3-Ethenylpyridine Measured in Urine of Active and Passive Smokers: A Promising Biomarker and Toxicological Implications

Article abstract of DOI:10.1021/acs.chemrestox.1c00064

In studies of tobacco toxicology, including comparisons of different tobacco products and exposure to secondhand or thirdhand smoke, exposure assessment using biomarkers is often useful. Some studies have indicated that most of the toxicity of tobacco smoke is due to gas-phase compounds. 3-Ethenylpyridine (3-EP) is a major nicotine pyrolysis product occurring in the gas phase of tobacco smoke. It has been used extensively as an environmental tracer for tobacco smoke. 3-EP would be expected to be a useful tobacco smoke biomarker as well, but nothing has been published about its metabolism and excretion in humans. In this Article we describe a solid-phase microextraction (SPME) GC-MS/MS method for determination of 3-EP in human urine and its application to the determination of 3-EP in the urine of smokers and people exposed to secondhand smoke. We conclude that 3-EP is a promising biomarker that could be useful in studies of tobacco smoke exposure and toxicology. We also point out the paucity of data on 3-EP toxicity and suggest that additional studies are needed.

Full text of DOI:10.1021/acs.chemrestox.1c00064

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Article
3‑Ethenylpyridine Measured in Urine of Active and Passive Smokers:
A Promising Biomarker and Toxicological Implications
Jia Liu, Neal L. Benowitz, Dorothy K. Hatsukami, Christopher M. Havel, Eduardo Lazcano-Ponce,
Andrew A. Strasser, and Peyton Jacob, 3rd*
Cite This: Chem. Res. Toxicol. 2021, 34, 1630−1639
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ABSTRACT: In studies of tobacco toxicology, including comparisons of different
tobacco products and exposure to secondhand or thirdhand smoke, exposure
assessment using biomarkers is often useful. Some studies have indicated that most
of the toxicity of tobacco smoke is due to gas-phase compounds. 3-Ethenylpyridine
(3-EP) is a major nicotine pyrolysis product occurring in the gas phase of tobacco
smoke. It has been used extensively as an environmental tracer for tobacco smoke. 3-
EP would be expected to be a useful tobacco smoke biomarker as well, but nothing
has been published about its metabolism and excretion in humans. In this Article we
describe a solid-phase microextraction (SPME) GC-MS/MS method for
determination of 3-EP in human urine and its application to the determination of
3-EP in the urine of smokers and people exposed to secondhand smoke. We
conclude that 3-EP is a promising biomarker that could be useful in studies of
tobacco smoke exposure and toxicology. We also point out the paucity of data on 3-
EP toxicity and suggest that additional studies are needed.
mainstream (MS) cigarette smoke (inhaled by the smoker)
are in the range ∼4−30 μg/cigarette. Concentrations in
sidestream (SS) cigarette smoke (emitted by the smoldering
cigarette) are much higher, ∼200−600 μg/cigarette (Table
1).1,4,18−21 Because 3-EP has high specificity for tobacco
smoke and is present in high concentrations in SS smoke, it has
been widely used as an environmental tracer for secondhand
smoke (SHS).4,22−29 It is mainly in the gas (vapor) phase of
tobacco smoke.1,14 3-EP has emission rates and distribution
characteristics that are similar to VOCs in tobacco smoke that
have other sources as well, and therefore it has been proposed
as an environmental tracer for VOCs derived from tobacco
smoke.4
A number of tobacco smoke VOCs have been measured as
metabolites in the urine of smokers31−33 and people exposed
to secondhand smoke (SHS),34 but none are tobacco-specific.
3-EP is tobacco-specific, and if 3-EP or its metabolites could be
measured in human biofluids, they would be promising
candidates for gas-phase biomarkers. In this article, we (1)
describe an analytical method for measuring 3-EP in human
INTRODUCTION
■
Numerous toxic substances are present in tobacco smoke.1,2
Tobacco smoke is an aerosol, a mixture of particles and gases
that include vapors of volatile organic compounds (VOCs).
Both the gas phase and the particulate matter contain harmful
substances that may be causative agents for the major diseases
associated with smoking, including lung and heart disease and
cancer.3−5 Some modeling studies suggest that most of the
toxicity of tobacco smoke is due to gas-phase substances.6−9
Because gas-phase compounds may distribute differently in the
environment10 than particle-phase compounds, and because
they may have different modes of absorption into the body,11
specific biomarkers and environmental tracers for both
particulate matter and the gas phase are desirable in studies
of tobacco smoke exposure and toxicity.12
3-Ethenylpyridine (3-EP) is a pyrolysis product of nicotine
and possibly other tobacco alkaloids (Scheme 1).13,14 The
presence of 3-ethenylpyridine in tobacco smoke has been
known for many years.15−17 Reported concentrations in
Scheme 1. Formation of 3-EP by Pyrolysis of Nicotine
Received: February 10, 2021
Published: May 17, 2021
https://doi.org/10.1021/acs.chemrestox.1c00064
© 2021 American Chemical Society
Chem. Res. Toxicol. 2021, 34, 1630−1639
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Article
PA), which has a wider inner diameter of 2 mm. A PAL 250 μm
PDMS SPME arrow (CTC Analytics AG, Zwingen, Switzerland) was
used for sample microextraction.
Table 1. Concentrations of 3-EP in Mainstream and
Sidestream Cigarette Smoke (μg/cig)
cigarette brand/
SS/
MS
Synthesis of 3-EP-d4 and Oxalate Salt. The method is based on
the Suzuki−Miyaura cross-coupling reactions of potassium vinyl-
trifluoroborate with 3-bromopyridine described by Molander and
study
type
mainstream sidestream
Koszowski et al.
3R4F reference
4.1
219
531
451
640
2550
53
a
200918
cigarette
Brown35 and Alacid and Najera.36 Argon-flushed isopropyl alcohol (5
́
a
Koszowski et al.
