Determination of major tobacco alkaloids in mainstream
cigarette smoking
C. C. Kurgat1, J. K. Kibet*1, P.
K. Cheplogoi1, S. C. Limo2, and P. M. Kimani1
1Department
of Chemistry, Egerton University P.O Box 536 - 20115, Egerton, Kenya
2Department
of Physics, University of Eldoret, P.O. Box 1125-30100, Eldoret, Kenya
*Corresponding Author E-mail: jkibet@egerton.ac.ke
The
popularity of tobacco use worldwide has kicked off one of the greatest clinical
debates on human toxicology and public health in general. Accordingly, this
study investigates some of the alkaloids in tobacco believed not only to be
addictive and carcinogenic but also as precursors for other related medical
problems. The characteristic behaviour, identification and product evolution of
β-nicotyrine and 3,5-dimethyl-1-phenylpyrazole in tobacco is reported
extensively in this study for the first time. Two commercial cigarette brands
coded SM1 and ES1 were explored for evolution of major alkaloids over a modest
temperature range of 200 – 700 ̊ C for a total pyrolysis time of 3 minutes
using a tubular quartz reactor, typically in increments of 100 ˚C using
nitrogen as the pyrolysis gas at a residence time of 2.0 seconds under 1
atmosphere pressure. The heating rate of the heater was ~ 20 ˚C s-1.
The pyrolysate was passed over 10 mL analytical grade methanol and analyzed
using a Gas-Chromatography hyphenated to a mass spectrometer (GC-MS) with a
mass selective detector (MSD). GC-MS results showed that nicotine was the major
alkaloid in both cigarettes reaching a maximum at ~ 400 ˚C (8.0 x 108
GC-Area counts) for ES1 cigarette and 500 ˚C (~2.7 x 108
GC-Area counts for SM1 cigarette. Clearly, the ratio of nicotine for ES1 to SM1
is approximately 3 indicating that ES1 cigarette is rich in nicotine. Based on
this data alone, ES1 cigarette was found to be more addictive.
KEYWORDS: Alkaloid, pyrolysate, toxicology, β-nicotyrine.
INTRODUCTION:
Smoking has serious effects on almost every organ in
the body, accounting for more than 10% of deaths from all causes and 30% of
deaths from cancer related cases worldwide1,2. According to the Global Burden of Disease study, smoking
causes incredible ill health, estimated at 2,276 disability-adjusted life years
(DALYs) per 100,000 tobacco consumers3. Accordingly, whereas studies
on nicotine is widely reported literature, research on β-nicotyrine and
3,5-dimethyl-1-phenylpyrazole alkaloids is yet to gain significant attention.
Therefore, this study is one of the first to explore the evolution of
β-nicotyrine
and 3,5-dimethyl-1-phenylpyrazole in a more detailed
approach. The alkaloid family in tobacco is known to be responsible for addiction
in cigarette smoking therefore their investigation is very important2,4.
Generally, Cigarette smoking is believed to be responsible for serious health,
and public health problems throughout the world 5. In addition,
cessation of cigarette smoking is particularly difficult because of the highly
addictive nature of nicotine 6. Among regular smokers, withdrawal
symptoms occur within two hours of the last cigarette, peaking within 24–48
hours and sometimes may last for weeks or in some cases even months3. These revelations are disturbing considering
that tobacco is the leading cause of preventable death in the world 7-9.
The symptoms of cigarette withdrawal are psychological
and include the desire to smoke, depression, sleeplessness, irritability,
anxiety, difficulty concentrating, restlessness, and weight gain 10.
Most smokers who quit smoking experience nicotine withdrawal symptoms due to
nicotine dependence 11. Usually the symptoms are most severe during
the first 3 days after cessation but may continue for weeks or even months 12.
The severity of these symptoms depends on the number of cigarettes smoked daily
and the duration of usage 2,4,13. Research has also proven that
women are unfortunately less successful in quitting smoking than men4,14
because the withdrawal symptoms of nicotine and possibly other alkaloids are
more severe in women and replacement therapy has proven to be less effective
15.
