ± 2.6380.5 ± 3.5
150420.5 ± 1.7465.3 ± 1.4392.7 ± 10.4422.6 ± 4.7
150391.7 ± 14.3
200479.8 ± 1.7697.4 ± 5.2457.5 ± 10.2540.5 ± 9.3
200461.3 ± 4.7546.0 ± 3.3
250496.7 ± 9.2109.1 ± 0.4595.8 ± 42.4102.4 ± 0.6
NAT (ng/g)
23566.7 ± 7.2566.7 ± 7.2469.1 ± 4.7469.1 ± 4.7
23566.4 ± 4.8566.4 ± 4.8447.9 ± 7.4447.9 ± 7.4
23457.9 ± 2.1457.9 ± 2.1
100602.8 ± 18.0645.0 ± 4.6520.0 ± 12.7492.6 ± 1.0
100517.5 ± 22.5483.6 ± 3.1
150693.2 ± 3.2836.3 ± 10.9529.3 ± 8.4618.3 ± 1.6
150520.9 ± 4.1
200909.8 ± 5.01366.4 ± 5.6649.3 ± 11.7871.2 ± 1.7
200657.5 ± 1.4885.8 ± 6.6
250945.6 ± 5.817.1 ± 0.91039.2 ± 30.610.9 ± 0.2
NAB (ng/g)
2331.9 ± 0.131.9 ± 0.134.8 ± 3.134.8 ± 3.1
2334.5 ± 0.234.5 ± 0.230.4 ± 0.230.4 ± 0.2
2332.7 ± 0.232.7 ± 0.2
10040.3 ± 0.538.0 ± 0.836.5 ± 0.536.9 ± 0.4
10038.4 ± 0.437.1 ± 0.5
15039.6 ± 0.246.4 ± 1.237.0 ± 0.243.8 ± 0.2
15038.2 ± 0.8
20051.1 ± 0.5101.0 ± 0.345.7 ± 2.472.3 ± 0.6
20048.6 ± 0.774.2 ± 0.6
25072.4 ± 0.641.1 ± 0.299.7 ± 5.743.5 ± 1.3
NNK (ng/g)
23270.1 ± 1.5270.1 ± 1.5225.3 ± 4.9225.3 ± 4.9
23296.1 ± 2.4296.1 ± 2.4218.0 ± 1.7218.0 ± 1.7
23239.0 ± 1.4239.0 ± 1.4
100318.5 ± 7.4314.5 ± 1.9242.8 ± 5.7235.0 ± 1.5
100236.2 ± 8.1225.6 ± 3.3
150341.0 ± 1.33343.9 ± 3.9240.6 ± 5.5225.4 ± 0.1
150231.4 ± 0.1
200352.7 ± 4.3658.7 ± 9.4249.1 ± 4.9349.1 ± 6.8
200259.0 ± 0.2340.3 ± 0.4
250640.0 ± 13.1269.7 ± 0.5430.6 ± 32.1216.4 ± 2.7

Variation of TSNAs in lower stalk Burley tobaccos upon heating (in ng/g).

Temperature (°C)Burley lower stalk, Bu-LoBurley upper stalk, Bu-Up
2 min5 min2 min5 min
NNN (ng/g)
234678.4 ± 262.04678.4 ± 262.04666.7 ± 65.84666.7 ± 65.8
234698.2 ± 40.44698.2 ± 40.44893.9 ± 15.24893.9 ± 15.2
1004715.8 ± 98.14701.7 ± 44.75458.9 ± 21.35753.9 ± 51.8
1504903.5 ± 219.25968.8 ± 69.26288.3 ± 4.47508.4 ± 7.5
2005930.8 ± 215.38541.1 ± 160.66953.1 ± 47.310030.8 ± 68.2
2006947 ± 42.4
2508961.2 ± 49.39383.9 ± 122.08525.8 ± 148.37077.4 ± 38.9
NAT (ng/g)
234611 ± 29.54611 ± 29.54430.1 ± 28.84430.1 ± 28.8
234622.5 ± 268.14622.5 ± 268.14894.2 ± 5.44894.2 ± 5.4
1004627.4 ± 5.14660.9 ± 7.95585.9 ± 6.15893.8 ± 86.0
1504812.6 ± 169.96485.5 ± 81.76386.6 ± 44.18067.5 ± 49.2
2006327.2 ± 160.110063.8 ± 169.110005.5 ± 0.010677.6 ± 59.8
2009543.5 ± 40.1
25010655.9 ± 86.34775.7 ± 27.28994.5 ± 161.91356.7 ± 3.7
NAB (ng/g)
23204.7 ± 10.8204.7 ± 10.8176.4 ± 2.1176.4 ± 2.1
23194.8 ± 0.8194.8 ± 0.8190.1 ± 0.8190.1 ± 0.8
100199.2 ± 1.4196.0 ± 1.2223.8 ± 0.1224.5 ± 1.7
150207.6 ± 9.3290.2 ± 1.6249.1 ± 2.7340.8 ± 0.5
200282.6 ± 7.1629.9 ± 7.1423.4 ± 2.4582.1 ± 2.9
200396.8 ± 3.1
250637.3 ± 2.51683.8 ± 11.1474.9 ± 6.51734.9 ± 15.4
NNK (ng/g)
23855.6 ± 32.3855.6 ± 32.3791.7 ± 1.7791.7 ± 1.7
23866.4 ± 0.4866.4 ± 0.4754.8 ± 0.0754.8 ± 0.0
100844.9 ± 9.5866.9 ± 6.7858.8 ± 13.7884.9 ± 1.8
150898.8 ± 29.31039.6 ± 11.1963.6 ± 3.11145.3 ± 1.3
2001040.9 ± 38.62551.8 ± 14.51298.4 ± 2.92065.1 ± 14.2
2001216.6 ± 13.5
2503517.8 ± 1.84749 ± 2.41711.0 ± 27.52916.5 ± 3.8

