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. 2023 Nov 28;36(12):2001–2009. doi: 10.1021/acs.chemrestox.3c00273

Evaluation of Six Aromatic Amines in the Mainstream Smoke of Commercial Cigars

Huihua Ji †,*, Zhenyu Jin †, Laura Fenton †, Stacey Slone ‡
PMCID: PMC10731631  PMID: 38014781

Abstract

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Aromatic amines are a class of carcinogenic compounds present in tobacco smoke that are listed on the U.S. Food and Drug Administration (FDA) list of harmful and potentially harmful constituents (HPHCs) in tobacco products and tobacco smoke. The yields of six aromatic amines (1-aminonaphthalene [1-AN], 2-aminonaphthalene [2-AN], 3-aminobiphenyl [3-ABP], 4-aminobiphenyl [4-ABP], ortho-toluidine [o-TOL], and o-anisidine [o-ANI]) in the mainstream smoke from 23 commercial filtered cigars, 16 cigarillos, and 11 large cigars were determined using solid-phase microextraction coupled to gas chromatography triple quadrupole mass spectrometry (SPME headspace GC–MS/MS). The commercial cigars were smoked under the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Recommended Method 64 using a linear cigar smoking machine. The aromatic amine yields in the mainstream smoke from 50 commercial cigars show high levels of variation within and between the products. The average yields of the aromatic amines in the filtered cigars, cigarillos, and large cigars were 108, 371, and 623 ng/cigar for o-TOL; 6, 14, and 22 ng/cigar for o-ANI; 65, 114, and 174 ng/cigar for 1-AN; 25, 59, and 87 ng/cigar for 2-AN; 6, 17, and 27 ng/cigar for 3- ABP; and 8, 11, and 17 ng/cigar for 4-ABP, respectively. The relationships between aromatic amines and (1) total particulate matter (TPM), (2) water-soluble proteins, and (3) water-insoluble proteins were evaluated. We found that the aromatic amines showed a good linear response with TPM on a per cigar basis and showed significant positive correlations with proteins. In addition, the water-insoluble proteins make a greater contribution to the formation of aromatic amines compared to the water-soluble proteins.

Introduction

Tobacco smoke is a complex mixture that contains thousands of compounds; more than 60 carcinogens were identified by the year 2000.1 Cigar smoking has been linked to a range of cancers that include oral, esophageal, laryngeal, bladder, and lung cancers, as well as other harmful health effects.2,3 In March 2012, the U.S. Food and Drug Administration (FDA) established a list of 93 harmful and potentially harmful constituents (HPHCs) in tobacco products and tobacco smoke.4 In August 2016, the “deeming rule” was finalized, extending the FDA’s authority to regulate all tobacco products, including cigars. The aromatic amines 1-aminonaphthalene (1-AN), 2-aminonaphthalene (2-AN), 4-aminobiphenyl (4-ABP), ortho-toluidine (o-TOL), and o-anisidine (o-ANI) are included in the FDA list of HPHCs. Aromatic amines make up a class of carcinogenic compounds. The International Agency for Research on Cancer (IARC) has classified 2-AN, 4-ABP, and o-TOL as Group 1 carcinogens, carcinogenic to humans, while o-ANI is classified in Group 2B, possible human carcinogens.5 Some researchers have studied aromatic amine yields in cigarette smoke;6−10 however, there are few published studies that mention aromatic amine yields in cigar smoke. Ai et al. reported levels of 1-AN, 2-AN, and 4-ABP in the smoke from 60 filtered cigars (also referred to as “little cigars”),11 but their research did not include aromatic amine yields in other types of cigars, such as cigarillos and large cigars. In this study, we determined the yields of six aromatic amines (o-TOL, o-ANI, 1-AN, 2-AN, 3-aminobiphenyl (3-ABP), and 4-ABP) (Figure 1) in three different types of cigars: little/filtered cigars, cigarillos, and large cigars.

Figure 1.

Figure 1

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Chemical structures of six aromatic amines.