The molecular structures of alkaloids explored in this
study are presented in Fig. 1. Nonetheless, other minor alkaloids such as
3-methyl pyridine, and 2-methyl pyrrole, have briefly been discussed in this
work from the mechanistic destruction of nicotine in high temperature smoking
regime.
Fig. 1: The
molecular structures of alkaloids investigated in this work
Considering
the complex nature of tobacco smoke, with more than 7,000 known constituents, and
its biological variety characterized by the presence of carcinogens, toxicants,
irritants, tumor promoters, co-carcinogens, and inflammatory agents, any
determination to identify individual compounds which cause lung cancer,
oxidative stress or cardiac arrest in smokers is difficult16,17.
Therefore, the concept of tobacco development, composition, and toxicity is no
doubt a very important area of research 18. Inhalation studies of
cigarette smoke have many difficulties which have been previously summarized,
all relating to the fact that laboratory animals do not voluntarily inhale
cigarette smoke, but rather try to avoid it 17,19. Nevertheless, a
number of relatively recent studies have demonstrated lung tumor induction in
both rats and mice exposed to cigarette smoke 16,19.
This
study focuses primarily on the evolution of major tobacco alkaloids at various
smoking temperatures using an in-line GC-MS at specific temperature at which
they are evolved in high amounts during cigarette smoking. The temperature at
which the concentration of alkaloids are released in high amounts is important
especially in designing modern cigarettes that can be smoked at lower
temperatures thus avoiding consumption of high levels of alkaloids by smokers.
Mechanistic description for the formation of free radicals from nicotine
considered biologically harmful has been explored quantum mechanically.
Furthermore, interesting conclusions have been drawn regarding the evolution of
nicotine from the two common cigarettes (ES1 and SM1). A toxicological survey
of these alkaloids based on our data and literature data has been briefly
discussed.
EXPERIMENTAL PROTOCOL:
Materials
The heater (muffle furnace) was purchased from Thermo
Scientific Inc., USA while the quartz reactor was locally fabricated in our
laboratory by a glass-blower. Commercial cigarettes SM1 and ES1 (for
confidentiality) were purchased from retail outlets and used without further
treatment. Methanol (purity ≥ 99%) used to dissolve cigarette pyrolysate
was purchased from Sigma Aldrich Inc. (USA).
Sample preparation
50 mg of tobacco was accurately weight to the nearest
mg and packed in a quartz reactor of dimensions: i.d. 1 cm x 2 cm (volume 
1.6 cm3). The
tobacco sample in the quartz reactor was placed in an electrical heater whose
maximum heating temperature is 1000 ˚C with a heating rate of ~ 20 ˚C
s-1. The tobacco sample was heated in flowing nitrogen pyrolysis gas
to maintain a residence time of 2.0 s and the smoke effluent was allowed to
pass through a silica coated transfer column and collected in 10 mL methanol in
a conical flask for a total pyrolysis time of 3 minutes and sampled into a 2 mL
crimp top amber vials for GC-MS analysis. This combustion experiment was
conducted under conventional pyrolysis described elsewhere 20 and
the evolution of alkaloids (nicotine, β-nicotyrine, 3,5-dimethyl-1-phenylpyrazole, pyrrole, and pyridine) were monitored between 200 and 700 ˚C.