Variation of TSNAs in Oriental tobaccos upon heating (in ng/g).

Temperature (°C)Oriental (1), Or(1)Oriental (2), Or(2)
2 min5 min2 min5 min
NNN (ng/g)
23156.9 ± 1.3156.9 ± 1.3130.2 ± 0.5130.2 ± 0.5
23156.3 ± 1.3156.3 ± 1.3127.0 ± 0.3127.0 ± 0.3
23122.0 ± 2.7122.0 ± 2.7
100203.7 ± 1.9218.0 ± 1.8146.2 ± 0.1147.6 ± 1.5
150211.4 ± 1.6273.4 ± 1.5194.1 ± 0.2225.3 ± 1.5
200282.1 ± 3.4489.2 ± 13.0205 ± 0.8325.3 ± 2.6
200307.0 ± 1.0319.5 ± 5.8
250386.7 ± 11.5146.2 ± 1.0289.1 ± 5.2
250298.3 ± 0.5
NAT (ng/g)
2355.0 ± 0.955.0 ± 0.933.3 ± 0.133.3 ± 0.1
2354.6 ± 0.754.6 ± 0.733.8 ± 1.333.8 ± 1.3
10069.7 ± 0.477.7 ± 1.239.0 ± 1.039.1 ± 0.7
15073.2 ± 1.5122.1 ± 0.253.5 ± 0.867.7 ± 2.0
200130.8 ± 1.5284.4 ± 4.564.7 ± 1.0121.7 ± 2.1
200139.3 ± 0.9118.6 ± 0.5
250207.3 ± 3.812.9 ± 0.4105.6 ± 2.7
250108.8 ± 1.7
NAB (ng/g)
236.2 ± 0.36.2 ± 0.34.9 ± 0.14.9 ± 0.1
236.1 ± 0.36.1 ± 0.34.7 ± 0.14.7 ± 0.1
1006.7 ± 0.37.1 ± 0.64.8 ± 0.15.1 ± 0.0
1506.9 ± 0.510.3 ± 0.15.7 ± 0.36.7 ± 0.0
20010.3 ± 0.228.0 ± 0.27.0 ± 0.018.1 ± 0.4
20011.2 ± 0.417.6 ± 0.0
25018.4 ± 0.425.6 ± 0.113.9 ± 0.7
25015.3 ± 0.1
NNK (ng/g)
2341.3 ± 0.641.3 ± 0.632.9 ± 0.432.9 ± 0.4
2344.7 ± 1.244.7 ± 1.232.4 ± 0.432.4 ± 0.4
10054.2 ± 0.854.7 ± 0.438.2 ± 2.038.2 ± 0.4
15055.2 ± 1.457.5 ± 0.445.2 ± 0.849.0 ± 1.5
20056.8 ± 2.0119.8 ± 0.448.4 ± 0.676.8 ± 0.1
20059.8 ± 0.776.8 ± 0.3
25085.5 ± 1.491.5 ± 2.862.0 ± 0.3
25065.9 ± 0.2

Each LC/MS/MS measurement for every experiment was performed in duplicate and the RSD% values are reported between repeated LC/MS/MS measurements.

The results of TSNA levels in heated flue-cured tobaccos FC-Lo and FC-Up are indicated in Table 8.

The results of TSNA levels in heated Burley tobaccos Bu-Lo and Bu-Up are indicated in Table 9. The results of TSNA levels in heated Oriental tobaccos are indicated in Table 10.

DISCUSSION

A typical level variation of TSNAs with oven temperatures as described in Tables 8 to 10, is shown in Figure 4 for NNN in FC-Up samples for 2-min heating intervals, and for 5-min heating intervals in Figure 5.

Figure 4

Variation of NNN in FC-Up sample with 2 min heating at different oven temperatures.

Figure 5

Variation of NNN in FC-Up sample with 5 min heating at different oven temperatures.

