Comparative Analysis of Particulate Content in Cigarette Smoke: Implications for Respiratory Health of Active and Passive Smokers

Cigarette smoke is a heterogeneous mixture of gases, uncondensed vapours and liquid particulate matter with millions of particles per cubic centimetre. Cigarette smoking can negatively impact the health of smokers and non-smokers. As such, there is a need to constantly assess the quantity of toxic substances emanating from smoking cigarettes. This study investigated the total amount of particulates to which smokers and non-smokers are exposed and identified the chemical compositions of total particulates in cigarette smoke from some selected cigarette brands obtained from Nagongera town council shops in Uganda. To achieve this, we made use of an apparatus that mimics the smoking process of a human being and a passive smoker by connecting this set to another set-up containing a cigarette to be burnt. Qualitative tests for chemical


INTRODUCTION
Cigarette smoking is a widespread habit in our society (Ussher et al., 2014) and the social, political, aesthetic and medical issues resulting from cigarette smoking have affected smokers and non-smokers in the same way (Gowing et al., 2015).According to the American Lung Association, cigarette smoking is the primary cause of lung cancer (Addolorato et al., 2016) and emphysema (Jou et al., 2019) which occurs when alveoli walls begin to break down.Also, tobacco smoke has been documented to cause numerous diseases such as asthma, bronchitis, heart diseases, high blood pressure, stroke, stomach ulcers and, many cancers such as cancer of the lungs, throat and mouth (Lei et al., 2023).Tobacco smoking can also increase complications of tuberculosis (TB), diabetes and HIV/AIDS (Mrigpuri et al., 2021).
In Uganda, 26% of deaths are due to cancers of the respiratory system and 14.0% of deaths due to other respiratory diseases were attributable to tobacco smoking (World Health Organization, 2011).Statistical data provided by WHO indicated that 17.2%, 2.6% and 9.9% of males, females and both sexes, respectively, in Uganda are currently smoking tobacco, respectively (World Health Organization, 2011).Further, it is important to highlight that many organic and inorganic chemicals in the aerosol (volatile, and particulate phases) of cigarette and tobacco smoke appear to contribute to the toxicity of the smoke to the respiratory system.Examples are hydrocarbons, aldehydes, ketones, organic acids, phenols, cyanides, acrolein, and nitrogen oxides (Hashizume et al., 2023).Some components contribute to the development of chronic mucus hypersecretion in the central airways, whereas others play a greater role in the production of small airway abnormalities and emphysematous injury to the peripheral air sacs.Oxidizing agents in smoke inhibit the enzymes that defend against the destruction of lung elastin (Hong et al., 2022).
In view of the prevalence of cigarette smoking in Uganda, there is a great need to provide information about aerosols in smoke by ascertaining the particulate content smokers and non-smokers are exposed to.Therefore, this study is targeted at determining the particulate content in cigarette smoke from selected cigarette brands common on the market in Uganda.Specifically, the study aimed to determine the particulate content in cigarette smoke and to test for the chemical composition of cigarette smoke particulates with carcinogenic effects.

Experimental Setup
The experimental setup consisted of a Humboldt vacuum aspirator, Welch vacuum pressure pump, Pyrex Buchner flasks, rubber tubing, glass funnel, glass tubing, filter papers, retort stand, and an analytical balance (Ohaus Pioneer).The diagram of the setup is shown in Figure 1.

Chemicals and Cigarettes
Reagent grade chemicals used in the study; Ammonia solution, chloroform, Mayer's reagent, Iron (III) chloride, sodium nitrite, 2,4dinitrophenylhydrazine, Fehling's solution (A+B), hydrochloric acid, nitric acid, and sulfuric acid.Additionally, four (4) commercially available cigarette brands on the Uganda market Safari, Sportsman, Super Match, Rex, and locally cured tobacco (hereafter called local), were used for the study, as shown in Figure 2. The locally cured tobacco was made by rolling 3 g of tobacco on a piece of paper.

