Introduction

Tobacco use, including smoking, causes over 8 million deaths each year, with over 7 million from direct tobacco use, 1.6 million from smoking, and 1.2 million from passive smoking1. On average, smoking shortens life expectancy by about 10 years2. Tobacco use is a major threat to public health; it kills around 2.3 million people every year in the World Health Organization (WHO) South-East Asia Region (SEAR)1. As per the fifth edition of the WHO global report on trends in the prevalence of tobacco use 2000–20303,4, the WHO SEAR is home to around 199 million tobacco smokers (20% of the global total) and 280 million smokeless tobacco users (77% of the global total). Tobacco is consumed in diverse forms, including smoked products such as cigarettes and bidis (a cheaper hand-rolled form of smoked tobacco), and a variety of smokeless tobacco. Comprehensive awareness of tobacco’s harmful effects is far from desirable among users due to culture-related customs and misbeliefs about their health effects3.

All forms of tobacco are harmful, and there is no safe level of exposure to tobacco. Cigarette smoking is the most common form of tobacco use worldwide. Other smoked tobacco products include waterpipe, cigars, cigarillos, heated tobacco products, roll-your-own tobacco, pipe tobacco, bidis, and kreteks. Around 80% of the 1.3 billion tobacco users worldwide live in low and middle-income countries4, where the burden of tobacco-related illness and death is heaviest. Tobacco use contributes to poverty by diverting household spending from basic needs such as food, education, and shelter to buying tobacco products5.

India is the second-largest consumer and producer of tobacco in the world. The Global Adult Tobacco Survey 2017 revealed that 28.6% (around 266 million) of adults in India, aged 15 and above, currently use tobacco in some form. Of these, 10.7% (around 99 million) currently smoke tobacco and 21.4% (around 199 million) use smokeless tobacco1,2. Tobacco products increase the risk of heart and lung diseases, as well as cancers and a variety of other adverse health outcomes4. India’s GDP is negatively impacted by tobacco use, and the direct medical expenses incurred in treating tobacco-related illnesses account for 5.3% of the nation’s yearly spending on both public and private health care5. It is estimated that the economic costs of tobacco-related diseases are ten times higher than the amount of money India receives from tobacco taxes5. This drains the public health system and the economy, which no country can afford. In the SEAR, Myanmar reports the highest tobacco use, with over half of adults (54%) using some form of tobacco. STEPS 2014 survey further revealed that 26% of adults in Myanmar were tobacco smokers and 43% used smokeless tobacco2,4. India and Myanmar can avert millions of preventable deaths as well as mitigate the damage of tobacco use on both society and the economy through the implementation of a comprehensive package of strong tobacco control policies outlined in the WHO Framework Convention on Tobacco Control (FCTC)5.

Nicotine in tobacco is responsible for its addictive nature1. Nicotine is a paradoxical substance because it has both stimulating and depressant effects. It affects the cardiovascular, skeletal, gastrointestinal, and peripheral nervous systems, among other systems5. At the molecular level, nicotine in tobacco mimics the neurotransmitter acetylcholine. Some of the effects of nicotine may be traced to its many receptors in cholinergic binding sites in the brain. Nicotine could be the main psychoactive component of tobacco due to its intricate pharmacological actions6. Several additional essences are purposefully added to tobacco products to make them attractive and to lessen their harshness and potential for dependence2. Many non-tobacco substances such as humectants, fragrances, and flavours are added to attract and appeal to consumers. The American Lung Association has released data indicating that there are roughly 600 ingredients in one cigarette. Almost 7000 chemicals are released when cigarettes burn, many of which are toxic. At least 69 of them are known to cause cancer7.

To ensure compliance with the WHO FCTC Articles 9 and 10, the contents and emissions of tobacco products must be monitored. The WHO Tobacco Free Initiative (TFI) established the tobacco testing laboratory network (TobLabNet) in 2005 as part of its global agenda. This is based on the recommendation of the WHO Study Group on Tobacco Product Regulation (TobReg)8 to build and strengthen tobacco product testing and research capacity by pursuing Articles 9 and 10 of the WHO FCTC9. Under WHO TFI initiative, TobLabNet develops and validates methods and standard operating procedures (SOPs) for testing the contents and emissions of tobacco products.