For other types of tobacco and other TSNAs it was common to obtain similar curves of variations although there were also some exceptions when the drop of the TSNA level for 5-min heating at 250 °C was less obvious. This can be explained by possible non-homogeneities in the heating of the specific sample. The typical increase in TSNA levels described in Tables 8 to 10 can be visualized by comparing Figure 6, which shows the initial TSNA levels with Figure 7, which shows the levels after 2-min heating at 200 °C.

Figure 6

Initial levels of TSNAs (data taken from Table 7).

Figure 7

Levels of TSNAs after heating for 2 min at 200 °C (data taken from Tables 8 to 10).

Also, as expected, the heating of tobacco for 5 min led to increased formation of TSNAs, as shown by comparing the results illustrated in Figure 7 with those illustrated in Figure 8 for heating at 200 °C for 5 min (all data taken from Tables 8 to 10).

Figure 8

Levels of TSNAs after heating for 5 min at 200 °C (data taken from Tables 8 to 10).

The heating of tobacco for 2 min at 250 °C continues in most cases to increase the TSNA level, as is illustrated by comparing Figure 7 with the data from Figure 9 which shows the results for heating the tobaccos for 2 min at 250 °C. However, by further heating the tobacco for 5 min at 250 °C, the levels of TSNAs were decreasing in most cases, as can be seen by comparing the results from Figure 9 with those from Figure 10 which shows the results for heating the samples for 5 min at 250 °C.

Figure 9

Levels of TSNAs after heating for 2 min at 250 °C (data taken from Tables 8 to 10).

Figure 10

Levels of TSNAs after heating for 5 min at 250 °C (data taken from Tables 8 to 10).

The results of this study indicate that moderate temperatures and extended heating time are factors leading to an increase in the TSNA levels in tobacco. They also show that this increase takes place up to a point as the temperature increases, and beyond this point, very likely, some TSNA’s decomposition starts to take place. In order to keep the TSNA levels as low as possible, unnecessary heating of tobacco at moderate temperatures must be avoided. However, tobacco heating cannot be avoided in many devices such as for example in the heat-not-burn type cigarettes, where certain temperatures are a precondition for the release of flavors and nicotine as well as humectants added to tobacco. As a result, the increase in TSNAs cannot be avoided. By selecting specific tobaccos already low in TSNAs, an excessive increase of TSNAs due to heating can be diminished. The decomposition of TSNAs that occurs when tobacco is heated beyond a specific temperature and length of time could probably be used for lowering the TSNA levels. Nevertheless a careful evaluation of this process must be performed with additional examination and control of the decomposition of other tobacco constituents such as sugars.

CONCLUSIONS

Six commonly used tobaccos, including two flue-cured, two Burleys and two Oriental tobaccos were analyzed for their TSNA levels. These tobaccos were heated in sealed glass containers at oven temperatures of 100, 150, 200 and 250 °C for intervals of 2 min or 5 min. The TSNA levels were found to increase up to a point during tobacco heating, reaching levels as high as double from the initial level. However, the TSNAs increase did not continue when further heating was performed due to some TSNA’s decomposition probably taking place beyond a specific point of heating.

Figure 1

Sealed tubes containing powdered tobacco (Burley lower stalk) before heating.
Sealed tubes containing powdered tobacco (Burley lower stalk) before heating.

Figure 2

Temperature variation inside a tobacco sample placed in a glass tube as a function of heating time of the tube. Heating was stopped at 5 min and the samples were exposed to room temperature.
Temperature variation inside a tobacco sample placed in a glass tube as a function of heating time of the tube. Heating was stopped at 5 min and the samples were exposed to room temperature.

Figure 3

Extracted ion chromatograms for NNN, NAT, NAB and NNK and their d4-labeled compounds measured for a flue-cured tobacco (upper stalk) heated at 200 °C for 5 min.
Extracted ion chromatograms for NNN, NAT, NAB and NNK and their d4-labeled compounds measured for a flue-cured tobacco (upper stalk) heated at 200 °C for 5 min.

Figure 4

Variation of NNN in FC-Up sample with 2 min heating at different oven temperatures.
Variation of NNN in FC-Up sample with 2 min heating at different oven temperatures.

Figure 5

Variation of NNN in FC-Up sample with 5 min heating at different oven temperatures.
Variation of NNN in FC-Up sample with 5 min heating at different oven temperatures.

Figure 6

Initial levels of TSNAs (data taken from Table 7).
Initial levels of TSNAs (data taken from Table 7).

Figure 7

Levels of TSNAs after heating for 2 min at 200 °C (data taken from Tables 8 to 10).
Levels of TSNAs after heating for 2 min at 200 °C (data taken from Tables 8 to 10).

Figure 8

Levels of TSNAs after heating for 5 min at 200 °C (data taken from Tables 8 to 10).
Levels of TSNAs after heating for 5 min at 200 °C (data taken from Tables 8 to 10).

Figure 9

Levels of TSNAs after heating for 2 min at 250 °C (data taken from Tables 8 to 10).
Levels of TSNAs after heating for 2 min at 250 °C (data taken from Tables 8 to 10).