Determination of Particulate Content in Cigarette Smoke
Two filter papers (Whatman no. 1) were carefully labelled as "1" for mainstream smoke and "2" for second-hand smoke.Each filter paper was weighed using an analytical balance (Ohaus Pioneer), with masses recorded.Filter paper 1 was attached to the Buchner flask connected to the smoker, while filter paper 2 was attached to the Buchner flask connected to the non-smoker.The flasks were sealed with rubber stoppers and connected to vacuum pumps (Welch vacuum pressure pump) via rubber tubing.Each cigarette brand was individually weighed, and a mark was made at 5 cm from the burning end using a pencil.The burning end of the cigarette was inserted into the funnel attached to the smoker's flask, which was connected to the vacuum aspirator.The cigarettes were lit and allowed to burn until reaching the marked point.During the burning process, the vacuum pump was alternated on for 10 seconds and off for 5 seconds.After the cigarettes burned up to the mark, the vacuum pump was disconnected, and the aspirator (Humboldt vacuum aspirator) was left on for 2 minutes to collect all the second-hand smoke before disconnecting the vacuum pump.The two filter papers were gently removed from the flasks, and their weights were measured.The remaining unconsumed portion of the cigarette was also weighed, and all relevant data were recorded.Proper disposal measures were taken for the filter papers and cigarette remnants, ensuring complete extinguishing of the cigarettes.The funnels were cleaned using tissue paper to remove any solid residue from the burned cigarettes.This process was repeated for each cigarette brand in the study.The percentage particulate content per cigarette brand, TPM in mg/g of tobacco or cigarette, percentage TPM in mg/ g of cigarette and total TPM in mg/g of cigarette are calculated using equations ( 1), ( 2), ( 3) and ( 4 Total TPM in mg/g of cigarette = mass of TPM for active smoker in mg/g + mass of TPM for passive in mg/g (4)

Analysis of Particulates in Cigarette Smoke
To analyse the particulates, present in the cigarette smoke, the following tests were conducted using previously described qualitative test protocols (Schmid et al., 1986;Sutoyo et al., 2021;TV & Table, 1921).

Test for Polycyclic Aromatic Hydrocarbons
A chloroform-aluminium test was performed by heating 0.2 g of AlCl3 in a boiling test tube until it sublimed and adhered to the tube's walls.Then, 2 mL of the sample in CHCl3 was carefully introduced along the sides of the test tube, and the resulting colour upon contact with AlCl3 was observed.The presence of red, orange, blue, or green colours indicated the presence of aromatic hydrocarbons.

Test for Alkaloids
Mayer's reagent was prepared by dissolving 0.136 g of mercuric chloride in 10 mL of distilled water (solvent A).Separately, 0.5 g of potassium iodide was dissolved in 1 mL of distilled water (solvent B).Solvent A and solvent B were mixed and allowed to stand.
For the alkaloid test, the test solution in a test tube was treated with ammonia solution and left to stand for a few minutes.Then, chloroform was added, and the mixture was shaken, filtered, and treated with Mayer's reagent.The formation of a cream-coloured precipitate indicated the presence of alkaloids.

Test for phenol
Drops of alcoholic iron (III) chloride solution were added to the test solution in a test tube.A small amount of the sample was added to a minute crystal of NaNO2 in a dry test tube and gently heated for a minute.After cooling, a few drops of concentrated H2SO4 were added from the sides.

Test for Carbonyl Compounds
Drops of 2,4-dinitrophenylhydrazine solution (in excess) were added to the test solution.The appearance of a red/orange precipitate indicated the presence of aldehydes or ketones.Additionally, drops of Fehling's solution (A+B) were added to the test solution and warmed in a water bath.The formation of a red precipitate indicated the presence of aldehydes, while the absence of a precipitate indicated the presence of ketones.

Test for Nitro Compounds
The test solution was dissolved in dilute hydrochloric acid in a test tube and added to a solution of NaNO2.The mixture was then boiled to remove nitrogen and allowed to cool.The formation of a white precipitate indicated the presence of aromatic amines.