Cigar smoking has increased in recent decades.1,12 In 2022, cigars were the most commonly used combustible tobacco product and the second most commonly used tobacco product among U.S. high school students.13 Cigars are manufactured in a wide range of sizes and shapes that include filtered/little cigars, cigarillos, large cigars, and premium cigars.14 Premium cigars were not included in our study because they are made by hand using long filler tobacco, and their weight can vary from 5 to 22 g.14 We focused on machine-made filtered cigars, cigarillos, and large cigars in this study. Filtered cigars have a filter and contain <1.36 g of tobacco per cigar for tax purposes.15 However, some manufacturers have altered their filtered cigar design to increase the cigar weight in order to meet the large cigar requirement and thereby benefit from the lower federal excise tax.16 Therefore, the weight of tobacco in some filtered cigars used in our study exceeded 1.36 g of tobacco per cigar. There are no established definitions for cigarillos and large cigars. It is generally accepted that cigarillos and large cigars do not have filters, and the weight ranges from 2.5 to 3.5 g for cigarillos and from 5 to 17 g for large cigars.14 The marketing terms were used to categorize the cigars used in this study; the 50 brands of cigars analyzed included 23 filtered cigars, 16 cigarillos, and 11 large cigars.

Aromatic amines make up a class of carcinogenic compounds, but there is limited understanding of the sources of aromatic amines in cigar smoke. Some previous studies have reported that proteins are the precursors of aromatic amines.17−20 The protein fractions include water-soluble proteins, such as most nitrogenous inorganic compounds and amino acids, and water-insoluble proteins, such as cell wall proteins.20−22 In this study, we determined the yields of six aromatic amines and total particulate matter (TPM) in the mainstream smoke of 50 brands of cigars. We also measured the amounts of water-soluble and water-insoluble proteins in the cigar filler tobacco. We examined the relationships between aromatic amine levels and (1) TPM, (2) water-soluble proteins, and (3) water-insoluble proteins. The results of this study provide valuable insight into the sources of aromatic amines in cigar smoke. In this study, we report aromatic amine yields in the mainstream smoke of various types of cigars and we also determined the influence of TPM and proteins on the yields of aromatic amines.

Methods and Materials

Cigars

A total of 50 brands of cigars (Table 1) sold in the US market were purchased from different cigar websites between February and November 2020. The purchased cigars include 23 filtered cigars, 16 cigarillos, and 11 large cigars. All cigars were machine-made and included cigars with natural wrappers and wrappers made from homogenized tobacco leaf (HTL). The machine-made cigars used were produced by the top five tobacco companies with the highest market shares and also included other brands.23 The purchased cigars were stored at −20°C before analysis.

Table 1. Commercial Cigar Product IDs, TPM (mg/cigar), and Contents of Water-Soluble and Water-Insoluble Proteins (mg/cigar).