Figure 10

Levels of TSNAs after heating for 5 min at 250 °C (data taken from Tables 8 to 10).
Levels of TSNAs after heating for 5 min at 250 °C (data taken from Tables 8 to 10).

Concentrations of deuterated TSNAs in the samples containing internal standard solution.

NNN-d4 (ng/mL)NAT-d4 (ng/mL)NAB-d4 (ng/mL)NNK-d4 (ng/mL)
40.66341.00910.37740.275

Concentrations of TSNAs in the standard solutions.

StandardNNN (ng/mL)NAT (ng/mL)NAB (ng/mL)NNK (ng/mL)
1162.65164.0441.51161.10
281.3382.0220.7580.55
340.6641.0110.3840.28
420.3320.505.1920.14
510.1710.252.5910.07
65.085.131.305.03
72.542.560.652.52

Solvent gradient employed for the HPLC-MS/MS analysis of TSNAs.

Time% A% BFlow rate (mL/min)
0.0100.00.00.6
1.7100.00.00.6
3.570.030.00.6
4.560.040.00.6
8.050.050.00.6
10.0100.00.00.6

Variation of TSNAs in flue-cured tobaccos upon heating (in ng/g).

Temperature (°C)Flue-cured lower stalk, FC-LoFlue-cured upper stalk, FC-Up
2 min5 min2 min5 min
NNN (ng/g)
23372.5 ± 2.1372.5 ± 2.1358.3 ± 3.9358.3 ± 3.9
23357.9 ± 0.5357.9 ± 0.5342.9 ± 1.7342.9 ± 1.7
23361.0 ± 2.9361.0 ± 2.9
100397.9 ± 6.9400.8 ± 0.2378.9 ± 9.1389.5 ± 5.3
100388.8 ± 2.6380.5 ± 3.5
150420.5 ± 1.7465.3 ± 1.4392.7 ± 10.4422.6 ± 4.7
150391.7 ± 14.3
200479.8 ± 1.7697.4 ± 5.2457.5 ± 10.2540.5 ± 9.3
200461.3 ± 4.7546.0 ± 3.3
250496.7 ± 9.2109.1 ± 0.4595.8 ± 42.4102.4 ± 0.6
NAT (ng/g)
23566.7 ± 7.2566.7 ± 7.2469.1 ± 4.7469.1 ± 4.7
23566.4 ± 4.8566.4 ± 4.8447.9 ± 7.4447.9 ± 7.4
23457.9 ± 2.1457.9 ± 2.1
100602.8 ± 18.0645.0 ± 4.6520.0 ± 12.7492.6 ± 1.0
100517.5 ± 22.5483.6 ± 3.1
150693.2 ± 3.2836.3 ± 10.9529.3 ± 8.4618.3 ± 1.6
150520.9 ± 4.1
200909.8 ± 5.01366.4 ± 5.6649.3 ± 11.7871.2 ± 1.7
200657.5 ± 1.4885.8 ± 6.6
250945.6 ± 5.817.1 ± 0.91039.2 ± 30.610.9 ± 0.2
NAB (ng/g)
2331.9 ± 0.131.9 ± 0.134.8 ± 3.134.8 ± 3.1
2334.5 ± 0.234.5 ± 0.230.4 ± 0.230.4 ± 0.2
2332.7 ± 0.232.7 ± 0.2
10040.3 ± 0.538.0 ± 0.836.5 ± 0.536.9 ± 0.4
10038.4 ± 0.437.1 ± 0.5
15039.6 ± 0.246.4 ± 1.237.0 ± 0.243.8 ± 0.2
15038.2 ± 0.8
20051.1 ± 0.5101.0 ± 0.345.7 ± 2.472.3 ± 0.6
20048.6 ± 0.774.2 ± 0.6
25072.4 ± 0.641.1 ± 0.299.7 ± 5.743.5 ± 1.3
NNK (ng/g)
23270.1 ± 1.5270.1 ± 1.5225.3 ± 4.9225.3 ± 4.9
23296.1 ± 2.4296.1 ± 2.4218.0 ± 1.7218.0 ± 1.7
23239.0 ± 1.4239.0 ± 1.4
100318.5 ± 7.4314.5 ± 1.9242.8 ± 5.7235.0 ± 1.5
100236.2 ± 8.1225.6 ± 3.3
150341.0 ± 1.33343.9 ± 3.9240.6 ± 5.5225.4 ± 0.1
150231.4 ± 0.1
200352.7 ± 4.3658.7 ± 9.4249.1 ± 4.9349.1 ± 6.8
200259.0 ± 0.2340.3 ± 0.4
250640.0 ± 13.1269.7 ± 0.5430.6 ± 32.1216.4 ± 2.7

Variation of TSNAs in lower stalk Burley tobaccos upon heating (in ng/g).