The regulation of maximum emission levels of tobacco product smoke, specifically tar, nicotine, and carbon monoxide (TNCO), is currently in progress in India. The Food Safety and Standards Authority of India (FSSAI) and the Central Drugs Standard Control Organization (CDSCO) have not yet formally mandated maximum TNCO limits in cigarettes or bidis. However, some tobacco manufacturers in India voluntarily adopt international standards such as 10 mg tar/1 mg nicotine/10 mg carbon monoxide (CO) per cigarette, following the European Union Directive 2001/37/EC10. A wide variety of smoked tobacco products are available in India and Myanmar; however, very little data on the emissions and addictiveness of smoked tobacco products are available from the region due to the infrastructure and technical expertise required for emission testing. This research aims to present primary scientific information on the amounts of nicotine, water, and benzo[a]pyrene (BaP) in tobacco emissions. Additionally, flavours and humectants were tested in fillers. Standardized and validated methods were employed.

Material and methods

This study was carried out in India from June 2023 to May 2024 at the Tobacco Testing and Drug Toxicology Laboratory, Centre for Addiction Medicine (CAM), National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore Karnataka. This tobacco testing facility is part of the WHO global network of tobacco testing laboratories.

All the chemicals used for analysis were of analytical grade. Nicotine, BaP, humectants (glycerol, propylene glycol, and triethylene glycol), flavours (methyl salicylate, ethyl salicylate, eugenol, diphenyl ether, cinnamaldehyde, menthol, coumarin, and camphor), internal standards (n-heptadecane, 3′,4′-methylenedioxyacetophenone, and 1,3-butanediol), and solvents (cyclohexane, propan-2-ol, benzene, and methanol) were procured through Sigma Aldrich, USA having a minimum purity of 99.0%.

Sample collection and storage

Cigarettes and bidis from India and Myanmar were selected based on market availability and consumer popularity at the time of sampling, with an emphasis on including both local and well-known brands. Sampling was conducted randomly within the limits of regional availability. Samples were randomly selected from the cigarette sales points. Four cigarettes and nine Bidi brand samples were procured locally from wholesale and retail outlets in and around Bangalore, India. Seven cigarette brands sourced from Myanmar were obtained through the WHO South-East Asia Regional Office (SEARO), New Delhi, India. Due to the small sample size and random selection, our results may be considered preliminary or indicative. Reference cigarettes (3R4F, 1R5F, and 2R4F) were obtained from the University of Kentucky, USA. Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Monitor (CM4 and CM6) reference cigarettes were received from CORESTA (Paris, France) (Table 3).

The samples were transported in air-tight sealed packs to the Tobacco Testing Facility, NIMHANS, Bengaluru. The labelled samples were stored in sealed packs within 10 days of receipt at –20 °C (Ultra-Low Deep Freezer, Vestfrost) in their original packaging. Before any analysis (e.g., TNCO, humectants, BaP, etc.), the samples were conditioned in a controlled environment as per the International Organization for Standardization (ISO) 3402, a critical pre-analytical step for reliable results11,13.

Cigarettes and bidis contain many chemicals, some occur naturally in tobacco, while others are formed during tobacco processing11. These products emit thousands of chemicals, including addictive substances (nicotine), toxicants (tar and CO), and carcinogens (BaP). A smoking machine is a valuable tool for researching human smoking patterns, even though it does not perfectly replicate every aspect of human smoking behavior. It allows for controlled, standardized smoke delivery, enabling researchers to isolate and study specific variables related to smoke exposure and its health effects. This machine simulates human smoking by controlling parameters such as puff volume, puff duration, puff frequency, and puff interval, allowing for the analysis of various factors13,14.

Quantitative analytical measurements

All quantitative methods were performed under a strict quality assurance (QA) and quality control (QC) protocol in accordance with ISO 17025. For quantifying emission ingredients from both bidis and cigarettes, globally accepted procedures from the ISO, the CORESTA, the TobLabNet, and the Centers for Disease Control and Prevention (CDC) were followed (Table 1). All reported data were within the lowest and highest calibration range of the respective SOP. Values lower than the lowest calibration point were reported as “not detected”. All QCs were verified using a modified Westgard protocol12. Any data failing QC were excluded, and the measurements were repeated.