GC-MS identification of tobacco alkaloids
GC-MS analysis was carried out using
an Agilent Technologies 7890A GC system coupled with an Agilent Technologies
5975C inert XL Electron Ionization/ Chemical Ionization (EI/CI) with a triple
axis mass selective detector (MSD), using HP-5MS 5% phenyl methyl siloxane
column (30 m x 250 µm x 0.25 µm). The
temperature of the injection port was set at set at 200 ˚C to vapourize
organic components to the gas-phase prior to MS analysis. The carrier gas was
ultra-high pure (UHP) helium (99.99%). The flow rate of the carrier gas (He)
was set at 3.3 mL/min at 1 atmosphere pressure and a residence time of 2.0
seconds. Temperature programming was applied at a heating rate of 15˚C for
10 minutes, holding for 1 minute at 200˚ C, followed by a heating rate of
25 ˚C for 4 minutes, and holding for 10 minutes at 300 ˚C. Electron
Impact ionization energy of 70 eV was used. To
ensure that the right alkaloid was detected, standards were run through the
GC-MS analytical system and the peak shapes and retention times compared with
the compounds of interest.The data was run through the NIST library database as
an additional tool to confirm the identity of compounds20.
GC-MS analysis Quality
Control (QC)
To
ensure consistency in GC-MS data the mass spectrometer was tuned to check for
leaks and water levels in the instrument which would affect the accuracy of the
data before any analysis was conducted. This procedure was very important in
order to prevent contamination, and extend the life of the EI filament.
Quantitative transport tests were initiated before any run was conducted to
ensure that there were no leaks in the GC-MS system and guarantee the pyrolysis
system is clean. The flow rate in the transfer line was monitored to make sure
that it was constant and did not fluctuate. If the flow rate was not
consistent, and the pressure was not stable when the transfer line was
connected to the GC-MS then leaks could be present in the system. This was
corrected before any experiment could begin. To correct for any leaks in the
system, a gas leak detector was used. Whenever leaks were detected along the
gas lines, transfer lines, or reactor-injection port interface, the connections
were tightened and quantitative transport experiment repeated to make sure no
leaks were in the system. A known concentration of nicotine was injected into
GC-MS system to monitor how much nicotine was recovered after analysis. This
was to test the efficiency of the GC-MS instrument. A recovery of ≥ 95%
was good enough in order to proceed with analysis.
Computational
methodology
In
order to investigate the molecular behaviour and energetics for formation of
free radicals from nicotine, thermochemical calculations were conducted using
Gaussian ’09 computational framework. Nicotine was used as the test compound
for selecting a suitable basis set (cf. scheme 1 and Figure 2). The geometries
were optimized at DFT/B3LYP using the 6-31G basis set 21. When using
DFT, however; the choice of basis set is considered to be inconsequential
because the convergence of DFT to the basis-set limit with increasing size of
basis set is relatively fast and thus small basis sets are used21, 22.
More often, diffuse functions on basis sets are not used for DFT calculations,
as these lead to linear dependencies and a bad convergence of the
self-consistent-field (SCF) Kohn– Sham equations for larger molecules 22.
RESULTS AND DISCUSSION:
In this study, it was established that the alkaloid
content varied widely among the two commercial cigarettes investigated as shown
in Fig, 2. Nonetheless, one thing was remarkable that most combustion alkaloids
were released in high quantities between 400 – 500 ˚C contrary to previous
biomass pyrolysis experiments which put the evolution of most by-products at
between 300 and 400 ˚C 20,23. Pyro-synthesis of alkaloids
during tobacco burning is therefore unique probably because of the high
energies involved during the formation of the C-N bonds. As a result, the
product evolution of major alkaloids is shifted to higher temperatures.
However, pyrrole for instance has a maximum release at ~ 450 ˚C which is
consistent with previous pyrolysis experiments on biomass pyrolysis 24.
It is important to note that whereas studies on product evolution agree on a
given temperature range of maximum release of reaction products, the pyrolysis
conditions and type of biomass under study may cause the pyrolysis products to
be released either at lower or higher temperature ranges. Nevertheless,
consensus of opinion agrees that most combustion by-products reach a maximum
between 400 and 500 ˚C 25.
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Fig. 2: Product distribution of nicotine and major alkaloids
in mainstream cigarette smoke determined from the burning of two commercial
cigarettes; ES1 (left) and SM1 (right).
|
Fig. 3:
Product yields of β-nicotyrine
and 3,5-dimethyl-1-phenylpyrazole (3,5-dmpp) for ES1 cigarette (pink) and SM1
(yellow).