Temperature (°C)Burley lower stalk, Bu-LoBurley upper stalk, Bu-Up
2 min5 min2 min5 min
NNN (ng/g)
234678.4 ± 262.04678.4 ± 262.04666.7 ± 65.84666.7 ± 65.8
234698.2 ± 40.44698.2 ± 40.44893.9 ± 15.24893.9 ± 15.2
1004715.8 ± 98.14701.7 ± 44.75458.9 ± 21.35753.9 ± 51.8
1504903.5 ± 219.25968.8 ± 69.26288.3 ± 4.47508.4 ± 7.5
2005930.8 ± 215.38541.1 ± 160.66953.1 ± 47.310030.8 ± 68.2
2006947 ± 42.4
2508961.2 ± 49.39383.9 ± 122.08525.8 ± 148.37077.4 ± 38.9
NAT (ng/g)
234611 ± 29.54611 ± 29.54430.1 ± 28.84430.1 ± 28.8
234622.5 ± 268.14622.5 ± 268.14894.2 ± 5.44894.2 ± 5.4
1004627.4 ± 5.14660.9 ± 7.95585.9 ± 6.15893.8 ± 86.0
1504812.6 ± 169.96485.5 ± 81.76386.6 ± 44.18067.5 ± 49.2
2006327.2 ± 160.110063.8 ± 169.110005.5 ± 0.010677.6 ± 59.8
2009543.5 ± 40.1
25010655.9 ± 86.34775.7 ± 27.28994.5 ± 161.91356.7 ± 3.7
NAB (ng/g)
23204.7 ± 10.8204.7 ± 10.8176.4 ± 2.1176.4 ± 2.1
23194.8 ± 0.8194.8 ± 0.8190.1 ± 0.8190.1 ± 0.8
100199.2 ± 1.4196.0 ± 1.2223.8 ± 0.1224.5 ± 1.7
150207.6 ± 9.3290.2 ± 1.6249.1 ± 2.7340.8 ± 0.5
200282.6 ± 7.1629.9 ± 7.1423.4 ± 2.4582.1 ± 2.9
200396.8 ± 3.1
250637.3 ± 2.51683.8 ± 11.1474.9 ± 6.51734.9 ± 15.4
NNK (ng/g)
23855.6 ± 32.3855.6 ± 32.3791.7 ± 1.7791.7 ± 1.7
23866.4 ± 0.4866.4 ± 0.4754.8 ± 0.0754.8 ± 0.0
100844.9 ± 9.5866.9 ± 6.7858.8 ± 13.7884.9 ± 1.8
150898.8 ± 29.31039.6 ± 11.1963.6 ± 3.11145.3 ± 1.3
2001040.9 ± 38.62551.8 ± 14.51298.4 ± 2.92065.1 ± 14.2
2001216.6 ± 13.5
2503517.8 ± 1.84749 ± 2.41711.0 ± 27.52916.5 ± 3.8

Parameters a, b and R 2 for the MS/MS quantitation.

AnalyteabR2
NNN33.51510.17980.9997
NAT40.85070.04790.9998
NAB35.15280.07680.9992
NNK35.76550.08890.9996

Initial TSNA levels in the tobacco samples in ng/g (tobacco as is).

Sample acronymNNN (ng/g)NAT (ng/g)NAB (ng/g)NNK (ng/g)
FC-Lo365.2566.533.2283.1
FC-Up354.1458.332.6227.4
Bu-Lo4688.34616.8199.7861.0
Bu-Up4780.34662.1183.2773.3
Or (1)156.654.86.1443.0
Or (2)126.433.54.7932.6

Tobacco samples, moisture (%), alkaloid level (%), and the conditions to which the samples were exposed.

Sample acronymTobacco descriptionMoisture (%)Total alkaloids (%)Temperatures (°C)Time of exposure (min)
FC-LoLower stalk flue-cured6.442.0710015020025025
FC-UpUpper stalk flue-cured6.712.8510015020025025
Bu-LoLower stalk Burley6.362.5110015020025025
Bu-UpUpper stalk Burley7.113.4610015020025025
Or (1)Oriental blend (1)6.641.1310015020025025
Or (2)Oriental blend (2)6.231.0210015020025025

Variation of TSNAs in Oriental tobaccos upon heating (in ng/g).