        TPM (mg/cigar)
 water-soluble protein
 water-insoluble protein
category product ID brand manufacturer mean STDEV mg/cigar STDEV mg/cigar STDEV
filtered cigars 1 Talon Regular Filtered Regular Scandinavian Tobacco Group 28.0 1.0 12.03 0.36 25.17 0.33
2 Phillies Filter Tipped 100 M Regular ITG Brands 29.3 1.5 11.46 0.29 21.85 0.47
3 Cheyenne Filtered Cigar Classic Cheyenne International 23.0 1.8 12.59 0.27 23.92 0.54
4 Cherokee Filtered Cigars Full Flavor Red 100 s Cherokee Tobacco Company 24.8 1.2 8.11 0.21 15.25 0.09
5 Swisher Sweets Little Cigars Regular Swisher International, Inc. 27.7 1.7 8.82 0.35 22.95 0.40
6 Santa Fe Filtered Cigars Original Swisher International, Inc. 31.7 1.2 12.19 0.38 26.67 0.53
7 Seneca Full Flavor Filtered Cigar Red Lake Erie Tobacco Company 27.7 1.6 11.89 0.33 28.35 0.53
8 Derringer Filtered Cigars Classic Cheyenne International, LLC 24.7 2.2 8.88 0.28 20.78 0.70
9 Captain Black Little Filtered Cigars Scandinavian Tobacco Group 23.5 0.5 11.90 0.21 20.86 0.34
10 King Edward Filtered Cigars Regular Swisher International 35.0 2.3 8.43 0.10 22.30 0.37
11 Winchester Little Cigars Classic King Box Scandinavian Tobacco Group, Tucker GA 22.2 1.0 14.12 0.32 18.63 0.28
12 Djarum Filtered Clove Cigars Special PT Djarum, Kudus, Indonesia 57.9 2.9 19.98 0.45 33.19 0.68
13 305’s Filtered Cigars Full Flavor Dosal Tobacco Corp., Miami, FL 36.6 3.6 12.23 0.21 19.03 1.04
14 Vaquero Filtered Cigars Original (Natural) Sunshine Tobacco, Miami, FL 20.8 3.3 13.38 0.50 12.46 0.56
15 Bella Orchid Filtered Cigars Full Flavor Sunshine Tobacco, Miami, FL 18.5 1.1 12.30 0.42 15.18 0.20
16 Clipper Filtered Cigars Full Flavor Global Tobacco LLC, Dallas, TX 26.4 2.1 9.86 0.12 20.35 0.40
17 Remington Filtered Cigars Full Flavor Good Times USA, LLC, Tampa FL Dominican Republic 22.5 1.5 10.96 0.39 22.08 0.38
18 Red Buck Filtered Cigars Full Flavor Distributed by Xcaliber International, Ltd., Pryor, OK 26.9 1.2 9.67 0.44 19.21 0.45
19 Supreme Blend Filtered Cigars Full Flavor Distributed by Global Tobacco LLC, Dallas, TX 21.9 1.3 10.32 0.23 18.79 0.36
20 Wrangler Filtered Cigars Full Flavor Sunshine Tobacco, Miami, FL 16.8 1.0 13.16 0.38 14.23 0.66
21 Racer Filtered Cigar Full Flavor Distributed by Global Tobacco LLC, Dallas, TX 22.9 1.7 11.43 0.46 19.65 0.61
22 Westport Filtered Cigars Original Distributed by Inter-Continential Trading USA, Inc., IL. 30.2 2.4 10.27 0.40 20.86 0.42
23 Richmonde Filtered Cigars Full Flavor 100 s Ohserase Manufacturing LLC, Akwesasne NY 26.8 1.8 8.60 0.36 18.94 0.39
cigarillos 1 Antonio Y Cleopatra Minis ITG Brands, LLC 64.6 11.17 38.10 0.41 107.25 1.36
2 Dutch Masters Cigarillos De Luxe ITG Brands, LLC 76.4 6.43 41.03 1.39 93.80 1.57