Table 1 Standard Operating Procedures applied for testing of cigarettes and bidis.
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Smoking machine regime

The Cerulean SM450i, a 20-channel semi-automatic linear smoking machine designed to replicate human smoking behavior, was used for cigarette, bidis analysis. It complies with the ISO 330813 standards, ensuring reliable and reproducible results (Table 1). The machine can handle a wide range of cigarettes and bidis without compromising efficiency or throughput. For bidi emission analysis, the ISO 1717514 protocol was applied. The smoking regime entailed puffing for two seconds at 30-s intervals. The Cerulean bidi holder (Part: 99301) and latex bidi sleeve (Part: KU0358) were used in conjunction with a handheld vacuum pump to smoke bidis (Table 2).

Table 2 Parameters of the smoking run using the Cerulean SM450i.
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Tar, Nicotine, CO, and water

The emissions were collected in bags, and the CO percentage by volume was measured using SOP 10 through the inbuilt non-dispersive infrared (NDIR) analyzer. Total Particulate Matter (TPM) was calculated as per ISO 3308 by weighing the holders before and after the smoking run13. The particulate matter collected on Cambridge Filter Pads (CFP, 44 mm) was extracted as per TobLabNet SOP 1015. Cigarette sampling was as per ISO 8243: 201316, and water analysis was performed using CORESTA SOP 1517. Nicotine quantification was carried out on an Agilent 8890 Gas Chromatograph (GC) with a Flame Ionization Detector (FID). Chromatographic separation was achieved on a CP-WAX 51 column (CP7405) in isothermal mode at 170º C, as shown in Fig. 1.

Fig. 1
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Chromatogram of nicotine with calibration curve.

Humectants

Humectants (glycerol, propylene glycol, and triethylene glycol) were determined in cigarette tobacco fillers, and sampling was done as per ISO 824316. The same SOP was followed for bidi analysis. For quantification, the TobLabNet SOP 618 was applied using an Agilent 8890 GC equipped with a FID. The humectants were separated on a DB Wax (3–7032) fused silica column, as shown in Fig. 2.

Fig. 2
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Chromatogram showing humectants.

BaP

BaP was quantified using TobLabNet SOP 519 on a 5975 Mass Selective Detector (MSD) coupled with an Agilent 7890 GC-MSD system. Chromatographic separation was achieved on a DB-5MS column (122-5532G). The mass spectra of ions produced by electron ionization were identified using the National Institute of Standards and Technology (NIST) library, as shown in Fig. 3.

Fig. 3
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Chromatogram, m/z, and calibration curve of benzo[a]pyrene(BaP).

Statistical analysis

IBM SPSS Statistics for Windows, Version 23.0 (2015; IBM Corp., Armonk, NY, USA) was used to perform the statistical analysis. Due to the small sample size, non-parametric statistical methods were applied to compare cigarettes and bidis between the two countries. The Mann–Whitney U test was employed for the analysis, with p < 0.05 considered statistically significant.

Results

Reference control samples (3R4F, 1R5F, 2R4F, CM4, and CM6) were studied in triplicate under comparable conditions, and the results are presented as means ( ~) in Table 3.

Table 3 Smoke constituents of reference cigarettes.
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Indian cigarettes vs Myanmar cigarettes

Tar, Nicotine, CO, and Water

The comparisons of nicotine, CO, water, TPM and tar between the cigarette samples received from Myanmar and India are summarized in Fig. 4. In Myanmar cigarettes, nicotine ranged from 0.59 to 1.08 mg/cigarette (median 0.83 mg/cigarette), while CO ranged from 7.77 to 18.96 mg/cigarette (median 12.49 mg/cigarette). In Indian cigarettes, nicotine ranged from 0.59 to 0.78 mg/cigarette (median 0.64 mg/cigarette), and CO ranged from 9.85 to 12.0 mg/cigarette (median 10.80 mg/cigarette). Although the nicotine and CO levels in Myanmar cigarettes were slightly higher than in Indian cigarettes, the differences were not statistically significant (p = 0.107 and p = 0.345, respectively). (see Supplementary filecigarette_beedi data F.xlsx).