Temperature (°C)Oriental (1), Or(1)Oriental (2), Or(2)
2 min5 min2 min5 min
NNN (ng/g)
23156.9 ± 1.3156.9 ± 1.3130.2 ± 0.5130.2 ± 0.5
23156.3 ± 1.3156.3 ± 1.3127.0 ± 0.3127.0 ± 0.3
23122.0 ± 2.7122.0 ± 2.7
100203.7 ± 1.9218.0 ± 1.8146.2 ± 0.1147.6 ± 1.5
150211.4 ± 1.6273.4 ± 1.5194.1 ± 0.2225.3 ± 1.5
200282.1 ± 3.4489.2 ± 13.0205 ± 0.8325.3 ± 2.6
200307.0 ± 1.0319.5 ± 5.8
250386.7 ± 11.5146.2 ± 1.0289.1 ± 5.2
250298.3 ± 0.5
NAT (ng/g)
2355.0 ± 0.955.0 ± 0.933.3 ± 0.133.3 ± 0.1
2354.6 ± 0.754.6 ± 0.733.8 ± 1.333.8 ± 1.3
10069.7 ± 0.477.7 ± 1.239.0 ± 1.039.1 ± 0.7
15073.2 ± 1.5122.1 ± 0.253.5 ± 0.867.7 ± 2.0
200130.8 ± 1.5284.4 ± 4.564.7 ± 1.0121.7 ± 2.1
200139.3 ± 0.9118.6 ± 0.5
250207.3 ± 3.812.9 ± 0.4105.6 ± 2.7
250108.8 ± 1.7
NAB (ng/g)
236.2 ± 0.36.2 ± 0.34.9 ± 0.14.9 ± 0.1
236.1 ± 0.36.1 ± 0.34.7 ± 0.14.7 ± 0.1
1006.7 ± 0.37.1 ± 0.64.8 ± 0.15.1 ± 0.0
1506.9 ± 0.510.3 ± 0.15.7 ± 0.36.7 ± 0.0
20010.3 ± 0.228.0 ± 0.27.0 ± 0.018.1 ± 0.4
20011.2 ± 0.417.6 ± 0.0
25018.4 ± 0.425.6 ± 0.113.9 ± 0.7
25015.3 ± 0.1
NNK (ng/g)
2341.3 ± 0.641.3 ± 0.632.9 ± 0.432.9 ± 0.4
2344.7 ± 1.244.7 ± 1.232.4 ± 0.432.4 ± 0.4
10054.2 ± 0.854.7 ± 0.438.2 ± 2.038.2 ± 0.4
15055.2 ± 1.457.5 ± 0.445.2 ± 0.849.0 ± 1.5
20056.8 ± 2.0119.8 ± 0.448.4 ± 0.676.8 ± 0.1
20059.8 ± 0.776.8 ± 0.3
25085.5 ± 1.491.5 ± 2.862.0 ± 0.3
25065.9 ± 0.2

Other parameters for the MS/MS detection.

CompoundIon for Q1Ion for Q3Retention time (min)DP1(V)CE2 (V)CXP3 (V)
NNN178.1148.14.7330159
NNN-d4182.1152.14.7030169
NAT190.1160.15.352815.510
NAT-d4194.1164.15.31281610
NAB192.1162.15.40261811
NAB-d4196.1166.15.37261710
NNK208.1122.15.163017.613
NNK-d4212.1126.15.15301612

U.S. Food & Drug Administration (FDA): Reporting Harmful and Potentially Harmful Constituents in Tobacco Products and Tobacco Smoke Under Section 904(a)(3) of the Federal Food, Drug, and Cosmetic Act; Issued by Center for Tobacco Products, Docket Number: FDA-2012-D-0049-0002, April 2012. Available at: https://www.fda.gov/TobaccoProducts/Labeling/RulesRegulationsGuidance/ucm297752.htm(accessed July 2020).U.S. Food & Drug Administration (FDA)Reporting Harmful and Potentially Harmful Constituents in Tobacco Products and Tobacco Smoke Under Section 904(a)(3) of the Federal Food, Drug, and Cosmetic ActIssued by Center for Tobacco Products, Docket Number: FDA-2012-D-0049-0002April2012Available at: https://www.fda.gov/TobaccoProducts/Labeling/RulesRegulationsGuidance/ucm297752.htm(accessed July 2020).Search in Google Scholar

International Agency for Research on Cancer (IARC): Agents Classified by the IARC Monographs, Volumes 1–127; IARC, Lyon, France. Available at: https://monographs.iarc.fr/agents-classified-by-the-iarc/ (accessed July 2020).International Agency for Research on Cancer (IARC)Agents Classified by the IARC Monographs1–127IARCLyon, FranceAvailable at: https://monographs.iarc.fr/agents-classified-by-the-iarc/ (accessed July 2020).Search in Google Scholar

Foster, M., C. Liu, M.G. Duke, K.G. McAdam, and C.J. Proctor: An Experimental Method to Study Emissions From Heated Tobacco Between 100–200 °C; Chem. Cent. J. 9 (2015) 20–30. DOI: 10.1186/s13065-015-0096-1.FosterM.LiuCDukeM.G.McAdamK.G.ProctorC.J.An Experimental Method to Study Emissions From Heated Tobacco Between 100–200 °CChem. Cent. J.92015203010.1186/s13065-015-0096-1Open DOISearch in Google Scholar

Uchiyama, S., M. Noguchi, N. Takagi, H. Hayashida, Y. Inaba, H. Ogura, and N. Kunugita: Simple Determination of Gaseous and Particulate Compounds Generated From Heated Tobacco Products; Chem. Res. Toxicol. 31 (2018) 585–593. DOI: 10.1021/acs.chemrestox.8b00024.UchiyamaS.NoguchiMTakagiN.HayashidaH.InabaY.OguraH.KunugitaN.Simple Determination of Gaseous and Particulate Compounds Generated From Heated Tobacco ProductsChem. Res. Toxicol.31201858559310.1021/acs.chemrestox.8b00024Open DOISearch in Google Scholar