3 Garcia Y Vega Game Cigarillo Black Swedish Match 74.9 8.78 34.51 0.72 60.55 1.29
4 King Edward Special Swisher International, Inc. 74.4 4.27 24.13 0.17 72.24 1.16
5 Optimo Cigars Sweet Swisher International, Inc. 77.1 5.83 28.65 0.64 63.50 3.69
6 Phillies Cigars Cigarillos Black ITG Brands, LLC 76.0 8.01 26.02 0.49 82.90 1.25
7 Phillies Cigars Cigarillos Sweet ITG Brands, LLC 70.6 6.51 27.59 1.16 83.11 1.35
8 Swisher Sweets Cigarillos Original Swisher International, Inc. 82.5 5.12 24.65 0.59 71.23 1.28
9 White Owl Cigarillos Black Swedish Match 77.6 3.45 29.41 0.97 73.32 1.64
10 White Owl Cigarillos Sweets Swedish Match 76.2 10.67 31.14 1.57 64.53 1.76
11 Backwoods Original ITG Brands, LLC 52.2 9.66 57.02 1.26 100.58 1.29
12 Pom Pom Sweet Swisher International, Inc. 74.4 2.90 39.99 1.09 85.36 1.01
13 Al Capone Sweets (Cognac Dipped) ICC Inter-Continental Cigar Corporation 44.7 4.27 17.83 0.63 29.87 1.09
14 Jackpot Sweets SMCI Holdings, Inc. 75.7 7.85 28.48 1.12 68.67 1.45
15 Black [Tc7]& Mild Original John Middleton Co. 90.7 6.08 61.18 1.65 68.85 1.71
16 Hav-A-Tampa Jewels Original ITG Brands, LLC 49.9 7.76 30.86 0.36 82.58 1.65
large cigars 1 Blunt Petite Corona Natural, Phillies ITG Brands, LLC 119.9 14.4 165.76 2.49 213.08 6.73
2 blunts natural petite corona sweet, Swisher Sweets Swisher International 120.6 22.8 135.80 4.84 193.94 2.73
3 Collection Palma Corona Natural, Dutch Masters ITG Brands, LLC 144.2 18.8 266.70 14.58 277.72 12.87
4 Corona Deluxe Natural, Dutch Masters ITG Brands, LLC 163.2 32.0 303.34 12.48 260.01 6.23
5 Coronas, Antonio Y Cleopatra ITG Brands, LLC 162.4 27.8 251.05 9.28 308.26 6.70
6 English Corona, Garcia Y Vega Swedish Match 105.9 33.6 208.17 5.87 207.15 6.71
7 Invincible Natural, White Owl Swedish Match 123.7 12.5 167.58 8.11 191.56 5.00
8 Perfecto, William Penn Swedish Match 107.8 6.3 139.41 8.18 188.09 4.19
9 Perfecto Natural Sweet, Swisher Sweets Swisher International 136.5 23.0 198.25 4.07 225.12 4.61
10 President, Dutch Masters ITG Brands, LLC 138.7 18.2 331.40 7.34 235.55 5.78
11 President, Garcia Y Vega Swedish Match 113.0 12.7 256.36 8.44 247.18 6.25
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Three types of reference cigars were used as a control in this study: filtered cigar (1C2), cigarillo (1C3), and large cigar with a natural wrapper (1C4). All of the samples were acquired from the Center for Tobacco Reference Products (CTRP) at the University of Kentucky (Lexington, KY). These cigars were used as quality control samples during the analyses of aromatic amines in cigar smoke. Data for aromatic amine levels in the control cigars were compiled from in-house data.