Statistical Analysis
Statistical analysis was conducted using MiniTab version 21 statistical software where One way ANOVA was conducted followed by post hoc analysis using Tukey at a 95% confidence level.

RESULTS
The particulate matter content in various cigarette brands was analysed for both smokers and passive smokers.Table 1 presents the average mass of particulates, total mass of particulates, and percentage particulate content for each cigarette brand.On the other hand, Table 2 shows the particulate chemical composition of cigarette smokers and non-smokers.
Our results demonstrated that all the cigarette brands examined contained aromatic compounds, alkaloids, phenols, and nitro compounds in their particulate content.Notably, carbonyl compounds were not detected in any of the brands, suggesting a unique chemical profile in the studied cigarettes.Regarding TPM levels, significant variations were observed among active smokers, ranging from 65.91 mg/g of cigarette (Safari) to 110.2 mg/g of cigarette (Sportsman).For passive smokers, the TPM content ranged from 7.15 mg/g of cigarette (local brand) to 25.66 mg/g of cigarette (Supermatch).However, statistical analysis revealed no significant differences in particulate content or the percentage of TPM across the different cigarette brands for both active and passive smokers.Moreover, the total particulate content (combining active and passive smoking) did not significantly differ among the brands.Nonetheless, active smokers exhibited significantly higher TPM levels compared to passive smokers, suggesting reduced exposure for the latter group.

DISCUSSION
The findings of this study shed light on the chemical composition (Table 2) and total particulate matter (TPM) levels in various cigarette brands, as well as the comparison between active and passive smokers.The presence of aromatic compounds, alkaloids, phenols, and nitro compounds in the particulate content of all examined cigarette brands aligns with previous research, confirming their consistent presence in tobacco smoke (Braun et al., 2019;Wen et al., 2022).An intriguing result of this study was the absence of carbonyl compounds in all the studied cigarette brands.Carbonyl compounds, known for their harmful effects, are commonly found in cigarette smoke (Díez-Izquierdo et al., 2018;Marcilla et al., 2022).The unique chemical profile lacking carbonyl compounds suggests that the studied cigarettes may differ from others in terms of their potential health risks.
Significant variations in TPM levels were observed among active smokers across different cigarette brands, indicating differences in the concentration of solid and liquid particles emitted during smoking.These variations can be attributed to several factors, including the composition of tobacco, the manufacturing process, and the design of the cigarettes themselves (Dai et al., 2022).Different brands may employ varying levels of additives, filters, and paper materials, which can influence the amount of particulate matter produced during combustion.Sportsman exhibited the highest TPM levels (110.2 mg/g of cigarette), while Safari had the lowest (65.91 mg/g of cigarette).These disparities highlight the importance of considering brand-specific characteristics when assessing the exposure of active smokers to particulate matter (Braun et al., 2019).Passive smokers, exposed to second-hand smoke, also displayed varying TPM content depending on the brand being smoked by active smokers nearby.The range of TPM levels for passive smokers was from 7.15 mg/g of cigarette (local brand) to 25.66 mg/g of cigarette (Supermatch).These findings emphasize the influence of the brand being smoked on the particulate matter exposure experienced by passive smokers.

Figure 1 :
Figure 1: Set-up used for the smoking apparatus ), respectively.The percentage particulate content per cigarette brand =         100 (1) TPM in mg/g of tobacco or cigarette = mass of TPM in mg mass of burnt cigarette in g (2) Percentage TPM in mg/ g of cigarette = mass of TPM in mg mass of cigarette burnt in mg  100 (3)

Table 1 : Particulate matter content in the cigarette brands for smokers and passive smokers Cigarette brand Average TPM per gram of tobacco (mg/g) Total TPM per gram of tobacco (mg/g) Percentage TPM in mg per g of cigarette
Statistical significance: a, b, c, d, e, f, g.All values are given as averages