200918
full flavor
9.5
56
mL) was added to a mixture of potassium vinyltrifluoroborate (805
mg, 6 mmol), tricyclohexylphosphine (100 mg 0.36 mmol),
palladium(0) bis(dibenzylideneacetone) (Pd(DBA)2) (100 mg 0.17
mmol), and 3-bromopyridine-d4, which was synthesized by the
method of Englert and McElvain37 (0.5 mL, 820 mg, 5 mmol) in an
argon-flushed 100 mL flask equipped with a reflux condenser and a
septum inlet and attached to a mineral oil bubbler. Potassium
carbonate (1.3 g,10 mmol) dissolved in 2 mL of argon-flushed water
was added. The mixture was refluxed with stirring for 2 h under a
static pressure of argon. Analysis of an aliquot by GC-MS indicated
that all of the 3-bromopyridine-d4 had reacted. Dichloromethane (5
mL) was added to the reaction mixture, which was filtered through
Celite that had been prewashed with isopropyl alcohol. The filter cake
was washed with 5 mL of dichloromethane, and 10 mg of butylated
hydroxytoluene (BHT) was added to the filtrate to inhibit possible
polymerization of the product. The lower aqueous layer was removed
and discarded, and the organic layer was distilled using a water bath
that was gradually heated to 50 °C under vacuum, increasing to ∼50
mmHg to remove the solvent. Subsequently, the remaining liquid was
distilled bulb-to-bulb (Kugelrohr) at 30 mmHg, and an air bath
temperature 115 °C to give 241 mg of colorless liquid, a 44% yield. A
considerable amount of yellow viscous liquid was left undistilled,
which was presumably a polymeric material. A 1 M solution of oxalic
acid dihydrate in isopropyl alcohol was prepared. To 201 mg of 3-EP-
d4 (1.84 mmol) was added 1.84 mL of the 1 M oxalic acid in a 20 mL
vial. The vial was vortexed, and the salt precipitated. This was diluted
with 2 mL of ether, and the product was filtered and washed with 2
mL of ether. The product was dried under suction, giving 200 mg of
white solid. This was recrystallized from 1.4 mL of ethanol and
washed with 2 mL of ethanol followed by 2 mL of ether. Air-drying
under suction provided 85 mg of white needles (mp 123−124 °C).
1H NMR (D2O): δ 6.81 (dd, J = 17.6, 10.8 Hz, 1H), 6.08 (d, J = 17.6
Hz, 1H), 5.65 (d, J = 10.8 Hz, 1H). 13C NMR (D2O): δ 164.7
(carbon in the oxalate group), 137.3, 129.8, 121.7. From GC-MS
analysis (isobutane CI), extracting the ion chromatogram correspond-
ing to 3-EP-d0 (m/z 106), no m/z 106 was detected, verifying its
suitability as a mass spectrometric internal standard for 3-EP.
a
Sakuma et al.
burley tobacco
13.2
23
34.2
28
198419
b
Brunnemann et al. U.S., unfiltered
197820
b
Brunnemann et al. U.S. cigar
30
85
197820
Kulshreshtha et al. full flavor
4.57
7−30
200321
c
Hoffmann et al.
unfiltered
20011
a
Hodgson et al.
reference 1R4F
680
19964
Hodgson et al.
19964
6 commercial
450−890
0.03−0.05
a
brands
d
Cancelada et al.
IQOS
2−3
201930
a
b
Both gas and particle phases. Measured as a mixture with 3,4-
c
d
lutidine. Vapor phase. Heat not burn product, μg/heatstick.
urine using headspace solid-phase microextraction (SPME) gas
chromatography−tandem mass spectrometry (GC-MS/MS);
(2) present data on the concentrations of 3-EP in the urine of
nonsmokers and of people using various combusted (cigarettes
and cigars) and noncombusted (smokeless and e-cigarettes)
tobacco products and secondhand-smoke exposure that
support its potential as a biomarker of tobacco smoke
exposure; and (3) propose that toxicological studies of 3-EP
are needed.
EXPERIMENTAL PROCEDURES
■
Chemicals. 3-Ethenylpyridine and 3-ethenylpyridine-d4 were
synthesized as described below. Both unlabeled 3-ethenylpyridine
and 3-ethenylpyridine-d4 can be purchased from Toronto Research
Chemicals (TRC), North York, ON, Canada, and other suppliers.
Reagents and solvents used for sample extractions and syntheses of
standards were of analytical reagent grade or high-performance liquid
chromatography (HPLC) grade. Unless otherwise specified, chem-
icals used in the synthesis of 3-ethenylpyridine and 3-ethenylpyridine-
d4 were from commercial vendors.
Synthesis of 3-EP. Unlabeled 3-EP and the oxalate salt were
prepared from 3-bromopyridine (10 mmol) as described earlier for 3-
EP-d4. The free base was obtained in 66% yield. Only trace impurities
1
were detected by GC-MS. The oxalate salt had mp 124−125 °C. H
NMR (D2O): δ 8.73 (br d, 1H), 8.59 (br d, J = 8.4 Hz, 1H), 8.56 (br
d, J = 6.0, 1H), 7.94 (dd, J = 8.4, 6.0 Hz, 1H), 6.81 (dd, J = 17.6, 10.8
Hz, 1H), 6.08 (d, J = 17.6 Hz, 1H), 5.65 (d, J = 10.8 Hz, 1H). 13C
NMR (D2O): δ 164.7 (carbon in the oxalate group), 143.2, 139.3,
138.8, 137.5, 129.9, 127.1, 121.7. The composition of the salt was
verified as being 1:1 by GC-MS comparison with freshly distilled 3-EP
free-base. For 3 aliquots of a solution extracted and analyzed, the
amount determined was within 5−10% of the specified amount, when
correcting the salt for the oxalic acid content, as being 53.8% base.