It
is evidently clear that of all the alkaloids determined in this study, nicotine
was found in high concentrations in the two cigarettes in the entire pyrolysis
temperature range. Nonetheless, while the maximum concentration of nicotine was
noted at 400 ˚C (~ 8.0 x 108 GC-area counts) for ES1 cigarette,
nicotine concentration peaked at ~500 ˚C (~2.7 x 108 GC-Area
Counts) for SM1 cigarette. The ratio of nicotine in ES1 cigarette to that in
SM1 cigarette was ~ 3 according to this study. This implies that ES1 cigarette
contains about 3 times the amount of nicotine than SM1 cigarette. These results
are remarkable and present the first intense study on two different commercial
cigarettes coded ES1 and SM1 popularly sold around the world. Even more
surprising is the fact that the maxima of the two cigarettes give a ratio of
~3. This validates the method of analysis used in this investigation because
virtually most of the reaction products of most biomass materials including
tobacco peak between 300 and 500 ˚C 20,26. From Fig. 2, it can
be observed that whereas pyridine is the key product of ES1 cigarette, the
other alkaloids (β-nicotyrine
and 3,5-dimethyl-1-phenylpyrazole, and pyrrole) have comparable concentrations
in the two cigarettes under investigation. For instance in SM1 the total
pyrrole concentration in the whole temperature range was found to be ~ 2.0 x 108 whereas in ES1 the total pyrrole concentration was
found to be ~1.5 x 108 in the same temperature range. Nevertheless,
the evolution of nicotine from ES1 is significantly high even at low
temperatures (≤ 300 ˚C) as can be observed in Fig. 2. On the
contrary, nicotine production from SM1 cigarette only becomes significant above
300 ˚C. In order to have a clear overview of variations in concentrations
between the other alkaloids explored in this work in the entire pyrolysis
temperature range, Fig.3 is presented.
Clearly, SM1 cigarette yields significantly
high concentrations of β-nicotyrine and 3,5-dimethyl-1-phenylpyrazole in
the whole pyrolysis range in comparison to ES1 cigarette (cf. Fig. 3). This
contrasts sharply with the high yields of nicotine and pyridine noted in ES1.
Based on these data alone, it can be noted that cigarette ES1 is very addictive
considering the high levels of nicotine it produces during smoking and possibly
the ‘darling’ of most cigarette smokers.
The overlay chromatograms indicating the
identification of alkaloid reaction products explored in this work is presented
in Fig. 4. The compounds numbered a and b represent β-nicotyrine and
3,5-dimethyl-1-phenylpyrazole respectively as presented in Fig. 1, vide
supra. It may be attractive to examine some of the other prominent peaks
shown in the Fig. 4 but the principal focus of this study was to investigate
the major tobacco alkaloids in a more thorough manner. The other reactions
products which are mainly oxygenated compounds and aromatics will be reported
in subsequent articles.
Fig. 4:
Overlapping chromatograms for ES1 (red line) and SM1 (blue line) at 300
˚C.
The proposed free radical formation from the thermal
degradation of nicotine
Quantum mechanical calculations are fundamental in
predicting new insights on feasible mechanisms on the thermal degradation of
environmental pollutants. Figure 5 shows the use of various basis sets for
formation of different radicals from the thermal degradation of nicotine. It is
evident that there are some slight differences between various basis sets. The
most consistent basis set chosen for this investigation is the 6-31G which
appears suitable for the tobacco alkaloids under investigation.
