Volume 12

Number 5
7 February 2020

Analytical
Pages 597–708

Methods
rsc.li/methods

ISSN 1759-9679

PAPER
Oluwabunmi M. Dada, Marie-Cecile G. Chalbot
and Ilias G. Kavouras
Functional characterization of flavorings in electronic
cigarette refill liquids by nuclear magnetic resonance
spectroscopy
Analytical
Methods
PAPER

Functional characterization of flavorings in

Cite this: Anal. Methods, 2020, 12, 611
electronic cigarette refill liquids by nuclear
magnetic resonance spectroscopy†
Oluwabunmi M. Dada,a Marie-Cecile G. Chalbot ab
and Ilias G. Kavouras *ac

Received 3rd October 2019
Accepted 9th December 2019
DOI: 10.1039/c9ay02147g
rsc.li/methods
The chemical content of electronic cigarette liquids (e-liquids) was determined by NMR spectroscopy. The
study aimed to quantify the abundance of propylene glycol, glycerol and nicotine and reconcile the
functional types of non-exchangeable hydrogen of flavorings in e-liquids. Propylene glycol, glycerol,
nicotine, menthol, vanillin and benzaldehyde were detected in e-liquids by 1D and 2D-NMR
spectroscopy. Short- and long-range coupling of carbonyl, aryl and alkyl hydrogen was also observed by
2D 1H–1H and 1H–13C NMR. Propylene glycol and glycerol (from 30 : 70 to 65 : 35) accounted for more
than 97% of e-liquids by mass with nicotine levels comparable to those reported by the manufacturer. E-
liquids were classified into five clusters based on the relative distribution of non-exchangeable hydrogen
types in flavorings. The mean relative abundance of flavorings ranged from 2.4 to 4.7% for the five
clusters. Alkyl hydrogen was dominant in clusters II–V, representing 35–55% of non-exchangeable
hydrogen, followed by saturated oxygenated hydrogen (32–33%). In cluster I, saturated oxygenated
hydrogen was predominant. The highest abundance of aryl hydrogen was estimated for clusters I and IV,
while carbonyls accounted for up to 1.5% of non-exchangeable hydrogen. Tobacco-flavored e-liquids
had an average flavoring molar ratio of 1.8% with saturated oxygenated hydrogen being the predominant
group with considerable quantities of aryl (6.8%) and carbonyl (1.5%) hydrogen atoms. Overall, this
analysis allowed for the qualitative and quantitative determination of flavorings in e-liquids. This is
needed to improve protocols aiming to determine the chemical composition of e-liquids and the
feasibility of toxicological investigations.

with a projected value of 47.11 billion U.S. dollars by 2025.8 E-cig

Introduction
There has been an exponential increase in electronic cigarette
(e-cigarette) use in the U.S. and worldwide.1 E-cigarette is
a battery-powered nicotine delivery system aiming to generate
nicotine vapors, the primary addictive agent, through the
controlled heating of a nicotine solution.2–4 They were intro-
duced as tobacco smoking cessation devices due to the elimi-
nation of tar and other harmful tobacco combustion
byproducts.5,6 Over the years, e-cigs evolved dramatically
including the content of e-liquids.5 Today, there are more than
400 brands available with over 15 000 different avors.7 The e-
cig market value has been increasing steadily from three
billion U.S. dollars in 2014 to ten billion U.S. dollars in 2017
a
Department of Environmental Health Sciences, University of Alabama at Birmingham,
Birmingham, AL 35233, USA
b
Department of Chemical and Biomolecular Engineering, New York University,
Brooklyn, NY 11201, USA
c
Department of Environmental, Occupational and Geospatial Health Sciences,
Graduate School of Public Health and Health Policy, City University of New York,
NY 10027, USA. E-mail: ilias.kavouras@sph.cuny.edu; Tel: +1-646-364-9628
† Electronic supplementary information (ESI) available. See
10.1039/c9ay02147g
use is widespread among children and adolescents with up to
20% of high school students and 5% of middle school students
in the U.S. using tobacco products with e-cig being the most
commonly used product.9
A typical e-liquid solution contains nicotine, propylene
glycol, glycerol, water and a wide range of chemical species as
avorings. Propylene glycol and glycerol account for more than
90% of e-liquids by weight and volume.4,10 Active pharmaco-
logical chemicals, tobacco specic nitrosamines, aldehydes,
metals and volatile organic compounds were also detected in e-
liquids.4,11–13 Development of analytical methodologies for
characterizing e-liquids is pivotal to determine the chemical
composition of e-liquids. Chromatography (gas and liquid)
coupled to mass spectrometry is the most frequently used
analytical technique due to its specicity and low detection
limit for target analytes.14,15 Complex methods are frequently
needed to clean up, elute and concentrate the target chemical
for analysis.16–18 The enhanced performance of chromato-
graphic methods is undercut by the complexity and variability
of e-liquids that contain propylene glycol and glycerol in high
DOI:
quantities and many unknown chemical species that vary by
brand and avor. This limits toxicological and risk assessment