Small amounts of 3-EP base were converted to salts that have been
previously reported.38 Hydrochloride, white powder, mp 115−116.5
°C (lit 114−115°); chloroaurate, yellow solid, mp 137−140° (lit
138−140°); chloroplatinate, orange solid, mp 155−157° dec (lit
158−160).
Instrumentation. For characterization of the standards, 3-EP and
3-EP-d4, GC-MS was carried out using an Agilent 6890 GC interfaced
with an Agilent 5973 MSD operated in the positive ion chemical
ionization mode using isobutene as the reagent gas. 1H NMR spectra
were recorded using a Bruker Avance III HD 400 instrument at 400
MHz, and 13C NMR spectra were recorded at 100 MHz. Chemical
shifts were reported in parts per million (ppm, δ). Proton-coupling
patterns are described as singlet (s), doublet (d), broad doublet (br
d), and doublet of doublets (dd). For the determination of 3-EP in
urine, GC-MS/MS analyses were carried out with a Trace1310 GC
coupled to a TSQ 8000 Evo triple quadrupole mass spectrometer
(Thermo Fisher Scientific, San Jose, CA). Solid-phase microextraction
was performed on a Thermo CTC TriPlus RSH autosampler
equipped with an accessory for SPME fiber/arrow conditioning and
for sample incubation and extraction. Conventional SPME fibers were
desorbed in splitless mode with a 0.75 mm i.d. SPME liner through a
standard inlet septum. A Merlin Microseal septum replacement and a
Merlin Microseal nut (Merlin Instrument Company, Half Moon Bay,
U.S.A.) were used in the injection port when SPME arrows were used.
The liner used for the SPME arrow is a splitless single taper
gooseneck with a wool Topaz liner (Restek Corporation, Bellefonte,
Analytical Method Development: SPME Phase Selection
and Optimization. Polydimethylsiloxane (PDMS)- and polydime-
thylsiloxane/divinylbenzene (PDMS/DVB)-coated SPME arrows
were compared for the coating selection. There are two types of
SPME coatings: polymeric films for absorption of analytes and
particles embedded in polymeric films for adsorption of analytes.
PDMS belongs to the first type and is suitable for relatively nonpolar
compounds. PDMS/DVB belongs to the second type and has a
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bipolar feature. Traditional SPME fibers were also evaluated to
compare their performance to SPME arrows and to optimize the
sensitivity. Two PDMS SPME fibers with a sorbent length of 10 mm
and sorbent film thicknesses of 30 and 100 μm, and two PDMS
SPME arrows with a sorbent length of 20 mm and sorbent film
thicknesses of 100 and 250 μm, were used. Two mL of 100 ng/mL 3-
EP and 100 ng/mL 3-EP-d4 solution saturated with K2HPO4 (pH ≈
9) in water was used for this evaluation. 3-EP is a weak base with a
calculated pKa of 4.86 at 25 °C.39 Adjusting the pH to >7 ensures that
3-EP is in the free-base form and can be released from the solution.
We found that the 250 μm PDMS SPME arrow provided the highest
peak response for 3-EP and 3-EP-d4, which indicated its superior
ability to absorb the analyte from the headspace vapor. Consequently,
a 120 μm PDMS/DVB SPME arrow was tested as a comparison to
the 250 μm PDMS SPME arrow. Two mL of 0.2 ng/mL 3-EP and 5
ng/mL 3-EP-d4 solution saturated with K2HPO4 in water was used for
this evaluation. Although a 120 μm PDMS/DVB SPME arrow
extracted three times more 3-EP compared to a PDMS 250 μm one
under the same conditions, it extracted more impurities as well,
resulting in no improvement in the S/N ratio. In an attempt to obtain
a cleaner extract and improve the S/N ratio resulting in better
sensitivity, a liquid extraction step was tried before SPME. Urine was
extracted with methyl tert-butyl ether after making it basic with
sodium hydroxide. The organic layer was acidified to convert 3-EP to
a nonvolatile salt, then evaporated to dryness, reconstituted with 2 mL
of water, and then analyzed by the SPME method. This resulted in
good recovery for 3-EP and 3-EP-d4 (50−100%), but the background
of the blank urine sample did not show a significant improvement
compared to the direct SPME method. Therefore, 250 μm PDMS
SPME arrows were selected for further method development by the
direct headspace SPME method.
Optimization of Extraction Conditions. The effect of
extraction temperature was studied from 60 to 80 °C. In these
experiments, 2 mL of 1 ng/mL 3-EP and 100 ng/mL 3-EP-d4 solution
saturated with K2HPO4 (pH ≈ 9) was used. The results showed that
heating at 80 °C resulted in the highest partitioning of the analytes
into the headspace, and thus this temperature was used. For extraction
time, three extraction times of 1, 2, and 5 min were compared. We
found that 2 min resulted in greater peak areas than 1 min, but 5 min
provided negligible improvement in the 3-EP peak area, demonstrat-
ing that an extraction time longer than 2 min did not improve the
sensitivity. Therefore, a 2 min extraction time was used. Sample
mixing is generally used in SPME to shorten the equilibrium time
needed for extraction.40 Sample mixing was tested in this study with
two different rates, 600 and 1200 rpm, with an extraction time of 2
min. Two mL of 1 ng/mL 3-EP and 100 ng/mL 3-EP-d4 solution
saturated with K2HPO4 was used. We found that 1200 rpm produced
a peak that was 2 times higher than that for 600 rpm. Salting-out is
generally used in SPME to decrease the analyte solubility and to keep
the ionic strength in real samples similar to standards.41 Base addition
was also applied to keep the pH well above the calculated pKa of 3-EP
(4.86),39 as discussed earlier. At a sample pH of ∼8, 3-EP should be
99.9% in the free-base form. Saturated K2HPO4 and saturated sodium
chloride with 50% K3PO4 aqueous solution (w/w) addition were
compared in these experiments. The result showed that 75 μL of 50%
K3PO4 solution is needed to adjust typical urine samples to a basic
range, and 150 μL of 50% K3PO4 solution is needed for the acidified
urine samples available in some of our studies. There was essentially
no difference in recovery between the two procedures. Therefore, we
chose saturated sodium chloride with 75 or 150 μL of 50% K3PO4
addition for our sample preparation.
which may cause a tailing peak as the desorption time increases.
