Chinese Journal of Analytical Chemistry 52.

9 (2024) 100430

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Chinese Journal of Analytical Chemistry

journal homepage: [Link]/locate/cjac

Determination of four tobacco-specific nitrosamines in electronic cigarette

liquids and aerosols by UPLC-QTOF-HRMS
Chunqiong WANG a,1, Wei LI a,b,1, Yanbo ZENG b, Yongliang WU c, Dan CHEN a,
Hongming MENG a, Ke ZHANG a, Xiaowei ZHANG a, Ganpeng LI b,∗, Kai LIU a,∗
a
Yunnan Tobacco Quality Supervision and Test Station, Kunming 650106, PR China
b
School of Ethnic Medicine, Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu
University, Kunming 650500, PR China
c
Guangdong Tobacco Quality Supervision and Test Station, Guangzhou 510175, PR China

a r t i c l e i n f o a b s t r a c t

Keywords: To elucidate the concentrations of four tobacco-specific nitrosamines (TSNAs) in commercial e-cigarettes, analyze
E-cigarette the correlation between TSNAs and Nicotine, and clarify the main sources of TSNAs in e-cigarettes. Ultra-high
E-liquids performance liquid chromatography with quadrupole time-of-flight high-resolution mass spectrometry (UPLC-
Aerosols
QTOF-HRMS) was employed to determine the concentrations of four TSNAs in thirty-two commercially available
TSNAs
e-cigarettes. The results demonstrated that the method exhibits excellent linearity and high mass accuracy. The
Concentration determination
limits of quantification (LOQ) of four TSNAs in e-cigarettes were 0.0010 –0.0165 (e-liquids) and 0.0032–0.0554
(aerosols) ng·g−1 . The limits of detection (LOD) of four TSNAs in e-cigarettes were 0.0011–0.0165 (e-liquids)
and 0.0033–0.0537 (aerosols) ng·20 puffs−1 . The recovery of four TSNAs ranged from 73.06 % to 109.95 %.
The e-liquids contained: 0–33.970 (NNN), 0.063–15.654 (NNK), 0–10.033 (NAT), and 0–0.251 (NAB) ng·g−1 .
The aerosols contained: 0–60.662 (NNN), 0.021–9.435 (NNK), 0.202– 29.866 (NAT), and 0–2.841 (NAB) ng·20
puffs−1 . The correlation analysis results have shown that there was no significant correlation between the con-
centrations of four TSNAs and nicotine in e-cigarettes. The concentrations of four TSNAs in e-liquid containing
tobacco extract were significantly higher than that in e-liquid without tobacco extract, suggested that the main
source of TSNAs was tobacco extract. This method is fast, simple, highly sensitive, and has low detection limits.
The approach taken can provide data support for the actual supervision of e-cigarettes, and evaluation of safety
components, contributing to effective quality evaluation systems.

1. Introduction TSNAs are a special class of nicotinoids, where the addition of nico-
tine extracted from tobacco may introduce a large number of nicoti-
The electronic cigarette (e-cigarette) is an electronic nicotine deliv- noids. The nicotinoid composition and concentration have a significant
ery system (ENDS) designed to deliver nicotine to the user’s lungs. The impact on the sensory response and the safety of tobacco. Nicotinic com-
entry of e-cigarettes into the Chinese domestic market dates from 2004, pounds can directly stimulate the human body’s central nervous system
with the introduction in the United States in 2006, and the European with consequent physiological reactions. Nicotinoids are readily metab-
market in 2007 [1,2]. Current research has shown that the chemical olized into reduced alkaloids in tobacco preparation and processing,
composition of e-liquids and aerosols are toxic to organs such as the hu- forming TSNAs exhibiting high levels of nitrite carcinogenicity [11,12].
man respiratory system, cardiovascular system, nervous system, liver, Eight TSNAs (as shown in Fig. 1) have been identified:
and kidneys [3–5]. Toxic substances reported in e-cigarettes include car- N-nitrosonornicotine (NNN), N’-nitrosoanatabine (NAT), 4-
bonyls, aldehydes, volatile organic compounds (VOCs), tobacco-specific (methylnitrosamino)−1-(3-pyridyl)−1-butanone (NNK), N-
nitrosamines (TSNAs), aromatic amines, polycyclic aromatic hydrocar- nitrosoanabasine (NAB), 4-(methylnitrosamino)−4-(3-pyridyl)−1-
bons (PAHs), and heavy metals. The concentrations of these chemical butanol (isoNNAL), 4-(methylnitrosamino)−1-(3-pyridyl)−1-butanol
substances differ in e-cigarettes [6–10]. (NNAL), 4-(methylnitrosamino)−4–3-pyridyl) butyric acid (isoNNAC),