Djordjevic, M.V., J. Fan, L.P. Bush, K.D. Brunnemann, and D. Hoffmann: Effect of Storage Conditions on Levels of Tobacco Specific N-Nitrosamines and N-Nitrosamino Acids in U.S. Moist Snuff; J. Agric. Food. Chem. 41 (1993) 1790–1794. DOI: 10.1021/jf00034a051.DjordjevicM.V.FanJBushL.P.BrunnemannK.D.HoffmannD.Effect of Storage Conditions on Levels of Tobacco Specific N-Nitrosamines and N-Nitrosamino Acids in U.S. Moist SnuffJ. Agric. Food. Chem.4119931790179410.1021/jf00034a051Open DOISearch in Google Scholar

Stepanov, I., S.G. Carmella, S.S. Hecht, and G. Duca: Analysis of Tobacco-Specific Nitrosamines in Moldovan Cigarette Tobacco; J. Agric. Food Chem. 50 (2002) 2793–2797. DOI: 10.1021/jf011552j.StepanovI.CarmellaS.GHechtS.S.DucaG.Analysis of Tobacco-Specific Nitrosamines in Moldovan Cigarette TobaccoJ. Agric. Food Chem.5020022793279710.1021/jf011552jOpen DOISearch in Google Scholar

Wu, W., D.L. Ashley, and C.H. Watson: Simultaneous Determination of Five Tobacco-Specific Nitrosamines in Mainstream Cigarette Smoke by Isotope Dilution Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry; Anal. Chem. 75 (2003) 4827–4832. DOI: 10.1021/ac030135y.WuW.AshleyD.LWatsonC.HSimultaneous Determination of Five Tobacco-Specific Nitrosamines in Mainstream Cigarette Smoke by Isotope Dilution Liquid Chromatography/Electrospray Ionization Tandem Mass SpectrometryAnal. Chem.7520034827483210.1021/ac030135yOpen DOISearch in Google Scholar

Jansson, C., A. Paccou, and B.G. Oesterdahl: Analysis of Tobacco-Specific N-Nitrosamines in Snuff by Ethyl Acetate Extraction and Liquid Chromatography-Tandem Mass Spectrometry; J. Chromatogr. A 1008 (2003) 135–143. DOI: 10.1016/S0021-9673(03)00981-6.JanssonC.PaccouAOesterdahlB.GAnalysis of Tobacco-Specific N-Nitrosamines in Snuff by Ethyl Acetate Extraction and Liquid Chromatography-Tandem Mass SpectrometryJ. Chromatogr. A1008200313514310.1016/S0021-9673(03)00981-6Open DOISearch in Google Scholar

Oesterdahl, B.G., C. Jansson, and A. Paccou: Decreased Levels of Tobacco-Specific N-Nitrosamines in Moist Snuff on the Swedish Market; J. Agric. Food Chem. 52 (2004) 5085–5088. DOI: 10.1021/jf049931a.OesterdahlB.G.JanssonCPaccouADecreased Levels of Tobacco-Specific N-Nitrosamines in Moist Snuff on the Swedish MarketJ. Agric. Food Chem.5220045085508810.1021/jf049931aOpen DOISearch in Google Scholar

Wagner, K.A., N.H. Finkel, J.E. Fossett, and I.G. Gillman: Development of a Quantitative Method for the Analysis of Tobacco-Specific Nitrosamines in Mainstream Cigarette Smoke Using Isotope Dilution Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry; Anal. Chem. 77 (2005) 1001–1006. DOI: 10.1021/ac048887v.WagnerK.A.FinkelN.HFossettJ.E.GillmanI.G.Development of a Quantitative Method for the Analysis of Tobacco-Specific Nitrosamines in Mainstream Cigarette Smoke Using Isotope Dilution Liquid Chromatography/Electrospray Ionization Tandem Mass SpectrometryAnal. Chem.7720051001100610.1021/ac048887vOpen DOISearch in Google Scholar

Stepanov, I., J. Jensen, D. Hatsukami, and S.S. Hecht: Tobacco-Specific Nitrosamines in New Tobacco Products; Nicotine Tob. Res. 8 (2006) 309–313. DOI: 10.1080/14622200500490151.StepanovI.JensenJHatsukamiD.HechtS.S.Tobacco-Specific Nitrosamines in New Tobacco ProductsNicotine Tob. Res.8200630931310.1080/14622200500490151Open DOISearch in Google Scholar