This journal is © The Royal Society of Chemistry 2020 Anal. Methods, 2020, 12, 611–619 | 611
Analytical Methods
investigations aiming to determine the short and long-term
health effects of e-cigarettes.
Nuclear magnetic resonance (NMR) spectroscopy is a non-
destructive method that requires no derivatization and
minimal sample preparation to analyze the e-liquid and the
resultant e-vapors, holistically. NMR spectra of organic
compound mixtures provide unique, robust and reproducible
qualitative and quantitative information on all chemical species
in the sample. This allows for the direct comparison of NMR
spectral ngerprints of e-liquids and e-vapors. On the other
hand, this is also a crucial limitation because NMR spectra may
contain both adequately resolved sharp and convoluted reso-
nances due to the range of concentrations (from ppt to ppm).
Two-dimensional NMR analysis may assist in resolving the
complexity of 1H-NMR spectra; however, its interpretation is not
trivial. NMR has been previously used to determine nicotine,
propylene glycol, glycerol and water in e-vapors and e-
liquids.4,19,20 Ethylene glycol was identied in e-liquids sold in
the European Union.21 Furthermore, nicotine was also detected
in nicotine-free e-liquids.4,11,15,21
The objective of this study was to develop and apply a NMR-
based approach to characterize the chemical content of rell e-
liquids. More specically, we aimed to determine both the
abundance of e-liquid major ingredients, namely, propylene
glycol, glycerol and nicotine, and the functional types and levels
of non-exchangeable hydrogen due to the presence of avoring
and other chemicals. Concentration diagnostic ratios reecting
the relative abundance of unoxidized (C–O) and oxidized (C]O)
oxygenated compounds and two dimensional homonuclear and
heteronuclear NMR spectroscopy were also applied to differ-
entiate e-liquids based on their compositional characteristics.
Experimental
Materials
A total of forty-two widely used e-liquids in the US market
manufactured by three U.S. providers, brand 1, brand 2 and
brand 3, were purchased through the internet and local
convenience stores: twelve (glacier mint, cherry, tobacco,
strawberry, Carolina bold, mint chocolate, gold leaf, menthol,
blueberry, caramel café, blueberry cobbler and vanilla) brand 1
e-liquids with 0.6 and 1.2% nicotine as reported by the manu-
facturer (or 6 and 12 mg mL
1), six (tobacco, cherry, menthol,
gourmet crème, berry mint and watermelon) brand 2 e-liquids
with 6 mg mL
1 of nicotine, and twelve (glacier mint, cherry,
tobacco, strawberry, Carolina bold, mint chocolate, gold leaf,
menthol, blueberry, caramel café, blueberry cobbler and
vanilla) brand 3 e-liquids with nicotine levels of 10 and 15 mg
mL
1. The purchased e-liquids were selected in order to obtain
a range of avors available in the market. Table S1 in the ESI†
shows the avor and nicotine content of the rell e-liquids.
Nicotine (3-(1-methyl-2-pyrrolidinyl)pyridine (N), C10H14N2,
99%), deuterium oxide (D2O, 99.8%), (trimethylsilyl)propionic
acid-d4 (2,2,3,3-d4-3-(trimethylsilyl)propionic acid sodium salt
(TSP), C6H13NaO2Si, 98%), sodium azide (NaN3, 99%), mono-
basic dehydrate sodium phosphate, (NaH2PO4$2H2O) and
disodium hydrogen phosphate (Na2HPO4, 98%) were purchased
612 | Anal. Methods, 2020, 12, 611–619
Paper
from Acros Chemicals (Pittsburg, PA). Propylene glycol (1,2-
propanediol (PG), C3H8O2, 99.5%), glycerol (2,3-propanetriol
(G), C3H8O3, 99+%) and ultrapure water (HPLC grade) were
acquired from Fisher Scientic (Waltham, MA).
Sample preparation
The e-liquid samples were prepared as follows: e-liquid
containers were vigorously shaken and 50 mL of e-liquid was
mixed with 350 mL of ultrapure water in 5.0 mm Norell NMR
tubes. The sample was further diluted to 2 : 3 by the addition of
200 mL of a D2O/H2O (30/70 v/v) phosphate buffer solution
(HPO42
/H2PO4
) to pH ¼ 7.4 containing 1 mM (trimethylsilyl)
propionic acid-d4 (TSP-d4, NMR internal standard) and 3 mM
sodium azide (NaN3). The solvent system was selected to
maintain the same experimental conditions for all e-liquids,
minimize changes in the chemical shi due to pH and facili-
tate compatibility with previous studies on the functional
composition of non-exchangeable hydrogen.22,23 At this pH,
nicotine is available in both its unprotonated and monoproto-
nated forms (pKa ¼ 8.01). Reference solutions of propylene
glycol (1.5 to 74 mg mL
1), glycerol (1.8 to 90 mg mL
1) and
nicotine (1.5 to 72 mg mL
1) were analyzed by 1H NMR spec-
troscopy to evaluate the performance of the method. Reference
samples were analyzed in triplicate.1D 1H and 2D 1H–13C NMR
spectra were obtained at 300 K using a Bruker Avance III spec-
trometer operating at 600.17 MHz with a proton-optimized
triple resonance “inverse” 5 mm cryogenic probe CP TCI600S3
H–C/N-D-05 Z tted with an actively shielded single axis z-
gradient and digital quadrature detection (DQD, 10 ms pre-
scan delay) using TopSpin 3.5/PL6 soware (Bruker BioSpin
Corp., Billerica, MA).22
NMR spectroscopy
1D 1H NMR spectra were collected using a gradient-based zgesgp
pulse sequence in 512 transients into 32 000 data points with
a spectral width of 16 ppm, with a relaxation delay of 1 s and an
acquisition time of 1.7 s. A Watergate pulse sequence was
applied for water suppression. The resultant 1H-NMR spectra
were processed with a 0.3 Hz exponential line broadening
factor, and phase and baseline corrected Advanced Chemistry
Development NMR Spectrus Processor soware (version 12.01;
ACDLabs, Toronto, Canada). All of the 1H-NMR spectra were
integrated into six regions representing different types of non-
exchangeable hydrogen as follows: (i) alkyl [H–C] dH: 0.70–
1.80 ppm; (ii) allylic [H–C–C]] dH: 1.80–3.20 ppm; (iii) satu-
rated oxygenated [H–C–O] dH: 3.20–4.70 ppm; (iv) vinylic [H–C]
C] and acetalic [O–CH–O] dH: 5.00–6.50 ppm; (v) aryl [H–Ar] dH:
6.50–9.00 ppm; and (vi) carbonyl [H–C]O] dH: 9.00–
12.00 ppm.23 The region corresponding to water (dH: 4.70–5.00
ppm) was removed. Quantication of propylene glycol, glycerol
and nicotine was done by integration of resonance signals at dH:
1.13 ppm, dH: 3.67 ppm and dH: 8.05 ppm, respectively. The
integrals of the remaining resonances of propylene glycol,
glycerol and nicotine were examined to evaluate the potential of
convoluted resonances. If a deviation was identied, the reso-
nances were manually integrated to resolve partially
This journal is © The Royal Society of Chemistry 2020
Paper Analytical Methods