∗
Corresponding authors.
E-mail addresses: ganpeng_li@[Link] (G. LI), 13888092288@[Link] (K. LIU).
1
These authors contributed equally to this work.

[Link]
Received 6 August 2024; Received in revised form 26 August 2024; Accepted 1 September 2024
Available online 2 September 2024
1872-2040/© 2024 The Authors. Published by Elsevier Ltd on behalf of Changchun Institute of Applied Chemistry Chinese Academy of Sciences. This is an open
access article under the CC BY-NC-ND license ([Link]
C. WANG, W. LI, Y. ZENG et al. Chinese Journal of Analytical Chemistry 52.9 (2024) 100430

Fig. 1. Chemical structures of eight TSNAs.

and 4-(methylnitrosamino)−4-(3-pyridyl)-butanal (NNA), in which deuterated N-nitrosoanabasine (NAB-d4 , TRC–N524252–1 mg); Nico-
NNN, NNK, NAB, and NAT are more abundant and more carcinogenic tine, Solanone, Tabanone (purity > 99 %) and burley tobacco leaves
[12–14]. were provided by Yunnan Tobacco Technology Center; Vegetable
TSNAs exist in a minimum amount in e-liquids, these levels are glycerin and propylene glycol were obtained from Procter & Gamble
comparable to those in nicotine pharmaceuticals and are much lower and Dow Co., and the H-MOR zeolite (SiO2 /Al2 O3 = 25) was supplied
than those of conventional cigarette smoke [15]. The determination of by Tianjin Nankai Catalyst Co., Ltd.; Watsons distilled water was
TSNAs is difficult requiring an analytical method that is sufficiently sen- utilized with ammonium acetate (HPLC grade, Shanghai Maclean’s
sitive and selective enough to detect all TSNAs [16]. Many of the ana- Biochemical Technology Co., Ltd.), methanol, acetonitrile and ethanol
lytical methods for the determination of TSNAs have been described (LC-MS grade, Fisher Company of United States).
in relation to tobacco and mainstream cigarette smoke [17–30]. The UPLC-QTOF-HRMS (Acquity UPLC I-Class Plus Xevo G2-XS Qtof-MS,
most widely used method for the analysis of TSNAs is GC-TEA [12]. Waters, United States) unit was employed with formic acid. Formic
Gas chromatography (GC) [17], gas chromatography-mass spectrom- acid, leucine enkephalin, and sodium hydroxide were all provided by
etry (GC–MS) [18–20], and high-performance liquid chromatography Waters. Analytic measurements employed an e-cigarette smoking ma-
(HPLC) [21,22] have also been utilized. These approaches are specific chine (SML 1000E, Hefei Zhongwo Instrument Technology Co., Ltd.);
to nitroso-containing compounds and have been successfully used for ultrasonic cleaner (JM-10D-40, Shenzhen Jiemeng Cleaning Equipment
many years in determining NNN and NNK in tobacco smoke. In recent Co., Ltd.); electronic analytical balances (ME204E, METTLER TOLEDO);
years, liquid chromatography-mass spectrometry (LC-MS/MS) has been Cambridge filters (44 mm, Whatman, United Kingdom); volumetric
used [23–30], and has shown very high sensitivity and reproducibility flasks (100, 10, and 5 mL, Chongqing Xinwei Glass Instrument Co., Ltd.);
with respect to TSNAs. Thirty-two commercially available e-cigarettes were tested (labelled
In this study, a targeted quadrupole time-of-flight (QTOF) method S1-S32), with S1-S10 being Brand A, S11-S20 (Brand B), S21-S25 (Brand
has been developed using a TOF multiple reaction monitoring (MRM) C), S26-S27 (Brand D), S28-S29 (Brand E), and S30-S32 (Brand F).
acquisition mode that serves to improve sensitivity and selectivity in de-
tecting four TSNAs, enabling a rapid analysis of four TSNAs in e-liquids 2.2. UPLC-QTOF-MS analysis method
and aerosols. The differences in the concentration differences of TSNAs
in 32 commercially available e-cigarettes have been evaluated, identi- Waters Acquity UPLC BEH C18 Column; The mobile phase system
fying potential sources of TSNAs production to provide data support for was acetonitrile (A)-water (B); The flow rate was 0.4 mL·min−1 ; The
the regulation, testing, and safety assessment of e-cigarettes. column temperature was 45 °C; The injection volume was 10 μL. Gradi-
ent elution conditions:

2. Experimental
Time/min A B Curve

2.1. Materials 0 1 99 6
6 90 10 5
7 90 10 5
N-nitrosonornicotine (NNN, TRC–N535001–10MG), deuter- 7.1 99 1 5
ated N-nitrosonornicotine (NNN-d4 , TRC–N535002–1 mg), 4- 8 1 99 5
(methylnitrosamino)−1-(3-pyridyl)−1-butanone (NNK, TRC-M325750– 10 1 99 5
10MG), deuterated 4-(methylnitrosamino)−1-(3-pyridyl)−1-butanone
(NNK-d4 , TRC-M325751–1 mg), N′-nitrosoanatabine (NAT, TRC– The ion source was an electrospray ionization source (ESI), and the
N524745–10 mg), deuterated N’-nitrosoanatabine (NAT-d4 , TRC- scanning mode was positive ion scanning. The detection mode was mul-
KIT8412–1 × 1 mL), N-nitrosoanabasine (NAB, TRC–N524250–10 mg), tiple reaction monitoring mode (MRM); Scanning time: 0.25 s; Capillary

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C. WANG, W. LI, Y. ZENG et al. Chinese Journal of Analytical Chemistry 52.9 (2024) 100430

Table 1
MRM ion pair information.

Precursor ions Cone voltage Collision energy

Compounds Rt (min) (m/z) Sub ions (m/z) (eV) (V)

NNN 1.91 178.0975 148.0986 24 10

105.0443∗ 24 16
NNK 2.04 208.1081 122.0594 28 12
148.0767∗ 28 32
NAT 2.34 190.0975 160.0983 30 10
106.0635∗ 30 26
NAB 2.44 192.1131 162.1145 30 12
133.0830∗ 30 20
NNN-d4 1.90 182.1226 152.1246 24 10
124.0934∗ 24 12
NNK-d4 2.04 212.1332 126.0846 28 16
110.0888∗ 28 32
NAT-d4 2.44 194.1226 164.1255 30 20
110.0853∗ 30 18
NAB-d4 2.44 196.1382 166.1395 30 26
137.1021∗ 30 25

Note: ∗ represents quantitative ion pairs.

voltage: 2.5 kV; Desolvation temperature: 550 °C Ion source tempera-

ture: 150 °C; Air curtain air flow rate: 100 L·h−1 ; The desolvation gas
flow rate: 1000 L·h−1 . Information regarding the selection of multiple
reaction monitoring ion pairs is provided in Table 1.