Under the condition of 1 min desorption, good sensitivity was
achieved with a peak width of 0.06 min.
Working Standards and Controls. A 1.00 mg/mL stock
standard solution of 3-EP, as the free base, was prepared in water
from 3-EP oxalate, corrected for the composition of 1:1 3-EP/oxalic
acid. The stock solution was then diluted successively with water to
form a set of 9 standards and quality control (QC) working solutions
ranging from 20 to 2000 ng/mL. One μg/mL of 3-EP-d4 in water was
used as the internal standard working solution. Nonsmokers’ urine
that was found to be free of 3-EP and 3-EP-d4 was used to prepare the
standards and QCs. Twenty μL aliquots of standard working solution
were spiked into 2 mL of urine to prepare the analytical run/
calibration standards and controls in the range of 0.2−20 ng/mL. The
final concentrations for the standards were 0.2, 0.5, 1, 5, 10, and 20
ng/mL, and the QCs were 0.2, 0.4, 2, and 8 ng/mL. The final
concentration for the internal standard is 5 ng/mL. Standards and
controls were freshly prepared before each use.
Sample Preparation. Ten μL of internal standard working
solution (10 ng of 3-EP-d4) was spiked into 2 mL of urine sample,
standard, or QC sample. One tablespoon of sodium chloride (∼700
mg) and 75 μL of 50% w/w potassium phosphate tribasic aqueous
solution were added. The amount of sodium chloride added was an
excess of what was necessary for reaching saturation, so the ionic
strengths of all samples were essentially the same. The final pH of the
urine samples was ∼8. Because some urine samples had been acidified
for stability purposes, a larger volume of 50% potassium phosphate
tribasic, 150 μL, was added to those samples.
Extraction Procedure. Samples were stored in the autosampler
tray at room temperature (23 °C). Prior to extraction, the SPME
fiber/arrow was preconditioned in the conditioning station at 250 °C
for 10 min under a stream of nitrogen at 5.0 mL/min. Before the
preconditioning began, the SPME tool including the fiber/arrow
transferred the sample from the autosampler tray to the incubation
station, where the autosampler mixed the samples with a rotating
motion at 600 rpm for 10 min at 80 °C. After the sample incubation
time, the sample was transferred to a stirring station where the sample
vials’ septa were pierced by the fiber/arrow and the sorption phase
was immersed into the sample headspace while the vial was
continuously mixed for 2 min at 1200 rpm to adsorb the analyte.
The sample vial penetration depth was set to 55 mm in order to
ensure constant and complete immersion of the sorption phase. After
the 2 min extraction period, the fiber/arrow was transferred into the
GC injector for thermal desorption. Subsequently, the fiber/arrow
was cleaned for 15 min in the corresponding conditioning station at
250 °C prior to adsorbing and injecting the following sample.
GC Chromatography. The analyte was desorbed from the fiber/
arrow in the injection port at 250 °C for 1 min. Analyte separation
was accomplished using a 30 m × 0.25 mm fused silica column, 0.25
μm HP-5MS stationary phase (Agilent Technologies, Palo Alto, CA).
Helium (99.995%, Airgas, Radnor, PA) was used as the carrier gas
with a flow rate of 1.2 mL/min. Nitrogen (99.999%, Airgas) acted as
the split and septum purge gas and also the gas for cleaning the SPME
fiber/arrow at the time of conditioning. A splitless injection mode was
used for the first 1 min. After a splitless time of 1 min, the split ratio
was set to 50:1. The oven temperature program was as follows: the
initial temperature was set at 40 °C and held for 1 min, followed by a
first temperature ramp of 20 °C min−1 to 150 °C, and a second ramp
of 80 °C min−1 to 280 °C, with a final time of 3 min.
Mass Spectrometry. Electron ionization at 70 eV was used. Data
were acquired in the selected reaction monitoring (SRM) mode. The
transitions 105 to 78 and 109 to 81 at a collision energy of 12 eV were
used for 3-EP and the internal standard 3-EP-d4, respectively, with
argon (99.998%, Airgas) as the collision gas. The transfer-line
temperature was set to 280 °C, and the ion source was set to 275 °C.
Instrument Calibration and Data Analysis. The XCalibur
software was used to generate calibration curves (linear regression, 1/
X weighting, ignore origin) and calculate concentrations using peak
area ratios of analyte/internal standard. Standard curves were linear
from 0.2 to 20 ng/mL for six concentrations spanning this range. Two
Optimization of Desorption and Injection Conditions. The
desorption time in the injector was set to 1 min because any longer
desorption time did not significantly increase the peak area. A splitless
injection mode was used so that all of the vaporized sample could be
applied to the column. After a splitless time of 1 min, the split ratio
was set to 50:1 for the purpose of septum and injector purge. The
column was kept at a low temperature of 40 °C for 1 min to focus all
3-EP on the head of the column. A longer desorption time requires a
longer splitless time and longer initial column temperature time,
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sets of standards and QCs were included in each run of 20−30 clinical
samples. Typically, one set of standards was injected at the beginning
of the run, and one set was injected following the injection of the
study samples. QC samples were run through the sequence among the
study samples. The equations and correlation coefficients for the
standard curves run with the study samples are as follows by date:
Scheme 2. Synthesis of 3-EP
modifications included the reaction solvent, reaction con-
ditions, and catalyst that resulted in a good yield in a short
period of time (2 h), obviating the need to carry out the
reaction for 22 h while being heated in a sealed tube, and
facilitated isolation of the product by a simple distillation
rather than column or flash chromatography. Because of the
instability of 3-EP during storage, we prepared known salts of
3-EP (hydrochloride, chloroplatinate, and chloroaurate),38 as
well as a new one, the oxalate, because amine salts are generally
more stable than the free bases. The oxalate was chosen as the
primary standard because the hydrochloride appeared to be
hygroscopic and because the expense of the precious metals
discourages their use. We should point out that 3-EP obtained
from commercial sources would be satisfactory if purified
before making working standards. A simple distillation should
suffice.