overlapping resonances. Because of the linear relationship
between the number of hydrogen atoms and resonance signal
intensity, TSP concentration was used to estimate propylene
glycol, glycerol, nicotine and non-exchangeable hydrogen
concentrations.24
Phase sensitive 2D 1H–1H double quantum lter correlation
spectroscopy (DQF-COSY) with gradient cosydfesgpph pulses and
solvent suppression using 1D excitation sculpting was per-
formed using acquisition times of 0.1420 s in F2 and 0.0355 s in
F1, 10.62 ms 90 excitation pulses (p1), 2 s relaxation delay (d1),
32 scans/512 experiments, 2k total data points and F1 States-
TPPI acquisition mode. The spectrum was computed to a 2048
 1024 matrix with 1 Hz (F2) and 0.3 Hz (F1) exponential
multiplication and squared shied sine bell in both dimensions
(P/9). 2D Heteronuclear 1H–13C correlation spectra were ob-
tained using phase-sensitive 1H–13C correlation via double
inept transfer HSQC spectra (hsqcetgp) using echo-antiecho
gradient selection (F1 detection mode) with decoupling
during acquisition under the following conditions: 16 scans in
the F2 dimension over 256 experiments in the F1 dimension,
1024 data points (TD, F2), 1J CH coupling constant of 145 Hz
(CNST2), 16 dummy scans (DS); F2 (1H) parameters: spectral
width (SW) of 15 ppm, frequency offset (O1P) of 4.7 ppm,
acquisition time of 40.1 ms, 12.61 ms 90 excitation pulses (p1),
1.5 s relaxation delay (d1); F1 (13C) parameters: SW ¼ 240 ppm;
O1P 110.0 ppm, acquisition time of 2.5 ms, garp composite

Fig. 1 600 MHz 1D 1H-NMR spectra of characteristic refill e-liquid solutions (nicotine percentage content provided by manufacturer within
parenthesis) for each e-liquid cluster. ([H–C]: alkyl, [H–C–C]]: allylic, [H–C–O]: saturated oxygenated, [H–C]C]: vinylic; [O–CH–O]: acetalic,
[H–Ar]: aryl, [H–C]O]: carbonyl, TSP-d4: internal standard, B: benzaldehyde, GL: glycerol, M: menthol, N: nicotine, PG: propylene glycol, and V:
vanillin).

This journal is © The Royal Society of Chemistry 2020 Anal. Methods, 2020, 12, 611–619 | 613
Analytical Methods Paper

Fig. 2 600 MHz (a) 1H–1H COSY and (b) 1H–13C HSQC NMR spectra of brand 1 menthol flavored e-liquid.

pulse 13C decoupling program (60 ms PCPD2). The 1H–13C Statistical analysis
HMBC spectra were acquired in magnitude-mode (ge-2D
Differences of the mean concentration of nicotine, propylene
HMBC) using a low-pass J-lter to suppress one-bond correla-
glycol, glycerol (reported in mg mL
1) and non-exchangeable
tions with hmbcgp gradient pulses for selection and purge pul-
hydrogen types (reported in mmolH L
1) were assessed by
ses before d1 as follows: 32 scans in the F2 dimension over 128
analysis-of-variance (ANOVA) at an a level of 90% using SPSS
experiments in the F1 dimension, 2048 data points (TD); F2 (1H)
(Version 25, IBM Statistics). The (carboxylic + carbonyl)-to-
parameters: spectral width (SW) of 15 ppm, frequency offset
aliphatics ([H–C–C]O]/[H-R]) and carbohydrate-to-aliphatic
(O1P) of 4.7 ppm, acquisition time of 80.3 ms, 12.61 ms 90 ([H–C–O]/[H-R]) carbon concentration diagnostic ratios were
excitation pulses (p1), 1.5 s relaxation delay (d1); F1 (13C) calculated using a H/C molar ratio of 2 for non-functionalized
parameters: SW ¼ 240 ppm; O1P 110.0 ppm, acquisition time of
saturated [H–C] (i.e. –(CH2)n– chains), 2 for allylic aliphatic
1.2 ms.
[H–C–C]] (average of CH3, CH2 and CH groups), 1.1 for