2.3. Preparation of standards

Mixed standard solution: 10 mg of NNN, NNK, NAT and NAB were

accurately weighed and placed in four 10 mL brown volumetric flasks,
and methanol was added and shaken well to obtain a single standard so-
lution of 1 mg·mL−1 . Then accurately pipette 1 mL of each single stan-
Fig. 2. The appearance of five types of e-liquid.
dard solution was pipetted into a 100 mL volumetric flask, with the
volume of methanol, and shake well to obtain a mixed standard solu-
tion of 10 μg·mL−1 . Mixed internal standard solution: 1 mg of NNN-d4 , as T2 (tobacco leaves not modulated and fermented), and T3 (tobacco
NNK-d4 , NAT-d4 , and NAB-d4 were accurately weighed and placed in a leaves modulated or fermented). The e-liquids with untreated tobacco
100 mL volumetric flask, methanol was added and shaken well to obtain extract added were denoted as T4 (tobacco leaves not modulated and
10 μg·mL−1 mixed internal standard solution. Series of standard work- fermented) and T5 (tobacco leaves modulated or fermented), as shown
ing solution: take the mixed standard solution and the mixed internal in Fig. 2.
standard solution, with 0.1 mol·L−1 ammonium acetate solution deliv-
ered 10, 5, 1, 0.5, 0.1 μg·L−1 solutions. Further dilution of the 0.1 μg·L−1 2.5. Sample extraction
sample yielded mixed internal standard solutions of 0.05, 0.01, 0.001,
and 0.0001 μg·L−1 . The addition of 10 μL mixed internal standard solu- A Cambridge filter with a diameter of 44 mm was placed in e-
tion resulted in a series of nine standard working solutions. cigarette smoking machine, and the rough side of the filter was facing
the incoming flue gas. The suction was carried out under the conditions
2.4. Preparation of e-liquid of a suction capacity of 55 mL, a puff of puff for 30 s, a continuous 3 s
for each time, and 20 puffs for each sample.
100 g of burley tobacco leaves was added to 1000 mL deionized wa- Weigh 1 g of e-liquid and take a Cambridge filter that captured
ter containing 12 g Vitamin C, soaked in the dark at 65 °C for 24 h, then aerosol, and place in 50 mL graduated centrifuge tube. Then, 20 mL 0.1
filtered, and the extraction solution diluted to 1000 mL. Then taking mol·L−1 ammonium acetate solution and 20 μL mixed internal standard
100 mL of the extraction solution in portions, 500 g sample of adsorbent solution were added and extracted by ultrasonic treatment for 40 min,
(H-MOR zeolite) was added to known volumes of the extraction solu- filtering through a 0.22 μm PTFE filter membrane to obtain the test so-
tion, stirring in a 30 °C water bath in the dark for 1 h, and centrifuging lution.
at 5000 r/min for 10 min to separate the supernatant for future use. The
supernatant (100 mL) was extracted three times using 300 mL ethyl ac- 2.6. Data analysis
etate, the extractants were combined and dried under reduced pressure
to concentrate, before diluting with concentrated solid:ethanol = 1:1 to SPSS Statistics 27.0 (IBM) was used for correlation analysis, where
obtain the tobacco extract. all data were standardized.
40 g glycerol, 45 g propylene glycol, and 5 g tobacco extract were
added and mixed to produce a mother liquor. Dissolve 1.5 g of nicotine 3. Results and discussion
and 200 mg of vitamin C were dissolved in 50 mL of deionized water,
and added them to the mother liquor to dissolve thoroughly to obtain 3.1. Optimization of analysis conditions
a mixed solution; 500 mg of cigarette essence and 500 mg flavoring
additive were added to the mixed solution with stirring, and aging for In contrast to a typical food sample matrix, e-cigarette oil was mainly
24 h to obtain the e-liquid. The e-liquid without tobacco extract was directly soluble in propylene glycol and glycerol from tobacco extracts,
denoted as T1, and the e-liquids with adsorbed TSNAs were denoted essence, and spices. This type of matrix must be diluted using a large

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C. WANG, W. LI, Y. ZENG et al. Chinese Journal of Analytical Chemistry 52.9 (2024) 100430

Table 2
Linear ranges, regression equations, correlation coefficients, limits of detection, and limit of quantitation of four TSNAs (n = 6).

Linear range LOD LOQ

Compounds (μg·L−1 ) Regression equation R LOD (ng·g−1 ) LOQ (ng·g−1 ) (ng·20puffs−1 ) (ng·20puffs−1 )

NNN 0.0001–5 Y = 50500X+19.7 0.9997 0.0024 0.0089 0.0072 0.0250

NNK 0.0001–5 Y = 295000X-43.2 0.9993 0.0011 0.0032 0.0011 0.0033
NAT 0.0001–10 Y = 33700X+5.45 0.9994 0.0165 0.0554 0.0160 0.0537
NAB 0.0001–1 Y = 40000X-70 0.9993 0.0030 0.0060 0.0060 0.0161

Table 3
LOD and LOQ values of four TSNAs analysis methods in the literature.