Method Development. Because of the volatility of 3-EP
and potential interference from extraction solvents, we
reasoned that headspace SPME would be more satisfactory
than methods involving liquid injection. A number of
parameters were evaluated, as described in the Experimental
Procedures section. These included the SPME fiber sorbent
types and film thicknesses, sorbent lengths, extraction times
and temperatures, and sample mixing rate. We found that 250
μm polydimethylsiloxane (PDMS) SPME arrows and an
extraction temperature of 80 °C provided the best results in
terms of sensitivity and chromatograms free from interfering
substances.
Method Validation. The analytical method was validated
according to protocols for bioanalytical method validation
generally applicable for pharmacokinetic studies and bio-
markers in drug development (Table 2).46−48 Standard curves
were constructed at six levels ranging from 0.2 to 20 ng/mL by
spiking 3-EP into a blank urine matrix. Precision, accuracy, and
lower limit of quantitation (LLOQ) were evaluated by
analyzing six different nonsmoker urines, from people who
had no known secondhand smoke exposure, spiked with 3-EP
in the same run and over 3−5 different runs. Intra-assay and
interassay precision (CV%) ranged from 0.6 to 8.7% for all
four concentration levels, and accuracy (percent of expected)
ranged from 92.1 to 104.5% (Table 2). The LLOQ was
determined to be 0.2 ng/mL on the basis of a CV < 20% and
an accuracy bias within 20%.
Specificity was determined by analyzing urine samples from
23 people who do not use tobacco products. All of them had 3-
EP concentrations below the limit of quantitation of 0.2 ng/
mL. Precision was also evaluated on a pooled smokers’ urine
sample, which was prepared by mixing equal amounts of urines
from three different smokers. Eighteen duplicate analysis were
carried out over three runs on three consecutive days with six
samples on each day. Intra-assay precision (CV%) ranged from
1.7 to 4.0%, and the interassay precision (CV%) is 6.1% (Table
2).
01/27/2020: Y = 0.0104749 + 0.329096X, r2 = 0.9985
02/06/2020: Y = 0.00915605 + 0.30328X, r2 = 0.9994
02/18/2020: Y = 0.00334046 + 0.331841X, r2 = 0.9997
06/17/2020: Y = 0.0661857 + 0.309408X, r2 = 0.9992
07/08/2020: Y = 0.0102504 + 0.288638X, r2 = 0.9992
09/06/2020: Y = 0.000327393 + 0.274545X, r2 = 0.9982
09/10/2020: Y = −0.00433334 + 0.279534X, r2 = 0.9991
Human Urine Samples. Urine samples were available from
previous studies.33,42−45 All studies received approval of the
appropriate institutional review boards. Sixteen urine samples were
obtained from cigarette smokers in a multisite, randomized clinical
trial.42 Participants were 18 years or older, smoked five or more
cigarettes per day, and had no current interest in quitting smoking. A
second set of 16 samples from cigarette smokers were from a
crossover study of dual users of small cigars and cigarettes in
Philadelphia (December 2012−December 2015) collected during the
cigarette-smoking arm.43 Samples from these same subjects during the
small cigar-smoking arm were also analyzed. Ten samples from water
pipe smokers were from a crossover study of water pipe and cigarette
smoking carried out by the Clinical Research Center at Zuckerberg
San Francisco General Hospital.33 These samples were 24-h
collections during ad libitum water pipe smoking, but participants
were asked to smoke a minimum of twice per day. Eight urine samples
were 24 h collections from e-cigarette users in San Francisco in a
crossover study of use of e-cigarettes, and combusted cigarettes were
collected during the vaping arm.44 This study was also carried out by
the Clinical Research Center at Zuckerberg San Francisco General
Hospital. The smokeless tobacco users (N = 11) were using their
usual brand of smokeless tobacco products. These samples were
obtained from the University of Minnesota Biorepository, which
contains biological samples from users of various tobacco products.
Urine samples from 9 nonsmokers exposed to SHS in a discotheque
were from nonsmoking adolescents, taken at baseline and after at least
4 h in a smoking-allowed disco.45 The 23 urine samples from
nonsmokers not exposed to SHS were obtained in San Francisco.
Smoking status and SHS exposure were by self-report and/or the
nicotine metabolite cotinine concentration being below the
established cutpoint for determining smoking status.
RESULTS
■
The goals of our study were (1) to develop an analytical
method with adequate sensitivity and specificity to measure 3-
EP in the urine of people exposed to tobacco smoke and (2) to
measure concentrations of 3-EP in the urine of people who
used various tobacco products to evaluate its utility as a
biomarker.