614 | Anal. Methods, 2020, 12, 611–619 This journal is © The Royal Society of Chemistry 2020
Paper
oxygenated aliphatic [H–C–O], and 0.4–1.0 for aromatic (H–Ar)
(average between the ratios for tri- and tetra-substituted
benzene rings) to convert the hydrogen to carbon concentra-
tions.25 The carboxylic + carbonyl (HC–C]O) fractions was
estimated as the amount of [H–C–C]] in excess of [H–Ar]. The
total aliphatic fraction (i.e. [H–R]) included the sum of [H–C],
[H–C–C]], [H–C–O] and [aliphatic C]O]. Decreasing ([H–C–
C]O]/[H-R]) and increasing [H–C–O]/[H-R] are indicative of the
presence of single bonded oxygenated compounds that may
undergo further oxidation yielding the production of carboxy/
carbonyl functional groups.
Results and discussion
1D and 2D NMR spectra
Fig. 1 shows the typical NMR spectra of rell e-liquids for each
cluster. The NMR spectra were dominated by sharp resonances
assigned to propylene glycol (PG in Fig. 1; d 1.15 ppm (d, 3H,
CH3), d 3.45 ppm and d 3.54 ppm (dd, 2H, CH2), and d 3.89 ppm
(m, 1H, CH)), glycerol (G in Fig. 1; d 3.56–3.59 ppm (m, 2H,
CH2), d 3.64–3.67 ppm (m, 2H, CH2), and d 3.79 ppm (tt, 1H,
CH)) and nicotine (N in Fig. 1; d 2.29 ppm (m, 2H, CH2),
d 2.39 ppm (m, 1H, CH2), d 2.55 ppm (m, 1H, CH2), d 2.66 ppm
(s, 3H, CH3), d 3.33 ppm (m, 1H, CH2), d 3.81 ppm (m, 1H, CH2),
d 4.30 ppm (dd, 1H, CH), d 7.59 ppm (dd, 1H, ]CH), d 8.03 ppm
(dt, 1H, ]CH), d 8.62 ppm (dd, 1H, ]CH), and d 8.64 ppm (d,
1H, N]CH)).
In addition, a limited number of chemical species were
identied by 1H-NMR spectroscopy using reference NMR
spectra.26 These were menthol (M in Fig. 1; d 0.77 ppm (d, 3H,
CH3), d 0.78 ppm (dd, 1H, CH2), d 0.89 ppm (d, 3H, CH3),
d 0.90 ppm (d, 3H, CH3), d 0.92 ppm (m, 2H, CH2), d 1.19 ppm
(m, 1H, CH), d 1.41 ppm (m, 1H, CH), d 1.62 ppm (dq, 1H, CH2),
d 1.64 ppm (dt, 1H, CH), d 1.94 ppm (ddd, 1H, CH2), d 2.13 ppm
(m, 1H, CH), and d 3.42 ppm (br.s, 1H, CH)), vanillin (V in Fig. 1;
d 4.0 ppm (s, 3H, CH3), d 6.96 ppm (s, 1H, CH), d 7.41 ppm (d,
1H, CH), d 0.7.50 ppm (d, 1H, CH), and d 9.52 ppm (s, 1H, HC]
O)), benzaldehyde (B in Fig. 1; d 7.65 ppm (t, 2H, CH),
d 7.78 ppm (tt, 1H, CH), d 7.96 ppm (dd, 2H, CH), and
d 9.96 ppm (s, 1H, HC]O)). Note that these compounds were
not quantied in this section as they are identied as avorings
in e-liquids.
The 1H–1H COSY and 1H–13C HSQC NMR spectra of
menthol-avored e-liquids in order to obtain more insights into
the molecular characteristics of e-liquids are presented in
Fig. 2a and b, respectively. COSY NMR spectra were dominated
by diagonal and cross peaks assigned to propylene glycol,
glycerol, nicotine, menthol and vanillin. Region A in COSY
(Fig. 2a) showed coupling of saturated oxygenated and aromatic
hydrogen (F1 dH 3.5–4.5 ppm/F2 7.5–8.5 ppm), which was
consistent with the cross-peak resonances in the dH/C 3.5–4.5/
140–150 ppm region (region (A)) in the HSQC spectra (Fig. 2b).
This was suggestive of non-exchangeable organic hydrogen
bonded to a saturated oxygenated group (such as methoxy
(–OCH3)) that is attached to an sp2-hybridized carbon. This
includes avorings such as methyl benzoate and vanillin
derivatives (note that vanillin is observed in menthol avored e-
This journal is © The Royal Society of Chemistry 2020
Analytical Methods
liquids (Fig. 1)). It is noteworthy that cross signals in the
aromatic region for both hydrogen and carbon were not
observed. Targeted or longer experiments would provide more
information on the structure of individual chemical species.
Region B in COSY (Fig. 2a) showed diagonal resonances in the
F2/F1 6.50–7.00 ppm region and a limited number of reso-
nances in the F1 dH 7.00–8.00 ppm/F2 6.50–7.00 region due to
couplings between aromatic and vinylic carbon. In the HSQC
spectra, these hydrogen atoms were associated with resonances
in the dC 120–130 ppm region ((B) in Fig. 2b). trans-Cinna-
maldehyde has been previously detected in e-liquids. Cross
peaks at F1 dH 1.30 ppm and F2 dH 8.2–8.9 ppm (region (C) in
Fig. 2a) are indicative of alkyl and aromatic hydrogen
couplings.27 It is noteworthy that 2D NMR spectroscopy may not
decisively identify the molecular structure of individual
compounds but can provide information on the functional
groups that can be subsequently applied to the developed tar-
geted and sensitive protocols to identify them by chromato-
graphic methods. Overall, qualitative 1D and 2D NMR
spectroscopy may provide insights into the types of chemical
species that are present in e-liquids to develop protocols for
targeted chromatographic and chemical analysis.
Propylene glycol, glycerol and nicotine
Table 1 shows the mean  standard error (percentage within
parentheses) of propylene glycol, glycerol and nicotine
(in mg L
1) grouped into ve clusters based on the functional
composition of non-exchangeable hydrogen. The limit of
detection (LOD) for propylene glycol, glycerol and nicotine was
90, 90 and 120 mg mL
1, respectively. The limit of quantication
(LOQ) for propylene glycol, glycerol and nicotine was 290, 300
and 420 mg mL
1, respectively. The percent residual standard
deviation was 6.5% for propylene glycol, 4.3% for glycerol and
1.1% for nicotine. The mean propylene glycol concentrations in
rell e-liquids varied from 462  28 mg mL
1 to 631  40 mg
mL
1. Glycerol concentration also varied from 543  38 mg
mL
1 to 678  44 mg mL
1. The propylene glycol to glycerol
ratio was approximately 50 : 50 for clusters II, IV and V and
40 : 60 for clusters I and III. A slightly higher mean nicotine
concentration was measured for cluster I (19  2 mg mL
1) as
compared to clusters II, III and V (12  1 mg mL
1 to 15  3 mg
mL
1). Lower nicotine levels were measured for cluster IV (6 
1 mg mL
1). In all cases, nicotine accounted for 0.5 to 1.5% by
mass. The measured nicotine concentrations by NMR (0–31 mg
mL
1) were, on average, comparable (ca. 4.8%) to those re-
ported by the manufacturer (0–24 mg mL
1). Trace quantities of
nicotine were also detected in nicotine-free e-liquids. The
measured nicotine levels for e-liquids of 6 to 15 mg mL
1 were
consistently lower than those reported, while the measured
nicotine levels were consistently higher for e-liquids of 24 mg
mL
1. Note that the difference of propylene glycol, glycerol and
nicotine levels among the ve clusters were not statistically
signicant at p < 0.05 level.
This analysis quantied the levels of propylene glycol, glyc-
erol and nicotine in rell e-liquid solutions. Manufacturers are
required to report only nicotine content in e-liquids. In
Anal. Methods, 2020, 12, 611–619 | 615
Analytical Methods Paper