Samples Method LOD LOQ Ref.

NNN NNK NAB NAT NNN NNK NAB NAT

Electronic cigarettes (μg·L−1 ) LC-MS/MS 0.02 0.02 0.02 0.01 0.06 0.07 0.06 0.04 12
Cigarette (ng·g−1 ) GPC-GC-MS2 0.07 0.03 0.22 0.23 0.24 0.09 0.73 0.76 19
Heated tobacco products (ng·mL−1 ) SPME-LC-MS/MS 0.040 0.049 0.068 0.024 13.1 16.2 22.4 8.1 24
Tobacco leaves (ng·g−1 ) LC-MS/MS 1.0 0.5 0.2 0.5 2.0 1.0 0.6 1.0 25
Cigarette mainstream smoke (ng cigarette−1 ) LC-MS/MS 0.3 0.1 0.1 0.1 0.6 0.2 0.2 0.2
Cigarette mainstream smoke (ng cigarette−1 ) SPE-LC-MS/MS 2.0 0.3 0.2 1.0 6.0 3.0 0.6 1.0 27
Tobacco leaves (ng·g−1 ) LC-MS/MS 18 6 15 15 75 24 60 60 29

Table 4
Precision test results (n = 3).

Compounds 1 2 3 4 5 6 RSD

NNN 301,023 310,499 327,425 315,139 350,493 337,189 5.66 %

NNK 788,495 782,336 824,764 831,810 832,298 789,826 2.93 %
NAT 134,884 131,351 136,790 140,875 141,665 133,254 3.03 %
NAB 170,862 160,835 178,012 182,103 178,177 191,120 5.80 %

volume of water as solvent before injection analysis. Dilution can affect three times the standard deviation of the results, and the limit of quan-
the concentration of the substance to be tested, thereby lowering the tification (LOQ) set at ten times the standard deviation; the results are
sensitivity of the detection method. Moreover, the four TSNAs under shown in Table 2. The linear range of the four TSNAs standard curves
consideration have similar polarities and cannot be effectively separated was 0.0001–10 μg·L−1 , which was wider than reported for existing meth-
by chromatography. Active monitoring of these harmful compounds at ods, and enables a quantitative analysis of samples with different con-
relatively low concentrations (ng·mL−1 or lower) is crucial and requires centrations. The LOD and LOQ values for the four TSNAs in e-liquid
the use of highly sensitive and selective analytical methods. Previous were also lower than those reported in the relevant literature (as shown
studies have mainly relied on LC-MS for detection and analysis. The in Table 3) [12,19,24,25,27,29] and satisfy the analysis requirements
detection limit of current analytical methods was relatively high, and for trace TSNAs in e-liquid and aerosol special matrix samples.
the sensitivity of LC-MS and other instruments was low, which does not
facilitate sufficiently accurate qualitative and quantitative analysis. 3.2.2. Precision test
After determining the retention time and optimal ion transport set- 10 μg·L−1 mixed standard solution was injected six times in accor-
tings, the TOF MRM acquisition mode was used to determine the par- dance with the analysis method. The calculated relative standard devi-
ent ion based on the molecular weight of each compound, and the Q1 ations (RSD) based on the peak areas for the four TSNAs are given in
full-scan adjustment parameters. Applying automatic optimization, two Table 4, demonstrating a high level of precision.
pairs of highly responsive and interference-free characteristic ions were
selected as target ion pairs, and the collision energy CE plasma source 3.2.3. Reproducibility test
parameters were optimized to develop a targeted QTOF method. This 1 g of e-liquids and six portions of Cambridge filter for capturing
method enables an enhancement of the m/z value for a specified com- aerosols were used to prepare the test solution, injected three times in
pound, accurately measuring the mass of all ions to improve analysis accordance with the analysis method. Calculating the RSD values of the
sensitivity and selectivity. The parameters required for setting the MRM peak areas of four TSNAs (Table 5) demonstrated good method repeata-
are shown in Table 2, and the MRM chromatograms associated with the bility.
four TSNAs are presented in Fig. 3.
3.2.4. Recovery test
3.2. Method validation In the spiked recovery test, the relatively low concentrations of four
TSNAs in the selected e-liquids and aerosols resulted in a large error in
3.2.1. Analysis of linearity determining the level of recovery. Consequently, standard samples were
The relevant instrumental parameters were used to analyze the added at concentrations close to the background of e-liquid or aerosols
mixed standard solution series, plotting the standard curve with the and six parallel measurements were conducted to determine the recov-
mass concentration of four TSNAs on the abscissa and the ratio of the ery levels. The recovery of e-liquid and aerosols was between 73.06 %
corresponding peak areas relative to the internal standard on the ordi- and 109.95 %, which meets the experimental requirements; sample re-
nate. As the corresponding signal-to-noise ratio was inaccessible when covery results were given in Table 6.
the UPLC-QTOF-HRMS system utilizes an internal standard injection, The concentration of NAT in e-liquid was approximately 0.5 μg·L−1 ,
the e-liquid blank solution and aerosol blank solution were measured with a relatively high concentration and recovery rate that was 80 %
eleven times during the analysis. The limit of detection (LOD) was set at lower than that of other compounds. This may be attributed to the low