Fig. 4
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Medians of nicotine, water, carbon monoxide (CO), tar, and total particulate matter (TPM) in cigarettes.

The water content in Indian cigarettes ranged from 3.68 to 5.18 mg/cigarette (median 4.45 mg/cigarette), while for Myanmar cigarettes it ranged from 1.09 to 7.35 mg/cigarette (median 4.25 mg/cigarette); the difference was not statistically significant (p = 0.705). The tar and TPM in Indian products ranged from 8.49 to 12.22 mg/cigarette (median 10.34 mg/cigarette) and 13.41 to 18.04 mg/cigarette (median 15.16 mg/cigarette), respectively. For Myanmar products, tar ranged from 7.88–18.60 mg/cigarette (median 10.90 mg/cigarette) and TPM from 9.56 to 24.21 mg/cigarette (median 14.93 mg/cigarette); however, the differences were not statistically significant (p = 0.705) (Fig. 4; see Supplementary file cigarette_beedi data F.xlsx).

BaP

As per our findings, BaP levels in Indian cigarettes ranged from 8.02 to 8.64 ng/cigarette (median 8.15 ng/cigarette), while Myanmar cigarettes showed higher BaP levels ranging from 9.72 to 14.90 ng/cigarette (median 11.95 ng/cigarette). The difference in BaP levels between Indian and Myanmar cigarettes was statistically significant (p = 0.008) (Fig. 5). Among the reference cigarettes, 3R4F, 1R5F, and 2R4F showed BaP levels ranging from 2.09 to 2.18 ng/cigarette (Table 3; see Supplementary file cigarette_beedi data F.xlsx).

Fig. 5
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Medians of benzo[a]pyrene(BaP) and humectants in cigarettes.

Humectants

Humectants are added to cigarettes to control and maintain the moisture content of the tobacco filler. In Indian cigarettes, glycerol levels ranged from 1.99 to 14.11 mg/g (median 12.50 mg/g), propylene glycol from 0.02 to 0.75 mg/gm (median 0.38 mg/g), and triethylene glycol from 0.00 to 0.09 mg/g (median 0.02 mg/g). In Myanmar cigarettes, glycerol levels ranged from 5.46 to 17.36 mg/g (median 6.94 mg/g), propylene glycol from 0.42 to 3.86 mg/g (median 3.32 mg/g), and triethylene glycol from 0.00 to 0.07 mg/g (median 0.01 mg/g).

On comparing all three humectants, no statistically significant variation was observed in glycerol (p = 1.000) and triethylene glycol (p = 0.766); however, propylene glycol levels showed a statistically significant difference (p = 0.023) (Fig. 5). Humectants were not detected in the reference cigarettes (Table 3, see Supplementary file cigarette_beedi data F.xlsx).

Cigarettes vs bidis

Bidis had nicotine levels ranging from 0.69 to 1.91 mg/bidi (median 1.70 mg/bidi) and CO levels from 11.28 to 18.69 mg/bidi (median 13.37 mg/bidi) (Fig. 6). The water content of bidis varied from 1.32 to 28.40 mg/bidi (median 17.14 mg/bidi). Tar levels ranged from 1.84- 29.24 mg/bidi (median 25.18 mg/bidi), and TPM levels from 25.35- 48.09 mg/bidi (median 41.15 mg/bidi).

Fig. 6
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Medians of water, total particulate matter (TPM), carbon monoxide (CO), tar, and nicotine in Indian cigarettes and bidis.

On comparing Indian cigarettes and bidis, nicotine and CO (p = 0.023), as well as water and TPM (p = 0.008), showed statistically significant differences. When comparing bidis and cigarettes from India and Myanmar, statistically significant differences were observed in nicotine (p = 0.041), water (p < 0.001), and TPM (p < 0.001) (see Supplementary file cigarettes_beedi data F.xlsx).