Wu, J., P. Joza, M. Sharifi, W.S. Rickert, and J.H. Lauterbach: Quantitative Method for the Analysis of Tobacco-Specific Nitrosamines in Cigarette Tobacco and Mainstream Cigarette Smoke by Use of Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry; Anal. Chem. 80 (2008) 1341–1345. DOI: 10.1021/ac702100c.WuJ.JozaPSharifiM.RickertW.S.LauterbachJ.H.Quantitative Method for the Analysis of Tobacco-Specific Nitrosamines in Cigarette Tobacco and Mainstream Cigarette Smoke by Use of Isotope Dilution Liquid Chromatography Tandem Mass SpectrometryAnal. Chem.8020081341134510.1021/ac702100cOpen DOISearch in Google Scholar

Moldoveanu, S.C. and M. Borgerding: Formation of Tobacco Specific Nitrosamines in Mainstream Cigarette Smoke; Part 1, FTC Smoking; Beitr. Tabakforsch. Int. 23 (2008) 19–31. DOI: 10.2478/cttr-2013-0845.MoldoveanuS.CBorgerdingMFormation of Tobacco Specific Nitrosamines in Mainstream Cigarette Smoke; Part 1, FTC SmokingBeitr. Tabakforsch. Int.232008193110.2478/cttr-2013-0845Open DOISearch in Google Scholar

Sleiman, M., R.L. Maddalena, L.A. Gundel, and H. Destaillats: Rapid and Sensitive Gas Chromatography-Ion-Trap Tandem Mass Spectrometry Method for the Determination of Tobacco-Specific N-Nitrosamines in Secondhand Smoke; J. Chromatogr. A 1216 (2009) 7899–7905. DOI: 10.1016/j.chroma.2009.09.020.SleimanM.MaddalenaR.LGundelL.A.DestaillatsH.Rapid and Sensitive Gas Chromatography-Ion-Trap Tandem Mass Spectrometry Method for the Determination of Tobacco-Specific N-Nitrosamines in Secondhand SmokeJ. Chromatogr. A121620097899790510.1016/j.chroma.2009.09.020Open DOISearch in Google Scholar

Xiong, W., H. Hou, X. Jiang, G. Tang, and Q. Hu: Simultaneous Determination of Four Tobacco-Specific N-Nitrosamines in Mainstream Smoke for Chinese Virginia Cigarettes by Liquid Chromatography-Tandem Mass Spectrometry and Validation Under ISO and “Canadian Intense” Machine Smoking Regimes; Anal. Chim. Acta 674 (2010) 71–78. DOI: 10.1016/j.aca.2010.06.011.XiongW.HouHJiangX.TangG.HuQ.Simultaneous Determination of Four Tobacco-Specific N-Nitrosamines in Mainstream Smoke for Chinese Virginia Cigarettes by Liquid Chromatography-Tandem Mass Spectrometry and Validation Under ISO and “Canadian Intense” Machine Smoking RegimesAnal. Chim. Acta6742010717810.1016/j.aca.2010.06.011Open DOISearch in Google Scholar

Kim, H.-J. and H.-S. Shin: Determination of Tobacco-Specific Nitrosamines in Replacement Liquids of Electronic Cigarettes by Liquid Chromatography-Tandem Mass Spectrometry; J. Chromatogr. A 1291 (2013) 48–55. DOI: 10.1016/j.chroma.2013.03.035.KimH.-JShinH.-SDetermination of Tobacco-Specific Nitrosamines in Replacement Liquids of Electronic Cigarettes by Liquid Chromatography-Tandem Mass SpectrometryJ. Chromatogr. A12912013485510.1016/j.chroma.2013.03.035Open DOISearch in Google Scholar

Zhang, J., R. Bai, X. Yi, Z. Yang, X. Liu, J. Zhou, and W. Liang: Fully Automated Analysis of Four Tobacco-Specific N-Nitrosamines in Mainstream Cigarette Smoke Using Two-Dimensional Online Solid Phase Extraction Combined with Liquid Chromatography-Tandem Mass Spectrometry; Talanta 146 (2016) 216–224. DOI: 10.1016/j.talanta.2015.08.057.ZhangJ.BaiRYiX.YangZ.LiuX.ZhouJ.LiangW.Fully Automated Analysis of Four Tobacco-Specific N-Nitrosamines in Mainstream Cigarette Smoke Using Two-Dimensional Online Solid Phase Extraction Combined with Liquid Chromatography-Tandem Mass SpectrometryTalanta146201621622410.1016/j.talanta.2015.08.057Open DOISearch in Google Scholar

Moldoveanu, S.C., J. Zhu, and N. Qian: Analysis of Traces of Tobacco-Specific Nitrosamines (TSNAs) in USP Grade Nicotine, E-Liquids, and Particulate Phase Generated by the Electronic Smoking Devices; Beitr. Tabakforsch Int. 27 (2017) 86–97. DOI: 10.1515/cttr-2017-0009MoldoveanuS.C.ZhuJQianNAnalysis of Traces of Tobacco-Specific Nitrosamines (TSNAs) in USP Grade Nicotine, E-Liquids, and Particulate Phase Generated by the Electronic Smoking DevicesBeitr. Tabakforsch Int272017869710.1515/cttr-2017-0009Open DOISearch in Google Scholar

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