Reagents

The chemical standards 1-AN, 3-ABP, 4-ABP, o-TOL, o-ANI, and N-methyl-bis(trifluoroacetamide) (MBTFA) were purchased from MilliporeSigma (St. Louis., MO). 2-AN was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). Isotopically labeled 1-AN-d7, 2-AN-d7, 4-ABP-d9, o-TOL-d9, and o-ANI-d7, the internal standards, were purchased from CDN Isotopes Inc. (Quebec, Canada). 3-ABP-d9 was purchased from Toronto Research Chemicals (Toronto, Canada). All other reagents were obtained from Fisher Scientific (Hampton, NH). The SPME fiber, polyacrylate 85 μm, was purchased from Supelco-MilliporeSigma (Bellefonte, PA). FOCUS Soluble and Insoluble Protein Extraction kits manufactured by G Biosciences were purchased from VWR International (Radnor, PA). The Bio-Rad Protein Assay Dye Reagent Concentrate and Bovine serum albumin (BSA) were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA).

Cigar Smoke Collection

A Borgwaldt LM5C linear cigar-smoking machine (Hamburg, Germany) was used to collect cigar mainstream smoke condensate. All cigars were conditioned for a minimum of 72 h at 22 °C and 60% relative humidity according to the CORESTA (Cooperation Centre for Scientific Research Relative to Tobacco) Recommended Method 46 (CRM-46).24 The cigars were smoked following CRM 64,25 in which the puff duration is 1.5 s, the puff frequency is 40 s, and the puff volume is based on the cigar diameter:

  • 20 mL when the cigar diameter is ≤12.0 mm

  • equal to 0.139 × d2 when the diameter is >12.0 mm

TPM in mainstream smoke was collected on 55 mm Cambridge filter pads. One cigarillo or large cigar was smoked per pad with CRM 64 while two filtered cigars were smoked per pad. Eight replicates were smoked for each cigar brand.

Aromatic Amines Analysis

Solid-phase microextraction coupled to gas chromatography triple quadrupole mass spectrometry (SPME headspace GC–MS/MS) was used to determine the aromatic amines in the mainstream smoke of cigars. The aromatic amine analyses were performed on an Agilent 7890B gas chromatograph equipped with a 7000C Triple Quad mass spectrometer system (GC/MS/MS) (Santa Clara, CA). The Agilent GC/MS/MS was coupled with a Gerstel Multipurpose sampler for SPME (Linthicum, MD). The full separation of derivatives of six aromatic amines was achieved using an Agilent DB-17 GC capillary column (30 m × 0.25 mm i.d.; 0.25 μm film thickness, Agilent Technologies) (Supporting Figure S1 and Table S1 from Ji and Jin paper).26 The 7000C Triple Quad mass spectrometer system was operated in the multiple reaction monitoring (MRM) mode.

The Cambridge filter pads were extracted with 10 mL of 0.1 M hydrochloric acid and the internal standards (the mixture of aromatic amine isotopes) and shaken for 1 h. After shaking, 1 mL of extract solution was transferred to a 10 mL headspace vial, and then 100 μL of 2.5 M sodium hydroxide and 25 μL of MBTFA were added to the extract solution. The MBTFA derivatives of the aromatic amine solutions were then ready for injection into the GC/MS/MS instrument with headspace-SPME mode for aromatic amine analysis. The six aromatic amines’ recoveries and coefficients of variation (CV%) were 90–112 and 2.1–6.6%, respectively. More details regarding the SPME headspace GC/MS/MS method for the determination of aromatic amines in cigar mainstream smoke were previously reported by Ji and Jin.26

Assays for Protein Quantification

Water-soluble and water-insoluble proteins in the cigar filler tobacco were extracted using the FOCUS Soluble and Insoluble Protein Extraction kits, respectively, following the manufacturer’s protocols.27 After extraction, the protein content in cigar filler was determined using the Bio-Rad Protein Assay, which is a colorimetric assay method based on the Bradford method. Bovine serum albumin (BSA) at 2 mg/mL was used as the protein standard. Samples (20 μL) of cigar protein extract were mixed with 1 mL of Bio-Rad Protein Assay Dye Reagent (diluted 5-fold) and the absorbance at 595 nm was then measured using a Beckman DU530 ultraviolet–visible (UV–vis) spectrophotometer (Brea, CA). Three replicates were performed for each brand of cigar filler tobacco.

Statistical Analysis

The experimental results were expressed as mean ± standard deviation. The relationship between aromatic amines and proteins was evaluated using the Spearman correlation coefficients (r) and p-values that were calculated with SAS Software 9.4 (Cary, NC). The significance level (α) was set at 0.05.