Table 1 The mean  standard error (percent contribution) concentrations of propylene glycol, glycerol, nicotine and non-exchangeable [H–C]
(alkyl), [H–C–C]] (allylic), [H–C–O] (saturated oxygenated), [H–C]C] (vinylic), [O–CH–O] (acetalic), [H–Ar] (aryl) and [H–C]O] (carbonyl)
non-exchangeable hydrogen and the flavoring molar ratio for clusters I, II, III, IV, and V and tobacco-flavored e-liquids

Cluster

Chemical species I II III IV V Tobacco avored

e-Liquid major ingredient concentration (mg mL
1)

Propylene glycol 471  35 (41.7) 561  46 (49.9) 462  28 (40.3) 547  23 (49.0) 631  40 (50.8) 434  42 (30.8)
Glycerol 652  52 (56.7) 543  38 (48.7) 678  44 (58.6) 566  30 (50.5) 596  35 (48.2) 731  171 (60.1)
Nicotine 19  2 (1.6) 15  3 (1.4) 14  03 (1.2) 6  1 (0.5) 12  1 (1.0) 16  8 (1.2)

Total non-exchangeable H concentration (mmolH L
1)

[H–C] 888  13 (27.1) 1421  194 (52.8) 726  190 (35.0) 1052  380 (45.2) 991  207 (42.1) 276  14 (21.0)
[H–C–C]] 699  11 (20.8) 461  68 (19.0) 432  84 (22.7) 262  24 (16.1) 502  44 (23.4) 382  200 (25.5)
[H–C–O] 1470  355 (39.8) 553  50 (22.1) 601  86 (32.7) 506  30 (33.3) 492  49 (22.6) 590  50 (44.4)
[O–CH–O], [H–C]C] 40  11 (1.0) 7  2 (0.3) 17  2 (1.0) 18  8 (1.4) 25  8 (1.2) 18  5 (1.3)
[H–Ar] 388  93 (9.8) 140  34 (5.3) 129  18 (7.4) 41  12 (3.1) 199  45 (9.9) 91  43 (6.3)
[H–C]O] 59  17 (1.5) 16  5 (0.6) 18  4 (1.2) 10  3 (0.8) 16  9 (0.8) 21  6 (1.5)
Flavorings molar ratio 0.047  0.007 0.035  0.003 0.026  0.004 0.024  0.006 0.027  0.002 0.018  0.002