Synthesis of Standards. The foundation of any analytical
method is the availability of a standard with documented
identity and purity. 3-EP is commercially available as the free
base, and purities of 95% or greater may be specified. However,
on receipt from more than one vendor, it was a black liquid,
which on distillation yielded a colorless distillate and a
considerable amount of tarry residue. One sample received as a
solid had apparently polymerized. Because of obvious stability
issues, and to make available reasonable quantities of both
unlabeled 3-EP and a stable isotope-labeled analogue needed
as a mass spectrometric internal standard, we modified and
optimized published methods for the synthesis of 3-EP via
Suzuki−Miyaura cross-coupling reactions of potassium vinyl-
trifluoroborate with 3-bromopyridine.35,36 (Scheme 2) The
The stability of 3-EP in urine was evaluated by testing two
pH conditions and three storage temperatures (Table 3). In
these experiments, two concentrations of 3-EP (0 and 2 ng/
mL) were utilized. Five samples for each concentration level
were prepared by spiking desired amounts of 3-EP into
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Table 2. Intra-assay and Interassay Precision and Accuracy for Determination of 3-EP in Urine
expected amount
(ng/mL)
intra-assay precision
(CV%), n = 6
intra-assay accuracy (% of
interassay precision (CV%), n = 18, interassay accuracy (% of expected), n = 18,
expected), n = 6
3−5 different runs
3−5 different runs
0.2
0.4
2
8
day 1, pool
day 2, pool
day 3, pool
3 days pool
4.1
3.4
2.9
0.6
4.0
3.7
1.7
104.5
92.1
97.6
92.4
6.8
8.7
4.4
3.7
100.5
98.8
97.5
92.8
a
NA ; mean = 4.94 ng/mL
a
NA ; mean = 5.48 ng/mL
a
NA ; mean = 4.91 ng/mL
a
6.1
NA ; mean = 5.11 ng/mL
a
NA, accuracy not applicable for pooled smokers’ urine.
Table 3. Stability of 3-EP in Acidified and Nonacidified
Urine Samples (Concentrations in ng/mL over Time)
smokers in a multisite, randomized clinical trial.42 The
concentrations of 3-EP in urine ranged from below the limit
of quantitation (BLQ) to 5.12 ng/mL, with of mean of 1.08
(SD = 1.31) and a detection frequency of 88% (Figure 1). The
second group was from a study of dual users of cigarettes and
small cigars in Philadelphia, in a crossover study in which the
participants either smoked cigarettes or small cigars in different
study blocks.43 This allowed us to compare 3-EP excretion in
the same people for the two different products. During the
cigarette-smoking arm, the concentrations of 3-EP in urine
ranged from BLQ to 2.56 ng/mL, with of mean of 0.45 (SD =
0.63) and a detection frequency of 63%. During the cigar-
smoking arm, the concentrations of 3-EP in urine ranged from
BLQ to 3.74 ng/mL, with of mean of 0.66 (SD = 0.87) and a
detection frequency of 88%. Concentrations in urine of 23
adults who did not use tobacco products were all below the
limit of quantitation (Table 4). Representative GC-MS/MS
chromatograms are presented in Figure 2.
a
condition
initial
24 h
48 h
7 days
30 days
RT, no acid
0
2
4.90
0
2
<0.2
2.22
4.42
<0.2
2.12
3.86
4.74
4.10
4.97
4.61
b
RT, acid
4.14
0
4 °C, no acid
2
4.90
0
2
4.14
0
2
4.90
0
2
4.14
4.74
4.54
4.72
4.47
b
4 °C, acid
<0.2
2.13
−20 °C, no acid
<0.2
2.04
We also analyzed urine from electronic cigarette users,44
water pipe (hookah) smokers,33 and daily smokeless tobacco
users. 3-EP concentrations were BLQ in the urine of all of the
8 e-cigarette users. 3-EP concentrations were BLQ in the urine
of all but 1 of the 10 water pipe smokers. 3-EP concentrations
were BLQ in the urine of all but 1 of the 11 smokeless tobacco
users (Table 4).
b
−20 °C, acid
<0.2
2.10
a
Concentrations of 4.90 and 4.14 are pooled smokers’ urine. The
b
others are concentrations spiked into nonsmokers’ urine. With solid
sodium bisulfate.
Concentrations of 3-EP in sidestream smoke are substantial
(Table 1), and exposure in nonsmokers exposed to SHS could
be significant. We measured concentrations of 3-EP in urine
from a study of SHS exposure in smoking-allowed Mexican
discotheques.45 For 9 nonsmoking participants prior to
entering the discos, 3-EP concentrations were below the
limit of quantitation. Following spending several hours in the
discos, 3-EP was detectable in the urine of 4 subjects, with a
mean for all 9 subjects of 0.21 ng/mL, with a range BLQ−0.38
ng/mL (Table 4).
nonsmoker’s urine, which were further divided into two groups
with three samples in the acidified group (pH 2−3, adjusted
with solid sodium bisulfate) and two samples in the
nonacidified group. The acidified group samples were stored
at three temperatures with different time periods, which were
room temperature for 24 h and 4 and −20 °C for 30 days. The
nonacidified group samples were stored at room temperature
for 24 h and −20 °C for 30 days, respectively. We also
analyzed pooled smokers’ urine samples, acidified and
nonacidified, over the course of 7 days. We did not find
significant increases or decreases in concentrations under any
conditions for the spiked nonsmoker’s urine or the pooled
smokers’ urine. The percent differences from initial concen-
trations ranged from −10% to +11%, which is within
acceptable precision for this type of analysis46 (Table 3).
Concentrations of 3-EP in Urine of People Using
Various Tobacco Products. Because cigarette smoking
remains the most prevalent form of tobacco use, the first
groups of samples analyzed were cigarette smokers and
nonsmokers who reported no significant exposure to SHS.
The goals were to determine whether measurable amounts of
3-EP are excreted in the urine of smokers and whether 3-EP is
present in the urine of nonsmokers. Two groups of cigarette
smokers were studied. The first, N = 16, were daily cigarette
DISCUSSION
■
In this article we provide data supporting 3-EP as a promising
biomarker for the gas phase of tobacco smoke. Previous studies
demonstrated that 3-EP has high specificity for tobacco
smoke.23,24 Undetectable amounts or low concentrations of
3-EP have been reported in venues where smoking has not
occurred as compared to venues where smoking takes place.
For example, in a study comparing VOC concentrations in the
homes of smokers and nonsmokers, mean 3-EP concentrations
of 0.08 μg/m3 (N = 24, median undetectable) were found in
nonsmokers’ homes compared to a mean of 1.28 μg/m3 (N =
25) in smokers’ homes. The authors also found a significant
correlation between 3-EP concentrations and the number of
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Figure 1. Concentrations of 3-EP in the urine of people using tobacco products and of non-tobacco users. Horizontal lines in columns are mean
concentrations, and the dashed line is the limit of quantitation. WP = water pipe, ST = smokeless tobacco, and NTU = non-tobacco user.