Results and Discussion

Aromatic Amine Yields in Commercial Cigar Mainstream Smoke

Tobacco combustion is the primary source of aromatic amine formation in the smoke.3,28 The mean values and standard deviations of the yields of six aromatic amines (o-TOL, o-ANI, 1-AN, 2-AN, 3-ABP, and 4-ABP) in the mainstream smoke of each product (commercial filtered cigars, cigarillos, and large cigars) are shown in waterfall plots in Figures 2–4. The data were sorted from the lowest to highest aromatic amine yield in mainstream smoke. For the filtered cigars, the mean ranges of aromatic amine yield for each product were 58–203 ng/cigar for o-TOL, 3–9 ng/cigar for o-ANI, 21–172 ng/cigar for 1-AN, 12–53 ng/cigar for 2-AN, 3–10 ng/cigar for 3-ABP, and 3–26 ng/cigar for 4-ABP. For the cigarillos, the mean ranges of aromatic amine yield for each product were 144–596 ng/cigar for o-TOL, 7–19 ng/cigar for o-ANI, 37–158 ng/cigar for 1-AN, 24–84 ng/cigar for 2-AN, 6–24 ng/cigar for 3-ABP, and 4–15 ng/cigar for 4-ABP. For the large cigars, the mean ranges of aromatic amine yield for each product were 238–985 ng/cigar for o-TOL, 13–35 ng/cigar for o-ANI, 91–242 ng/cigar for 1-AN, 41–112 ng/cigar for 2-AN, 21–35 ng/cigar for 3-ABP, and 12–23 ng/cigar for 4-ABP. In all tested cigars, the amount of o-TOL was the highest of all six aromatic amines detected, ranging from 58 to 985 ng/cigar. The results for the commercial cigars had high variability in some individual cigars within a product line. The relative standard deviations (RSD) of each aromatic amine yield within the product in the filtered cigars were 2–30% for o-TOL, 3–22% for o-ANI, 6–56% for 1-AN, 3–40% for 2-AN, 2–27% for 3-ABP, and 4–26% for 4-ABP. For cigarillos, the RSDs of each aromatic amine yield were 5–38% for o-TOL, 8–33% for o-ANI, 5–25% for 1-AN, 7–24% for 2-AN, 4–31% for 3-ABP, and 6–25% for 4-ABP. For the large cigars, the RSDs of each aromatic amine yield were 17–35% for o-TOL, 14–40% for o-ANI, 13–39% for 1-AN, 12–36% for 2-AN, 21–35% for 3-ABP, and 11–31% for 4-ABP.

Figure 2.

Figure 2

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Aromatic amine yields in the mainstream smoke of the filtered cigars (n = 8). The product IDs are listed in Table 1. The bars with the cross-hatching on each graph represent the mean aromatic amine values in filtered cigar mainstream smoke.

Figure 4.

Figure 4

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Aromatic amine yields in large cigar mainstream smoke (n = 8). The product IDs are listed in Table 1. The bars with the cross-hatching on each graph represent the mean aromatic amine values in the large cigar mainstream smoke.

Figure 3.

Figure 3

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Aromatic amine yields in cigarillo mainstream smoke (n = 8). The product IDs are listed in Table 1. The bars with the cross-hatching on each graph represent the mean aromatic amine values in cigarillo mainstream smoke.

The averages of each aromatic amine yield in the filtered cigars, cigarillos, and large cigars were 108, 371, and 608 ng/cigar for o-TOL; 6, 14, and 22 ng/cigar for o-ANI; 64, 114, and 170 ng/cigar for 1-AN; 25, 59, and 85 ng/cigar for 2-AN; 6, 17, and 27 ng/cigar for 3-ABP; and 8, 11, and 17 ng/cigar for 4-ABP, respectively. The average tobacco weights in the tested filtered cigars, cigarillos, and large cigars were 1.1, 2.7, and 8.0 g (Supporting Table S2), respectively. On a per cigar basis, the more tobacco contained in the cigar, the more aromatic amines were generated. On a per mass of tobacco smoked basis, most of the ratios of aromatic amines per gram of tobacco smoked (ng/g) vs tobacco weight were consistent in each cigar category. As an example, the figures for o-TOL are shown in Supporting Figure S2. A comparable amount of aromatic amines was generated when equal amounts of tobacco were smoked. Therefore, large cigars produced the highest aromatic amine yield per cigar, while the filtered cigars had the lowest aromatic amine yield in mainstream smoke (Supporting Figure S3). The results from the commercial cigars were highly variable between products in each cigar category. The RSD of each aromatic amine yield between products in the filtered cigar, cigarillo, and large cigar categories were 33, 33, and 39% for o-TOL, 22, 23, and 38% for o-ANI, 63, 26, and 31% for 1-AN, 39, 26, and 29% for 2-AN, 27, 27, and 20% for 3-ABP, and 83, 26, and 23% for 4-ABP, respectively. This is consistent with the study of Ai et al. that showed high variation of 1-AN, 2-AN, and 4-ABP in the smoke from 60 filtered cigars. Stabbert et al. reported the o-TOL, o-ANI, 2-AN, and 4-ABP levels in eight commercial cigarettes that also showed high variations. Because cigars are agricultural products, the chemical composition of cigar tobacco leaves is affected by how and where the tobacco plants are grown and harvested, as well as the conditions in which the leaves are cured and fermented.29−31