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C. WANG, W. LI, Y. ZENG et al. Chinese Journal of Analytical Chemistry 52.9 (2024) 100430

Fig. 3. MRM chromatograms of four TSNAs.

Note: NNN (a); NNK (b); NAT (c); NAB (d).

solubility of NAT, leading to incomplete dissolution in ammonium ac- on the Cambridge filter, and leading to a loss during the extraction
etate during ultrasonic extraction. As a result of the interference of the process.
e-liquid matrix, the loss in the extraction process was large. The main
reasons for the low recovery rate of NAB in aerosols are as follows: 3.3. Analysis of commercial samples
○1 the addition of excess standard substances leads to a large error; ○ 2
the background value of NAB was relatively low with a consequent re- Using the established detection method, the concentration of four
duction in recovery rate; ○ 3 a minor component of NAB was adsorbed TSNAs in 32 commercially available e-liquids and aerosols was deter-

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C. WANG, W. LI, Y. ZENG et al. Chinese Journal of Analytical Chemistry 52.9 (2024) 100430

Fig. 3. Continued

mined. The correlation analysis was conducted by importing the spec- 60.662 (NNN), 0.021–9.435 (NNK), 0.202–29.866 (NAT), and 0–2.841
ified nicotine concentration values into IBM SPSS Statistics 27.0. The (NAB) ng·20 puffs−1 , respectively.
corresponding results are presented in Tables 7 and 8. There are sig- The nicotine concentration in S1–S25 and S30–S32 is 18 mg·g−1 ,
nificant differences in the concentrations of four TSNAs in 32 different whereas the concentration in S26 and S27 is 20 mg·g−1 , and 10 mg·g−1
samples of e-liquid and aerosol. The concentrations of the four TSNAs in in S28 and S29. There is no significant correlation between the con-
e-liquids fall within the ranges 0–33.970 (NNN), 0.063–15.654 (NNK), centrations of four TSNAs in e-liquid and nicotine, indicating that the
0–10.033 (NAT), and 0–0.251 (NAB) ng·g−1 . The aerosols contained: 0– differences in nicotine concentration do not influence the concentra-

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C. WANG, W. LI, Y. ZENG et al. Chinese Journal of Analytical Chemistry 52.9 (2024) 100430

Table 5
Reproducibility test results (n = 3).