Participants in the disco study were non-tobacco users. If below the limit of quantitation (BLQ), LLOQ/√2 was used.
a
Table 4. 3-Ethenylpyridine Concentrations in Urine of People Using Tobacco Products, People Exposed To Secondhand
Smoke (SHS), and People Without SHS Exposure Who Did Not Use Tobacco Products
b
3-EP urine concentration, ng/mL
cotinine, ng/mL
c
product use or exposure
cigarette smokers
mean (N)
median
SD (range)
detection frequency
p
mean
1.08 (16)
0.45 (16)
0.66 (16)
BLQ (10)
BLQ (8)
BLQ (11)
BLQ (9)
0.21 (9)
0.61
0.27
0.42
BLQ
BLQ
BLQ
BLQ
0.14
BLQ
1.31 (BLQ-5.12)
0.63 (BLQ-2.56)
0.87 (BLQ-3.74)
0.09 (BLQ-0.42)
all BLQ
0.02 (BLQ-0.22)
all BLQ
0.08 (BLQ-0.38)
all BLQ
88%
63%
88%
10%
0%
9%
0%
44%
0%
0.01
0.07
0.03
0.34
0.99
0.34
0.99
0.05
5400 (total)
3800 (total)
3500 (total)
920 (total)
1400 (total)
5100 (total)
0.38 (free)
20 (free)
d
cigarette smokers
d
cigar smokers
water pipe smokers
e
e-cigarette users
smokeless tobacco users
f
nonsmokers pre-disco
f
nonsmokers post-disco
non-tobacco users
BLQ (23)
a
b
Lower limit of quantitation (LLOQ) = 0.2 ng/mL. If below the limit of quantitation (BLQ), LLOQ/√2 was used. Total cotinine is the sum of
cotinine glucuronide and unconjugated cotinine. Free cotinine is unconjugated cotinine. About equal amounts of free cotinine and cotinine
glucuronide are excreted by most smokers.49 p-values are for differences between tobacco product use and non-tobacco users by t test. These
c
d
participants were dual users of cigarettes and small cigars in a crossover study in which participants smoked either cigarettes or small cigars in
e
f
different study blocks.43 These samples were analyzed in duplicate. These participants were nonsmokers who spent several hours in smoking-
allowed discotheques.45
cigarettes smoked.50 In our studies, the concentrations of 3-EP
in the urine of nonsmokers (N = 23) were below the limit of
quantitation. 3-EP concentrations were measurable in most
urine samples from combustible tobacco users, confirming its
specificity for tobacco smoke (Table 4).
Specific biomarkers for both the particle phase and the gas
phase are desirable because the two phases distribute
differently in the environment,10 and compounds in them
may have different modes of absorption in the respiratory
tract.11 3-EP exists primarily in the gas phase of cigarette
smoke, a requisite for it to be a useful marker for gas-phase
compounds.1 The validity of 3-EP as a biomarker for gas-phase
components is supported by studies showing that 3-EP is a
useful tracer for VOCs derived from tobacco smoke. 3-EP has
been utilized for source apportionment to estimate the
contribution of SHS (ETS) to concentrations of VOCs in
indoor air.4,50
Because 3-EP is produced by pyrolysis at high temper-
ature13,14 and does not appear to be naturally occurring in
tobacco, it would not be expected to be present in the urine of
people using e-cigarettes or smokeless tobacco. That indeed
was found to be the case (Table 4). 3-EP was not detected in
the urine of 8 electronic cigarettes users. For 11 users of
smokeless tobacco, 3-EP was detected in the urine of only one
study participant, and the concentration (0.22 ng/mL) was just
above the LLOQ, possibly the result of exposure to SHS. For
10 study participants who smoked a water pipe on a research
ward, 3-EP was detected in the urine of just one participant at
a concentration of 0.42 ng/mL. Because these participants
excreted substantial concentrations of nicotine metabolites,33
we conclude that the temperatures achieved during water pipe
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wallboard in extreme cases, as well as avoiding venues where
THS is present.10 Unlike nicotine and other less-volatile
substances in tobacco smoke, which have a strong affinity for
surfaces and can persist for long periods indoors after smoking
ceases,3 3-EP has a relatively low affinity for surfaces29 and due
to its volatility is removed fairly rapidly by ventilation.
Therefore, 3-EP should be a selective marker for SHS
exposure, and its concentrations in biofluids could potentially
be used to distinguish SHS exposure from THS exposure.
Concentrations of 3-EP in tobacco smoke are substantial
(Table 1). Because of its structural similarity to styrene, a
probable human carcinogen (Figure 3), consideration of the
Figure 3. Structures of 3-EP and styrene.
potential toxicity of 3-EP is warranted.52 Surprisingly, very
little has been published on its potential toxicity. In a study
published in 1992,53 it was reported that 3-EP was not
mutagenic in Salmonella typhimurium strains TA 1535, TA
1538, TA 98, and TA 100, nor was it genotoxic in the rat
hepatocyte DNA-repair test. No significant incidence of lung
adenoma or of any other type of tumors was found after
intraperitoneal injection in A/J mice. However, in this same
study, styrene was not found to be mutagenic or genotoxic and
likewise did not lead to a significant incidence of any type of
tumor. Subsequent studies have determined that styrene is
carcinogenic, and it is considered a probable human
carcinogen by the International Agency for Research on
Cancer (IARC).54 We are unaware of any studies of 3-EP
toxicity other than the 1992 study, which suggests that further
studies are warranted.