Bidis had glycerol levels ranging from 0.53 to 2.38 mg/g (median 0.74 mg/g), propylene glycol from 0.00 to 0.15 mg/g (median 0.13 mg/g), and triethylene glycol from 0.00 to 0.07 mg/g (median 0.06 mg/g). BaP levels in bidis ranged from 9.27 to 12.35 ng/bidi (median 9.89 ng/bidi). On comparing Indian bidis and cigarettes, glycerol and BaP levels were found to be statistically significant (p = 0.009 and p = 0.008, respectively), as shown in Fig. 7.

Fig. 7
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Medians of benzo[a]pyrene (BaP) and humectants in Indian cigarettes and bidis.

All cigarette and bidi emissions were tested for flavours (methyl salicylate, ethyl salicylate, eugenol, eucalyptol, diphenyl ether, cinnamaldehyde, menthol, coumarin, and camphor) using the CDC TL-Method 06011. None of these flavours were detected in the samples received from either country.

Discussion

Cigarettes are expertly crafted, mass-produced tobacco products available worldwide. They are filled with processed tobacco and wrapped in paper. Bidis, in contrast, are small, hand-rolled tobacco products made of sun-dried and processed tobacco wrapped in Tendu or Temburni leaves and secured a with cotton thread20. India has the world’s second-largest number of adult smokers. Bidi is the most commonly smoked tobacco product, used by an estimated 71.8 million adults1,2.

Tobacco smoke is a complex mixture of over seven thousand volatile and semi-volatile compounds. Particulate matter (PM) generated from tobacco smoking can be coarse (≤ 10 µm), fine (≤ 2.5 µm), or ultrafine (< 0.1 µm)20. The particle size of PM determines both its ability to adsorb toxic organic compounds and the depth to which it can enter the respiratory system. Protonated nicotine is rapidly absorbed by the body. Other constituents of tobacco smoke include humectants, volatile aldehydes, nitrogen oxides, and polycyclic aromatic hydrocarbons. Hoffmann et al.21 identified N-nitrosonornicotine in unburned tobacco using gas chromatography–mass spectrometry (GC–MS) and reported it as a potential carcinogen at concentrations ranging from 0.12 to 3.7 µg/cigarette. Many harmful and potentially carcinogenic substances are present in mainstream smoke. When inhaled, high levels of carbon monoxide, especially from bidis, enter the bloodstream and bind with hemoglobin to form carboxyhemoglobin. This results in tissue ischemia, a major contributor to cardiovascular diseases common among bidi smokers20,22.

The Government of India signed the WHO FCTC in 20049 and enacted the Cigarettes and Other Tobacco Products Act (COTPA) in 2003. However, COTPA does not impose any specific limits on tobacco product emissions or nicotine content. In its advisory note on the Global Nicotine Reduction Strategy, the TobReg8 stated that the risk of dependence on cigarettes can be reduced by lowering their nicotine content to very low levels. Accordingly, the nicotine content should be maintained as low as is technically feasible.

According to WHO’s global review of national laws2,4,10 on maximum permissible emission levels of tar, nicotine, and CO, as of 31 December 2022, eighty-three countries had established maximum permissible nicotine levels per cigarette, with sixty of these (72%) allowing up to 1 mg of nicotine per cigarette. Furthermore, sixty-seven countries permit 10 mg of tar per cigarette, while eighty-five countries have set maximum permissible tar levels per cigarette. Additionally, fifty-seven countries have set a CO limit of 10 mg per cigarette. Within the WHO SEAR, only Myanmar expressly forbids the display of emission levels, whereas Timor-Leste has established limits of 10 mg for tar, 1 mg for nicotine, and 10 mg for CO per cigarette10.

The cigarettes tested displayed nicotine concentrations between 0.59 and 1.08 mg per cigarette, with a median value of 0.77 mg per cigarette; all measured levels were within the regulatory threshold of 1.0 mg per unit22. For bidis, nicotine levels ranged from 0.69 to 1.91 mg per bidi (median 1.70 mg per bidi), which were well above 1.0 mg per unit20,22. Cigarette samples received from Myanmar were found to contain carbon monoxide (CO) levels exceeding 10 mg per cigarette. For bidis, CO levels ranged from 11.28 to 18.69 mg per bidi (median 13.37 mg per bidi). Under standard smoking machine conditions, bidis exhibited higher nicotine delivery, suggesting greater exposure potential. Research has shown that the non-porous nature and higher moisture content of Tendu leaf wrappers in bidis lead to higher levels of CO and tar in their smoke compared with regular cigarettes20. The presence of harmful and carcinogenic chemicals in mainstream bidi smoke can pose serious risks to human health12.