Scheme 1: The
formation of various radicals from the thermal degradation of nicotine
Scheme 1, vide supra depicts the formation of
various radicals in the proposed thermal degradation of nicotine in high
temperature cigarette smoking. Clearly, reaction 3 proceeds with a lower energy
in comparison to reactions 1 and 2. Reaction 3 therefore is a very important
reaction because it leads to the formation of intermediates which can transform
to other combustion by-products including pyridine (when pyridinyl radical
reacts with H radical in the combustion system) or methylated pyrrolidine when
pyrrodinyl radical reacts with H radical. Nonetheless, reactions of the free
radical in reaction 3 with methyl radical to form 3-methyl pyridine is known
but according to literature, the reactivity of H radical occurs by several
magnitudes in comparison to the methyl radical 27,28. This
corroborates our experimental investigation in this study in which low yields
of 3-methyl pyridine and aniline were detected. Pyrrole however, was reported
in high yields in this study (Fig. 4) and may be attributed to the scission of
the phenyl C-C linkage in β-nicotyrine and possible other pyrosynthetic
reactions during tobacco burning. The formation of free radicals and transient
intermediates is considered the major reaction in tobacco burning. Free
radicals are responsible for severe health conditions such as cardiac arrest,
oxidative stress, tumours, and cancer related illnesses therefore understanding
the energetics of these species are fundamental in demystifying cigarette
smoking 29,30.
Figure 5: A plot of
enthalpy change for reactions 1-3 (scheme 1) at various basis sets
Based
on the thermodynamic data presented in Fig. 5, it is apparent that reactions 1
and 2 proceed with high energy and therefore not the major mechanistic pathways
for the thermal decomposition of nicotine. A detailed mechanistic studies of β-nicotyrine and
3,5-dimethyl-1-phenylpyrazole will be treated thoroughly in our next article
from a theoretical perspective.
The toxicological consequences of cigarette smoking
There is no doubt that inhaled toxicants such as
alkaloids in cigarette smoke can cause both irreversible alterations to the DNA
and serious variations in the genetic landscape which include changes in the
DNA methylation and chromatin alteration state 31,32. Clearly, from
this study, alkaloids and their corresponding free radicals are potential
candidates for diseases that are believed to comprise genetic and epigenetic
perturbations such as lung cancer, chronic obstructive pulmonary disease
(COPD), and cardiovascular disease (CVD), all of which are strongly linked
epidemiologically to cigarette smoking 31. Lung cancer development
involves various genomic aberrations, such as point mutations, deleterious
effects, and gene amplifications31. Direct DNA damage by cigarette
toxicants including nicotine, pyrrole, and pyridine has been linked to
cardiovascular disease 31,32. Additionally, Reproductive function and
fertility are thought to be compromised by behaviors such as cigarette smoking2,32.
Although very little information is available in literature on the effects of
β-nicotyrine and 3,5-dimethylpyrazole, it is evident from this study that
their molecular nature as well as their intermediate radicals may pose critical
impacts to the biological structures of cigarette smokers. The molecular
structure of nicotine and other alkaloid related compounds investigated in this
work may covalently bond to the DNA, lipids, nuclei acids, and body cells
before metabolizing to harmful by-products that are potentially risk to the
human health.
CONCLUSION:
It was established in this study that most of the
alkaloids were evolved between 400 and 500˚C. Clearly, this is a region
that should be avoided in the design of modern cigarettes because most organic
toxics are released in this temperature range. Although cigarette research has
dominated critical discussions in addiction and inhalation science with no
conclusive remedy on sight, this study has presented interesting findings that
may assist cigarette manufacturers to design cigarettes that can be smoked at
lower temperatures and thereby avoid inhaling high levels of tobacco toxins
usually smoked at high temperature smoking regimes. Therefore, cigarettes that
can be smoked at a lower temperature would be beneficial to the general
cigarette smoking community. Tobacco additives which may results in increased
levels of organic toxins must be regulated. Evidently from this study, the
commercial cigarette ES1 is very addictive considering the high levels of
nicotine it emits during smoking.
ACKNOWLEDGEMENT:
This work was partially funded by the Directorate of
Research and Extension (R&E) at Egerton University (Njoro). The Department
of Chemistry at Egerton University is appreciated for providing the
computational resources used in this work.
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