addition, a limited number of chemical species associated with concentration was 2598  353 mmolH L
1 with [H–C]
characteristics avors were also identied. accounting for most than half (52.8%). [H–C–O] represented
only 22% of non-exchangeable hydrogen. The aromatic fraction
Functional composition accounted for about 5% with trace quantities of [H–C]O] and
[O–CH–O] + [H–C]]. The total non-exchangeable hydrogen
This section describes the functional composition of avorings
concentration for cluster III was 1922  384 mmolH L
1, with
in rell e-liquid solutions excluding propylene glycol, glycerol
equal quantities of [H–C] and [H–C–O] groups and increased
and nicotine to determine their compositional features and the
abundance of [H–C–C]], [H–Ar] and [H–C]O]. It included
potential to be clustered based on them. The six functional
brand 1 tobacco (1.2 and 2.4% nicotine), vanilla (0, 1.2 and
groups of non-exchangeable hydrogen were alkyl [H–C], unsat-
2.4%) mint (1.2% nicotine) and strawberry mint (2.4%). In
urated aliphatic [H–C–C]], saturated oxygenated [H–C–O],
addition, brand 2 strawberry mint and cherry (6 mg mL
1) were
vinylic and acetalic ([H–C]C] + [O–CH–O]), aryl [H–Ar] and
included in this cluster.
carbonyl [H–C]O] (Fig. 1). Note that a single chemical species
Flavors of brand 2 (gourmet coffee, menthol, tobacco and
may have resonances of more than one hydrogen type; thus, the
watermelon (6 mg mL
1 nicotine)) and brand 3 (blood orange
calculated concentrations of non-exchangeable hydrogen re-
with 10 mg mL
1) were included in cluster IV. The lowest non-
ected the abundance of functional groups rather than chem-
exchangeable hydrogen concentration was measured (1889 
ical species in e-liquids. However, understanding the relative
457 mmolH L
1) with [H–C] and [H–C–O] accounting for up to
abundance of functional groups may be physiologically appro-
80% of that. These e-liquids had also the lowest, in both abso-
priate because the biological fate of individual chemical species
lute and relative, concentrations of [H–Ar], [H–C–C]] and [H–
may be related to functional groups.
C]O] groups. Cluster V consisted of brand 3 blood orange
Rell e-liquids were grouped into ve clusters based on the
(15 mg mL
1) and pomegranate, peach tea, single malt scotch
relative abundance of functional groups. Table 1 also shows the
and vanilla bean (10 and 15 mg mL
1). The alkyl fraction ([H–C],
mean  standard error (percentage within parentheses) of
501  457 mmolH L
1) accounted for approximately 40% of the
avorings' non-exchangeable hydrogen types (in mmolH L
1) of
total non-exchangeable hydrogen concentration (2225  362
e-liquids classied into ve clusters.
mmolH L
1) with equal contribution from unsaturated [H–C–
Cluster I comprised brand 1 rell e-liquids with 1.2% and
C]] and oxygenated [H–C–O] hydrogen (502  422 mmolH L
1
2.4% nicotine with cherry, berry cobbler, blueberry, caramel
and 492  49 mmolH L
1, respectively). The highest relative
café, and Carolina bold avors. The mean total non-
abundance of aromatic hydrogen [H–Ar] was observed for these
exchangeable hydrogen, [H–C–O], [O–CH–O] + [H–C]C], [H–
e-liquids (9.9%), albeit their concentrations were substantially
Ar] and [H–C]O] concentrations (3544  487, 699  11, 1470 
lower than those measured for cluster I e-liquids.
355, 40  11, 388  93 and 59  17 mmolH L
1, respectively)
The molar concentration ratio of avorings' non-
were the highest among the ve clusters. The saturated
exchangeable hydrogen to the total concentration (nicotine,
oxygenated fraction accounted for approximately 40% of the
propylene glycol, and glycerol) may be interpreted as a metric of
avorings' non-exchangeable hydrogen, followed by [H–C] and
the relative abundance of organic species in e-liquids. It is
[H–C–C]]. The aromatic and carbonyl groups represented up
noteworthy that the molar ratio does not refer either to the
to 10% and 1.5% respectively. Cluster II included brand 1 gold
quantity (by mass or volume) or the number of individual
leaf, glacier mint, menthol, and mint chocolate avors with 0%,
chemical species because multiple resonances may be
1.2% and 2.4% nicotine. The total non-exchangeable hydrogen