The tar levels in Myanmar cigarettes ranged from 7.88 to 18.60 mg per cigarette (median 10.90 mg per cigarette), exceeding the highest permissible limit of 10.0 mg per cigarette set by several countries. Indian bidis exhibited tar levels ranging from 1.84 to 29.24 mg per bidi (median 25.18 mg per bidi), demonstrating substantially higher values compared to typical manufactured cigarette standards. Humectants, propylene glycol, and glycerol were present in all samples. Among Indian cigarettes, propylene glycol levels did not exceed 10.0 mg/g, while glycerol levels were 10 mg/g or higher, with the exception of one brand. Triethylene glycol was detected only in two samples, ranging from 0.04 to 0.09 mg/g.

Yan Xizheng et al. tested twenty-seven popular cigarette brands in the United States of America (USA) for humectants. Only four brands showed no detectable amounts of humectants, while the remaining brands contained glycerol or 1,2-propylene glycol with concentrations ranging from 1.66 to 3.57% for glycerol and 0.23 to 1.35% for 1,2-propylene glycol. In general, all manufacturers used higher amounts of glycerol than 1,2-propylene glycol as a humectant, which may act as a precursor for the formation of harmful carbonyl compounds23.

BaP is a marker of carcinogenic activity of polycyclic aromatic hydrocarbons (PAHs)24. PAHs are present in cigarette smoke emissions in small quantities, typically < 10 ng per cigarette. BaP levels in Indian cigarettes were ≤ 10 ng per cigarette. In the Myanmar samples, BaP ranged from 9.72 to 14.90 ng per cigarette (median 11.95 ng per cigarette), which may be harmful depending on frequency and duration of use. Pakhale S.S. et al. analyzed mainstream smoke from popular Indian tobacco products using a standard smoking machine and reported BaP levels ranging from 85 to 114 ng per cigarette, while for bidis, levels ranged from 108 to 144 ng per bidi25. The lower BaP levels observed in the current study may be attributed to improved analytical techniques, differences in smoking machine regimens, and variations in product design or combustion conditions. Despite containing less tobacco, bidis deliver significantly higher levels of harmful and carcinogenic chemicals, including nicotine, CO, and PAHs25. Analysis of mainstream smoke in cigarettes from Nigeria20 revealed BaP levels between 0 and 22.7 ng per cigarette. The most prevalent PAH detected across all tested products was naphthalene, which ranged from 210.7 to 460.34 ng per cigarette24. Unlike conventional cigarettes, for which more than 6500 tobacco-specific chemicals have been identified and studied in detail25, research on bidi smoke remains limited. Therefore, more studies are required to characterize the complete chemical profile of bidis and assess their health impact.

Conclusion

Cigarettes and bidis pose a significant public health challenge. Their wide availability and continued appeal may overshadow the serious health risks associated with these products. Quantitative analysis of emissions in mainstream smoke is necessary to assess the harmful effects of smoking. In the present study, we quantitatively analyzed nicotine, CO, water, tar, TPM, and BaP in mainstream smoke from randomly selected cigarette (India and Myanmar) and bidi (India) samples. These samples were analyzed using globally accepted SOPs developed by the ISO, CORESTA, and TobLabNet. Statistically significant differences were observed in BaP (p = 0.008) and propylene glycol (p = 0.023) levels between Indian and Myanmar cigarettes. When comparing Indian cigarettes with bidis, nicotine and CO (p = 0.023), water and BaP (p = 0.008), and glycerol (p = 0.009) showed statistically significant variation.

The contents and emissions were measured using globally approved protocols. The results of this study can be utilized to inform policy formulation, educate the public about the dangers of smoking, and potentially incentivize tobacco companies to produce less harmful products by adhering to established emission standards for toxic constituents.

Limitation

Analysis of tobacco-specific nitrosamines could not be performed due to non-availability of reference standards. Also, the overall findings of this study should be considered preliminary or indicative, given the limited sample size.