Aromatic amines are formed during cigar smoking, and they are found mainly in the particle phase that was collected in the TPM. The plots showing the aromatic amine yield and TPM from all cigar products are presented in Figures 2–4 and Table 1, respectively. The figures for o-TOL as an example are shown in Supporting Figure S4. Across all cigar products, on a per cigar basis, the total amount of aromatic amines increased with increased TPM (Supporting Figure S4A). The aromatic amines showed a good linear response (R2 > 0.8348) with TPM except for 1-AN and 4-ABP, which had R2 values of 0.6693 and 0.4315, respectively. On a per mass of TPM basis, the ratios of aromatic amines and TPM were similar and consistent for all three categories (Supporting Figure S4B). Therefore, a comparable amount of aromatic amines was generated on equal TPM delivery. The six aromatic amines in the mainstream smoke of the reference cigarettes 1R6F and 2R5F and CORESTA monitor CM8 were determined following the CRM 64 in our previous research.26 The same method was used for current cigar smoking. Comparing the level of the six aromatic amines in the mainstream smoke of cigars and cigarettes, on a per test unit basis, the cigars generated more aromatic amines in the mainstream smoke than cigarettes (Supporting Figure S4A); on a per mass of TPM basis, the aromatic amines generated by cigars are compatible with that of cigarettes (Supporting Figure S4B).

Relationships between Aromatic Amines and Proteins

It has been reported that proteins are precursors for aromatic amine formation in tobacco. Therefore, we measured the water-soluble and water-insoluble protein contents in commercial cigar fillers. There were three replicates for each brand. Data are presented in Table 1. The Spearman correlation coefficients between the six aromatic amines (o-TOL, o-ANI, 1-AN, 2-AN, 3-ABP, and 4-ABP) and the water-soluble and water-insoluble proteins are provided in Supporting Table S3.

For the large cigars, the aromatic amines (o-TOL, o-ANI, 1-AN, and 4-ABP) showed the highest significantly large positive correlations with the water-insoluble proteins (r > 0.531) with p-values < 0.002. 2-AN and 3-ABP showed significantly medium positive correlations with the water-insoluble proteins (r = 0.350 and 0.446, respectively) with p-values < 0.05. The aromatic amines were positively correlated with the water-soluble proteins, although with lower correlation coefficients compared to the water-insoluble proteins. The two exceptions were o-ANI and 3-ABP, which have higher coefficients than water-insoluble proteins.

We measured the aromatic amines and protein contents in 16 commercial cigarillos; however, two cigarillos (Black & Mild original and Hav-A-Tampa Jewels original) have a plastic tip on the mouth end of the cigarillos. These tips captured some of the aromatic amines produced during the smoking. The aromatic amines in the tips were measured (data not shown here). Therefore, these two cigarillo data sets were excluded when the Spearman correlation coefficients were calculated due to their different designs. The aromatic amines showed large positive correlations with the water-insoluble proteins (r > 0.303) with p-values < 0.05 except for 2-AN and 3-ABP, which had small but nonsignificant positive correlations with the water-insoluble proteins in cigarillos.