616 | Anal. Methods, 2020, 12, 611–619 This journal is © The Royal Society of Chemistry 2020
Paper
attributed to a single compound. Yet, the ratio may be used to
compare the abundance of additives in rell e-liquids.
Furthermore, liable hydrogen (OH, COOH) was not quantied.
It varied from 0.011 (for nicotine-free brand 1 vanilla) to 0.082
(for brand 1 berry cobbler with 1.2% nicotine) with a mean
molar ratio of 0.033  0.001. The avorings' molar ratio values
were comparable for the same avors with variable nicotine
levels across all brands. For the ve clusters, the avorings'
molar ratio varied from 0.024  0.006 for cluster IV to 0.047 
0.007 for cluster I indicating the presence of more avorings.
Recently and following a cascade of respiratory illnesses and
deaths of e-cigarette users,28 state agencies adopted rules and
regulations to control the sale of avored e-liquids including
menthol with a history of targeting young people and minori-
ties.29 The variety of avors had a signicant impact in the rapid
growth of e-cigarette users among the youth and adolescents.29
The U.S. Food and Drug Administration upheld existing rules
that limit the sale of avored e-liquids until manufacturers
demonstrate that they are appropriate for the protection of
public health.30 Meanwhile, tobacco avored e-liquids may be
available. Three tobacco-avored e-liquids were included in this
study, grouped into clusters III and IV. Table 1 shows the
concentrations of the major ingredients in e-liquids and non-
exchangeable hydrogen for the tobacco-avored e-liquids, too.
Fig. 3 shows the 1D 1H-NMR plots for the three tobacco avored
e-liquids. There were notable differences including the detec-
tion of vanillin and resonances in the vinylic/acetalic region in
brand 1 tobacco avored e-liquids. Glycerol is the dominant
solvent (60%) as compared to e-liquids in clusters I–V. The
mean additive molar ratio (0.018  0.002) was lower than those
Fig. 3 600 MHz 1D 1H-NMR spectra of brand 1 and 2 tobacco-flavored e-liquids. ([H–C]: alkyl, [H–C–C]]: allylic, [H–C–O]: saturated
oxygenated, [H–C]C]: vinylic; [O–CH–O]: acetalic, [H–Ar]: aryl, [H–C]O]: carbonyl, TSP-d4: internal standard, B: benzaldehyde, GL:
glycerol, M: menthol, N: nicotine, PG: propylene glycol, and V: vanillin).
This journal is © The Royal Society of Chemistry 2020
Analytical Methods
computed from cluster I–V but the relative abundances of
carbonyl, aryl, vinylic/acetalic non-exchangeable hydrogen were
higher than those of most of the rell e-liquids. These results
indicate that tobacco-avored e-liquids may contain chemical
species found in other avored e-liquids.
Fig. 4 shows the (carboxylic + carbonyl)-to-aliphatic ([H–C–
C]O]/[H-R]) and carbohydrate-to-aliphatic ([H–C–O]/[H-R])
carbon concentration diagnostic ratios.31 The [H–C–C]O]/[H-
R] ratio ranges from 0.189 to 0.749 with a mean of 0.414 
0.021, while [H–C–O]/[H-R] varied from 0.000 to 0.501 with
a mean of 0.218  0.020. E-liquids with high [H–C–O]/[H-R] are
most likely to contain hydroxyl, methoxy and ether functional
groups (sp3-hybridized C–O) that may undergo oxidation to
carbonyl, keto, carboxyl and ester functionalities (i.e. H–C–C]
O, sp2-hybridized C]O) at different reaction rates during
heating in electronic cigarettes modifying the chemical
composition of e-vapors.
On the other hand, rell e-liquid solutions with high [H–C–
C]O]/[H-R] values suggest the presence of aldehydes, ketones,
carboxylic acids and esters in the solution. Rell e-liquids with
a high value of the diagnostic ratio (e.g. [H–C–O]/[H-R] > 0.500)
typically have low values for the [H–C–C]O]/[H-R] diagnostic
ratio and consist predominantly of [H–C–O] (49%) non-
exchangeable hydrogen. E-liquids with moderate [H–C–O]/[H-
R] ratios (0.250 to 0.500) also have comparable [H–C–C]O]/
[H-R] ratios and contain a large fraction of [H–C] (44%) to [H–
C–O] (26%). For samples with low [H–C–O]/[H-R] ratios (<0.250),
[H–C–C]O]/[H-R] was high (>0.500) and are characterized by
a strong alkyl signature [H–C] (up to 65%), with approximately
15% of [H–C–O] and trace quantities of [H–Ar] (2%). The
Anal. Methods, 2020, 12, 611–619 | 617
Analytical Methods
Fig. 4 The distribution of (carboxylic + carbonyl)-to-aliphatic ([H–C–
C]O]/[H-R]) and carbohydrate-to-aliphatic ([H–C–O]/[H-R]) carbon
concentration diagnostic ratios of refill e-liquid solutions.
variability of the diagnostic ratios offers additional insights into
the molecular proling of e-liquids and potential molecular
changes during vaping because of heating.
Overall, the 1H-NMR spectra demonstrated that e-liquid
composition may vary substantially across brands and avors
both qualitatively and quantitatively. This is suggestive of the
presence of many chemical species in e-liquids as avors and
additives with different functional groups. This variability
complicates efforts to characterize exposure to e-vapors and
their effects in epidemiological investigations, particularly in
cases where specic chemical species are targeted. At the same
time, quantication of non-exchangeable hydrogen for different
functional groups allows for the clustering of e-liquids and may
be advantageous in controlled toxicological investigations
aiming to elucidate the cellular responses to e-vapors.
Conclusions
The functional characteristics of electronic cigarette rell e-
liquids have been studied. Using 1H-, 1H–1H and 1H–13C NMR
ngerprints and concentration diagnostic ratios, the abun-
dance of major ingredients and avorings and other chemicals
in e-liquids was estimated. E-liquids were reconciled in ve
clusters with distinct compositional proles. Cluster I was
characterized by elevated non-exchangeable hydrogen levels
accounting for up to 5% of total non-exchangeable hydrogen
(including propylene glycol, glycerol and nicotine) with satu-
rated oxygenated functional groups being the predominant one.
The highest abundances of aryl and carbonyl hydrogen were