Samples Compounds 1 2 3 4 5 6 RSD

E-liquid NNN – – – – – – –
NNK 360 398 396 393 348 372 5.56 %
NAT 16,930 16,331 16,581 16,794 17,163 17,662 2.76 %
NAB – – – – – – –
Aerosol NNN 172,688 174,076 175,012 176,827 177,732 179,162 1.38 %
NNK 4913 4712 4469 4431 4406 4322 4.93 %
NAT 12,592 12,430 12,357 11,906 11,841 11,337 3.89 %
NAB 60 61 61 57 56 55 4.56 %

Note: “–” indicates not detected.

Table 6
Recovery rates of four TSNAs (n = 3).

Fortification Measured concentration

Samples Compounds Background (μg·L−1 ) level (μg·L−1 ) (μg·L−1 ) Recovery (%) RSD

E-liquid NNN 0.000, 0.000, 0.000 0.010 0.009, 0.010, 0.010 92.93, 95.11, 103.03 5.12 %
0.000, 0.000, 0.000 0.009, 0.009, 0.010 90.16, 91.35, 98.67
NNK 0.002, 0.002, 0.001 0.010 0.011, 0.011, 0.011 90.00, 93.90, 91.56 2.69 %
0.001, 0.001, 0.000 0.010, 0.011, 0.010 90.00, 93.25, 96.37
NAT 0.502, 0.484, 0.492 0.500 0.920, 0.910, 0.900 83.55, 85.01, 81.61 5.29 %
0.498, 0.509, 0.524 0.917, 0.917, 0.889 83.76, 81.59, 73.06
NAB 0.000, 0.000, 0.000 0.010 0.010, 0.010, 0.010 98.75, 100.00, 96.75 4.26 %
0.000, 0.000, 0.002 0.011, 0.009, 0.010 106.75, 94.25, 100.75
Aerosol NNN 2.033, 1.863, 2.079 2.000 4.027, 4.047, 4.058 99.71, 109.23, 98.95 4.22 %
2.115, 2.133, 2.161 4.060, 4.131, 4.154 97.25, 99.91, 99.62
NNK 0.017, 0.016, 0.015 0.020 0.036, 0.038, 0.037 97.46, 109.37, 107.03 6.27 %
0.015, 0.014, 0.014 0.037, 0.033, 0.035 109.95, 94.44, 106.41
NAT 0.373, 0.369, 0.367 0.300 0.676, 0.661, 0.658 90.88, 97.42, 97.27 5.75 %
0.353, 0.351, 0.336 0.622, 0.578, 0.535 89.48, 85.48, 86.08
NAB 0.000, 0.003, 0.003 0.010 0.008, 0.011, 0.011 80.25, 78.00, 73.50 7.91 %
0.003, 0.000, 0.003 0.011, 0.009, 0.011 78.25, 92.25, 78.75

Table 7
Correlation analysis between four TSNAs and nicotine in e-liquids.

Compounds Nicotine NNN NNK NAT NAB

Nicotine Pearson correlation 1 0.083 0.039 −0.017 −0.026

Significance – 0.653 0.831 0.924 0.888
NNN Pearson correlation 0.083 1 0.890∗ ∗ 0.219 0.511∗ ∗
Significance 0.653 – <0.001 0.229 0.003
NNK Pearson correlation 0.039 0.890∗ ∗ 1 0.289 0.526∗ ∗
Significance 0.831 <0.001 – 0.108 0.002
NAT Pearson correlation −0.017 0.219 0.289 1 −0.241
Significance 0.924 0.229 0.108 – 0.183
NAB Pearson correlation −0.026 0.511∗ ∗ 0.526∗ ∗ −0.241 1
Significance 0.888 0.003 0.002 0.183 –

Table 8
Correlation analysis between four TSNAs and nicotine in aerosols.