For the filtered cigars, o-TOL, 1-AN, 2-AN, and 3-ABP showed large or medium significant positive correlations with the water-insoluble proteins (r > 0.486) with p-values < 0.0001. The aromatic amines showed medium or small negative correlations with the water-soluble proteins. The filtered cigars have filters that trap some of the aromatic amines during smoking.8 Most cigar filters are made of cellulose acetate; however, some filters contain clay-like or couscous-like granules to increase the cigar weight. The clay-like materials are most likely sepiolite.32,33 The lengths and ventilation properties of cigar filters also differ among cigar brands. All of these factors cause varying amounts of aromatic amines to be trapped in the filters. As a result, the aromatic amine levels in the smoke that were captured on the pad did not represent that generated by the whole cigar during smoking. Consequently, it affected the correct evaluation of the correlation between aromatic amines and proteins in the filtered cigars.

Overall, aromatic amines are positively correlated with most proteins. The notable exception is between aromatic amines and water-soluble proteins in filtered cigars which had negative correlations; however, it is important to note that the results might be affected by the type of filter and the level of ventilation of the cigar. We found that the water-insoluble proteins had a greater contribution to the formation of aromatic amines compared to the water-soluble proteins (Supporting Table S3). This conclusion is consistent with previously published results.18,20 Yoshida et al. conducted a pyrolysis experiment on the formation of polycyclic aromatic amines. Their results support our findings.20

We also analyzed the correlations between the six aromatic amines. For the large cigars and cigarillos, Spearman correlation coefficients indicated a significant positive correlation between each aromatic amine (r > 0.544) with p-values < 0.001; the exception was o-ANI, which had slightly less significant positive correlations with 2-AN and 3-ABP with r > 0.462 and p-values < 0.002 (Supporting Table S4) in the cigarillos. The correlations between each aromatic amine in the filtered cigars showed more variation due to the differences in the filtered cigar design, the type of filter, and the ventilation of the cigars.

Conclusions

In this study, we determined the yields of six aromatic amines (o-TOL, o-ANI, 1-AN, 2-AN, 3-ABP, and 4-ABP) in mainstream smoke from 23 commercial filtered cigars, 16 cigarillos, and 11 large cigars. The aromatic amine yields in the mainstream smoke showed high levels of variation within and between products. The relationships between aromatic amines and (1) TPM, (2) water-soluble proteins, and (3) water-insoluble proteins were evaluated. The aromatic amines showed a good linear response with TPM on a per cigar basis. The aromatic amines were positively correlated with proteins. The water-insoluble proteins have a greater contribution to the formation of aromatic amines compared to the water-soluble proteins.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.chemrestox.3c00273.

  • Mass spectrometric parameters for the quantification and confirmation of the MBTFA derivatives of aromatic amines in the multiple reaction monitoring (MRM) modes (Table S1); physical parameters of the commercial cigars (Table S2); Spearman coefficients and p-values between aromatic amines and water-soluble or water-insoluble proteins (Table S3); Spearman coefficients and p-values between aromatic amines (Table S4); typical chromatographic separation of the MBTFA derivatives of six aromatic amines, pure aromatic amine standard mixture, and the reference cigar smoke extract (Figure S1); yield of o-toluidine (ng/g) in mainstream smoke per tobacco smoked in the filtered cigars, cigarillos, and large cigars (Figure S2); averages of each aromatic amine yield in the filtered cigars, cigarillos, and large cigars (Figure S3); yield of o-toluidine in mainstream smoke from the reference cigarettes, filtered cigars, cigarillos, and large cigars: o-toluidine (ng/cigar) and o-toluidine (ng/mg TPM) (Figure S4) (PDF)

This project was supported by the U.S. Food and Drug Administration through grant UC2FD006890. The views expressed in this paper do not necessarily reflect the official policies of the Department of Health and Human Services, nor does any mention of trade names, commercial practices, or organization imply endorsement by the United States Government.

The authors declare no competing financial interest.

Supplementary Material

tx3c00273_si_001.pdf (556.9KB, pdf)

References

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