also measured for this cluster. For clusters II through IV, the
highest concentrations were measured for alkyl hydrogen. The
relative abundance of aryl and carbonyl hydrogen varied from
3.1–7.4% and 0.6–1.2%, respectively. For cluster V, alkyl and
allylic hydrogen accounted for, on average, 65% of non-
exchangeable hydrogen concentration with aryl hydrogen
accounting for up to 10%. Overall, we characterized the func-
tional content of e-liquids including both their major
618 | Anal. Methods, 2020, 12, 611–619
Paper
ingredients and trace quantities of avorings and additives. We
showed that NMR provides qualitative and quantitative infor-
mation on the compositional features of e-liquids. Finally, the
relative distribution of non-exchangeable organic hydrogen
functional groups appeared to have similar ngerprints for e-
liquids with different avors or from different brands.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
OMD thanks the UAB Ryals School of Public Health for a grad-
uate research fellowship.
References
1 B. J. Ingebrethsen, S. K. Cole and S. L. Alderman, Inhalation
Toxicol., 2012, 24, 976–984.
2 P. Callahan-Lyon, Tob. Control, 2014, 23, ii36–ii40.
3 R. Grana, N. Benowitz and S. A. Glantz, Circulation, 2014,
129, 1972–1986.
4 J. Hahn, Y. B. Monakhova, J. Hengen, M. Kohl-Himmelseher,
J. Schüssler, H. Hahn, T. Kuballa and D. W. Lachenmeier,
Tob. Induced Dis., 2014, 12, 23.
5 D. Hammond, O. A. Wackowski, J. L. Reid and
R. J. O'Connor, Nicotine Tob. Res., 2018, nty237.
6 R. M. Pellegrino, B. Tinghino, G. Mangiaracina, A. Marani,
M. Vitali, C. Protano, J. F. Osborn and M. S. Cattaruzza,
Ann. Ig., 2012, 24, 279–288.
7 G. Hsu, J. Y. Sun and S. H. Zhu, J. Med. Internet Res., 2018, 20,
e80.
8 E-Cigarette and Vaping Market Size Worth $47.11 Billion by
2025: Grand View Research, Inc, https://
www.prnewswire.com/news-releases/e-cigarette-and-vaping-
market-size-worth-47-11-billion-by-2025-grand-view-
research-inc–876278791.html, accessed September 16, 2019.
9 T. W. Wang, A. Gentzke, S. Sharapova, K. A. Cullen,
B. K. Ambrose and A. Jamal, MMWR Morb. Mortal Wkly
Rep., 2018, 67, 629–633.
10 R. Tayyarah and G. A. Long, Regul. Toxicol. Pharmacol., 2014,
70, 704–710.
11 M. E. Hadwiger, M. L. Trehy, W. Ye, T. Moore, J. Allgir and
B. Westenberger, J. Chromatogr. A, 2010, 1217, 7547–7555.
12 A. Bhatnagar, L. P. Whitsel, K. M. Ribisl, C. Bullen,
F. Chaloupka, M. R. Piano, R. M. Robertson, T. McAuley,
D. Goff and N. Benowitz, Circulation, 2014, 130, 1418–1436.
13 J. Margham, K. McAdam, M. Forster, C. Liu, C. Wright,
D. Mariner and C. Proctor, Chem. Res. Toxicol., 2016, 29,
1662–1678.
14 J. Singh, E. Luquet, D. P. T. Smith, H. J. Potgieter and
P. Ragazzon, Sci. Prog., 2016, 99, 351–398.
15 T. Cheng, Tob. Control, 2014, 23, ii11–ii17.
16 F. Dal Bello, V. Santoro, V. Scarpino, C. Martano, R. Aigotti,
A. Chiappa, E. Davoli and C. Medana, Drug Test. Anal., 2016,
8, 730–737.
This journal is © The Royal Society of Chemistry 2020
Paper
17 J. Dai, K.-H. Kim, J. E. Szulejko and S. H. Jo, Microchem. J.,
2017, 133, 237–245.
18 S. Barhdadi, M. Canfyn, P. Courselle, V. Rogiers,
T. Vanhaecke and E. Deconinck, J. Pharm. Biomed. Anal.,
2017, 142, 218–224.
19 A. L. Duell, J. F. Pankow and D. H. Peyton, Chem. Res.
Toxicol., 2018, 31, 431–434.
20 M. D. Crenshaw, M. E. Te, S. S. Buehler, M. C. Brinkman,
P. I. Clark and S. M. Gordon, Magn. Reson. Chem., 2016, 54,
901–904.
21 C. Hutzler, M. Paschke, S. Kruschinski, F. Henkler, J. Hahn
and A. Luch, Arch. Toxicol., 2014, 88, 1295–1308.
22 M.-C. G. Chalbot, P. Chitranshi, G. Gamboa da Costa,
E. Pollock and I. G. Kavouras, Atmos. Environ., 2016, 128,
235–245.
23 M.-C. G. Chalbot and I. G. Kavouras, Nat. Prod. Commun.,
2019, 14, 1934578X19849972.
24 S. K. Bharti and R. Roy, Trends Anal. Chem., 2012, 35, 5–26.
25 S. Fuzzi, S. Decesari, M. C. Facchini, E. Matta, M. Mircea and
E. Tagliavini, Geophys. Res. Lett., 2001, 28, 4079–4082.
26 D. S. Wishart, C. Knox, A. C. Guo, R. Eisner, N. Young,
B. Gautam, D. D. Hau, N. Psychogios, E. Dong, S. Bouatra,
R. Mandal, I. Sinelnikov, J. Xia, L. Jia, J. A. Cruz, E. Lim,
C. A. Sobsey, S. Shrivastava, P. Huang, P. Liu, L. Fang,
J. Peng, R. Fradette, D. Cheng, D. Tzur, M. Clements,
A. Lewis, A. De Souza, A. Zuniga, M. Dawe, Y. Xiong,
D. Clive, R. Greiner, A. Nazyrova, R. Shaykhutdinov, L. Li,
H. J. Vogel and I. Forsythe, Nucleic Acids Res., 2009, 37,
D603–D610.
This journal is © The Royal Society of Chemistry 2020
Analytical Methods
27 M.-C. G. Chalbot, S. V. Pirela, L. Schifman, V. Kasaraneni,
V. Oyanedel-Craver, D. Bello, V. Castranova, Y. Qian,
T. Thomas, I. G. Kavouras and P. Demokritou, Environ.
Sci.: Nano, 2017, 4, 2144–2156.
28 US FDA and CDC Joint Statement on federal and state
collaboration to investigate respiratory illnesses reported aer
use of e-cigarette products, https://www.fda.gov/news-events/
press-announcements/statement-federal-and-state-
collaboration-investigate-respiratory-illnesses-reported-
aer-use-e, accessed September 16, 2019.
29 US FDA News release Trump Administration Combating
Epidemic of Youth E-Cigarette Use with Plan to Clear Market
of Unauthorized, Non-Tobacco-Flavored E-Cigarette Products,
https://www.fda.gov/news-events/press-announcements/
trump-administration-combating-epidemic-youth-e-
cigarette-use-plan-clear-market-unauthorized-non, accessed
September 16, 2019.
30 US FDA New Release: FDA warns JUUL Labs for marketing
unauthorized modied risk tobacco products, including in
outreach to youth, https://www.fda.gov/news-events/press-
announcements/fda-warns-juul-labs-marketing-
unauthorized-modied-risk-tobacco-products-including-
outreach-youth, accessed September 16, 2019.
31 S. Decesari, E. Finessi, M. Rinaldi, M. Paglione, S. Fuzzi,
E. G. Stephanou, T. Tziaras, A. Spyros, D. Ceburnis,
C. O'Dowd, M. Dall'Osto, R. M. Harrison, J. Allan, H. Coe
and M. C. Facchini, J. Geophys. Res.: Atmos., 2011, 116,
D22210.
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