Compounds Nicotine NNN NNK NAT NAB

Nicotine Pearson correlation 1 0.054 0.129 −0.15 −0.556∗ ∗

Significance – 0.771 0.48 0.413 0.001
NNN Pearson correlation 0.054 1 0.156 0.187 0.023
Significance 0.771 – 0.394 0.306 0.901
NNK Pearson correlation 0.129 0.156 1 0.758∗ ∗ 0.242
Significance 0.48 0.394 – <0.001 0.183
NAT Pearson correlation −0.15 0.187 0.758∗ ∗ 1 0.659∗ ∗
Significance 0.413 0.306 <0.001 – <0.001
NAB Pearson correlation −0.556∗ ∗ 0.023 0.242 0.659∗ ∗ 1
Significance 0.001 0.901 0.183 <0.001 –

tion of TSNAs. It is proposed that the response is caused by differences total concentrations of TSNAs in cigar leaves. However, under the ex-
in various additives, particularly tobacco extracts, in the e-liquid for- perimental conditions, there was no significant correlation between the
mulation. There was a negative correlation between NAB and nicotine concentrations of NNN, NNK, and NAT in e-cigarettes and the nicotine
in aerosols, and no significant correlation between other components. concentration, with a negative correlation in the case of NAB. Further
The results of related studies [31] have suggested that the conversion research is required to account for these effects. The concentration of
of nicotine in cigars results in an increase in nicotine concentrations, NNN in the e-liquid is positively correlated with the concentration of
which is a major contributor to the increase in NNN concentrations and NNK and NAB, and the NNK concentration is positively correlated with

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C. WANG, W. LI, Y. ZENG et al. Chinese Journal of Analytical Chemistry 52.9 (2024) 100430

Table 9 centrations measured under experimental conditions and those actually

Concentrations of four TSNAs in 5 e-liquids (x ± s, n = 3). inhaled by users during the act of smoking e-cigarettes.
Concentration (ng·g−1 )
E-liquids Ethical approval
NNN NNK NAT NAB

T1 0.001±0 – 0.019±0.001 0.003±0.002 This study did not conduct any research on animals or humans. The
T2 0.518±0.011 0.141±0.003 0.703±0.023 0.314±0.030
T3 0.724±0.044 0.215±0.008 0.885±0.024 0.373±0.020
authors confirm compliance with relevant ethical guidelines.
T4 1.036±0.044 0.208±0.004 1.413±0.013 0.499±0.023
T5 1.573±0.024 0.282±0.013 2.391±0.021 0.570±0.015 Declaration of interests

The authors declare that they have no known competing financial

the concentrations of NAB. Moreover, there is a positive correlation be- interests or personal relationships that could appear to influence the
tween NNK and NAT concentrations in the aerosols. The concentration work reported in this paper.
of NAT was also positively correlated with NNK and NAB concentration.
CRediT authorship contribution statement
3.4. Analysis of 5 e-liquid samples supplemented with different tobacco
extracts Chunqiong WANG: Data curation. Wei LI: Writing – original draft,
Software, Methodology, Formal analysis, Data curation, Conceptualiza-
In order to explore the possible sources of TSNAs, e-liquids contain- tion. Yanbo ZENG: Supervision. Yongliang WU: Project administra-
ing different tobacco extracts were prepared, and the concentrations of tion, Data curation. Dan CHEN: Writing – original draft. Hongming
the four TSNAs were determined (Table 9). The results have shown that MENG: Supervision. Ke ZHANG: Methodology, Data curation. Xiaowei
the concentration of TSNAs in e-liquids without the tobacco extract is ZHANG: Funding acquisition. Ganpeng LI: Writing – review & editing.
lower, whereas the concentrations in e-liquids with untreated and ad- Kai LIU: Funding acquisition.
sorbed tobacco extract are relatively higher. In addition, the concentra-
tion of TSNAs in the samples of tobacco extract and e-liquid prepared Acknowledgments
from modulated and fermented tobacco leaves is slightly higher than
those in the samples of unmodified and fermented tobacco leaves. This Financial support was provided by the Major Science and Technol-
further indicated that TSNAs in e-cigarettes mainly come from tobacco ogy Program of the Yunnan Branch of China Tobacco Corporation (No.
extracts and the content is related to the preparation and fermentation 2023530000241029).
of the tobacco leaves.
Supplementary materials
4. Conclusions
Supplementary material associated with this article can be found, in
This study described a method for detecting TSNAs in e-cigarettes has the online version, at doi:10.1016/[Link].2024.100430.
been established that delivers the required precision, repeatability, and
is simple to operate, practical, and suitable for regulatory and quality References
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