Figure 2. GC-MS/MS selected reaction monitoring (SRM)
chromatograms of 3-EP in a smoker’s urine and a nonsmoker’s
urine. Parent-to-product ion transitions are indicated in the upper left
corners of the chromatogram panels.
smoking generally are not high enough to convert nicotine to
3-EP. Therefore, a potential application of 3-EP is a biomarker
to distinguish combusted tobacco use from the use of other
tobacco products. We should note that typical water pipe use
does not involve tobacco combustion per se. It involves placing
a piece of burning charcoal on top of a moist mixture of fruit
and tobacco, and the smoker inhales the resulting aerosol
without actual combustion of the smoking product. Our results
are consistent with those of a study of water pipe smoke
contamination indoors, in which increased concentrations of
nicotine and benzene were measured, but 3-EP concentrations
were below the detection limit before and after water pipe
smoking.51 Distinguishing combusted tobacco use from the use
of other products is of interest in studies comparing exposure
and health effects in people using different or multiple
products.
Another possible application of 3-EP is as a biomarker for
secondhand smoke (SHS) exposure and to distinguish
secondhand smoke exposure form thirdhand smoke (THS)
exposure. From a toxicological standpoint, this is important
because modes of exposure are different and strategies for
reducing exposure are different. SHS consists of airborne
particles and gases, exposure is primarily by inhalation, and
strategies for reducing exposure include room ventilation and
avoiding venues where people smoke. THS consists of the
residues that remain and react with other substances in the
environment and substances that can be reemitted from
surfaces. Exposure to toxic substances in THS can be by
transdermal absorption, ingestion of dust and hand-to-mouth
behavior by young children, and inhalation. Strategies for
reducing THS exposure include remediation of the venue by
thorough cleaning or even replacing carpets, furniture, and
CONCLUSIONS AND FUTURE STUDIES
■
We have demonstrated, we believe for the first time, that 3-EP
is present and measurable in the urine of smokers and that it
may have utility as a biomarker of gas-phase toxicants in
studies of tobacco smoke exposure and toxicity. There are
some limitations to our studies. Larger numbers of samples
from people using various product types and samples from
people of different demographics will be needed to properly
determine 3-EP concentrations on a population basis. Because
3-EP was not detected in all smoking participants, improve-
ments in the sensitivity of the analytical method would be
desirable. Nothing is known about the half-life of 3-EP, and if it
is short 3-EP might not be detectable if sufficient time had
elapsed between smoking and the time of sampling for
concentrations to fall below the LLOQ. It is possible that
metabolites of 3-EP may be present in higher concentrations
than 3-EP and might be amenable to more sensitive detection.
Studies on the metabolism of 3-EP are underway in our
laboratory.52 Our study provides proof of concept that, with
improvements in method sensitivity and/or identification of a
more abundant metabolite, 3-EP could become a generally
useful gas-phase biomarker in smokers and in nonsmokers
exposed to tobacco smoke. Our study also calls attention to the
significant exposure in both smokers and nonsmokers exposed
to SHS and the fact that the paucity of toxicity data on 3-EP
suggests the need for further toxicological studies.
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AUTHOR INFORMATION
ABBREVIATIONS
■
■
3-EP, 3-ethenylpyridine; SPME, solid-phase microextraction;
VOC, volatile organic compound; MS cigarette smoke,
mainstream cigarette smoke; SS cigarette smoke, sidestream
cigarette smoke; PDMS, polydimethylsiloxane; Cy3P, tricyclo-
hexylphosphine; DBA, dibenzylideneacetone; LLOQ, lower
limit of quantitation; BLQ, below the limit of quantitation;
SHS, secondhand smoke; THS, thirdhand smoke
Corresponding Author
Peyton Jacob, 3rd − Clinical Pharmacology Program, Division
of Cardiology, Department of Medicine, University of
California, San Francisco, California 94143, United States;
orcid.org/0000-0003-3366-2775; Phone: (415) 282-
9495; Email: peyton.jacob@ucsf.edu; Fax: (415) 206-
5080
REFERENCES
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Jia Liu − Clinical Pharmacology Program, Division of
Cardiology, Department of Medicine, University of California,
San Francisco, California 94143, United States;
orcid.org/0000-0003-0512-2026
Neal L. Benowitz − Clinical Pharmacology Program, Division
of Cardiology, Department of Medicine, University of
California, San Francisco, California 94143, United States
Dorothy K. Hatsukami − Department of Psychiatry and
Behavioral Sciences and Masonic Cancer Center, University
of Minnesota, Minneapolis, Minnesota 55455, United States
Christopher M. Havel − Clinical Pharmacology Program,
Division of Cardiology, Department of Medicine, University of
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Eduardo Lazcano-Ponce − School of Public Health of Mexico,
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Andrew A. Strasser − Tobacco Center of Regulatory Science,
Department of Psychiatry and Abramson Cancer Center,
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.chemrestox.1c00064
Author Contributions
P.J. and J.L. conceived and designed the study and carried out
data analysis. J.L. developed the analytical method and
performed the urine analyses. P.J. synthesized the standards.
C.M.H. assisted with method development and data
acquisition. N.L.B., D.K.H., E.L.-P., and A.A.S. designed and
directed the studies that generated the urine samples from
tobacco product users. All authors contributed to writing the
manuscript and approved the final version.
Funding
Financial support from the California Tobacco-Related Disease
Research Program (TRDRP), 28PT-0077 (P.J.) and 28PT-
0013 (N.L.B.), and from the National Institutes of Health, P30
DA012393 (P.J.), R01 CA141531 (D.K.H.), and U54
CA229973 (A.A.S.), is gratefully acknowledged. The content
is solely the responsibility of the authors and does not
necessarily represent the official views of the TRDRP, the
National Institutes of Health, or the U.S. Food and Drug
Administration that provided funding for U54 CA229973.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Lisa Yu, Kristina Bello, Ti Won, and Marlene
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study, and we thank Newton Addo for statistical analyses and
figure preparation.
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NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was published on May 17, 2021, with an incorrect
column heading in Table 2. The corrected version was
reposted on May 17, 2021.
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Chem. Res. Toxicol. 2021, 34, 1630−1639