Quantitative Analysis of Six Heterocyclic Aromatic Amines in Mainstream Cigarette Smoke Condensate Using Isotope Dilution Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry (original) (raw)
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Emergency Response and Air Toxicants Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA
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Emergency Response and Air Toxicants Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA
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Clifford H. Watson, Ph.D.
Emergency Response and Air Toxicants Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA
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Received:
13 October 2010
Accepted:
16 November 2010
Published:
20 December 2010
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Liqin Zhang, David L. Ashley, Clifford H. Watson, Quantitative Analysis of Six Heterocyclic Aromatic Amines in Mainstream Cigarette Smoke Condensate Using Isotope Dilution Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry, Nicotine & Tobacco Research, Volume 13, Issue 2, February 2011, Pages 120–126, https://doi.org/10.1093/ntr/ntq219
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Abstract
Introduction:
Heterocyclic aromatic amines (HAAs) represent an important class of carcinogens in mainstream cigarette smoke. Accurate HAA quantification is challenging because of their relative low abundances and numerous chemical interferences that arise naturally from thousands of the constituents present in cigarette smoke. We have developed and validated a straightforward high-throughput method to quantify HAA levels in mainstream cigarette smoke and demonstrated the applicability by analyzing select research and domestic cigarette brands.
Methods:
Machine-smoked cigarette condensate collected under both standard and intensive smoking regimens was examined. Mainstream smoke particulate from individual cigarettes trapped on a glass fiber filter pad was spiked with an appropriate internal standard solution and subsequently solvent extracted. The extract was quantitatively analyzed by high-performance liquid chromatography and tandem mass spectrometry.
Results:
Method validation data showed excellent accuracy, reproducibility and high throughput; it is suitable for the routine analysis of HAAs in cigarette smoke condensate delivered under a wide of differing smoking conditions. The smoking machine deliveries of HAAs are strongly influenced by cigarettes’ physical design, filler blend, and smoking regimen.
Conclusions:
A quick and accurate method has been developed for the analysis of 6 HAAs in mainstream cigarette smoke condensate. Results provided a good mean to access the ranges of HAAs in commercial products and evaluate the relative contribution of cigarette design, filler blend, and smoking regimen on delivery. Such data are vital in helping provide exposure ranges for potential human exposure estimates.
Introduction
An estimated 438,000 people die prematurely each year in the United States from smoking or secondhand smoke exposure (Centers for Disease Control and Prevention [CDC], 2005). Despite these risks, approximately 45.1 million American adults continue to smoke cigarettes (CDC, 2007). Tobacco smoke is an exceedingly complex matrix consisting thousands of compounds. So far, more than 4,000 chemicals have been identified among which more than 50 of the chemicals are known carcinogens (Hoffmann & Hoffmann, 1997), including tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, aromatic amines, aldehydes and volatile hydrocarbons, as well as heterocyclic aromatic amines (HAAs). Studies have reported that HAAs have a potentially important impact on human health and, particularly, on cancer risk due to frequent exposure (Felton, Knize, Salmon, Malfatti, & Kulp, 2002; Gorlewska-Roberts, Green, Fares, Ambrosone, & Kadlubar, 2002; Sugimura, Wakabayashi, Nakagama, & Nagao, 2004; Totsuka, Takamura-Enya, Nishigaki, Sugimura, & Wakabayashi, 2004; Wakabayashi, Nagao, Esumi, & Sugimura, 1992). HAAs in cigarette smoke are primarily generated from the combustion of tobacco. The HAAs considered to be carcinogenic in cigarette smoke by the International Agency for Research on Cancer (IARC) (2004) include norharman, 9H-pyrido[3,4-b]indole; harman, 1-methyl-9H-pyrido[3,4-b]indole; Trp-P-1, 3-amino-1, 4-dimethyl-5H-pyrido [4,3-b ]indole; Trp-P-2, 3-amino-1-methyl-5H-pyrido [4,3-b] indole; AC, 2-amino-9H-pyrido[2,3-b] indole; MeAC, 2-amino-3-methyl-9H-pyrido[2,3-b] indole; Glu-P-1, 2-amino-6-methyldipyrido[1,2-A:3’,2’-D]imidazole; Glu-P-2, 2-aminodipyrido[1,2-α:3’,2-D]imidazole; IQ, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline; and PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (Figure 1). Harman and norharman are not mutagenic in the Ames test but have been shown to be comutagens and to enhance or induce the mutagenicity of other HAAs (Nagao, Yahagi, Kawachi, et al., 1977; Nagao, Yahagi, & Sugimura, 1978; Totsuka et al., 2004). Thus, they are important contributors when assessing HAAs and are therefore frequently included for analysis. It is essential to accurately identify and determine HAAs in cigarette smoke under a variety of smoking conditions in order to help estimate the potential human health exposure associated with smoking.
Figure 1.
Structures of heterocyclic aromatic amines: norharman, 9H-pyrido[3,4-b]indole; harman, 1-methyl-9H-pyrido[3,4-b]indole; Trp-P-1, 3-amino-1, 4-dimethyl-5H-pyrido [4,3-b ]indole; Trp-P-2, 3-amino-1-methyl-5H-pyrido [4,3-b] indole; AC, 2-amino-9H-pyrido[2,3-b] indole; MeAC, 2-amino-3-methyl-9H-pyrido[2,3-b] indole; Glu-P-1, 2-amino-6-methyldipyrido[1,2-A:3’,2’-D]imidazole; Glu-P-2, 2-aminodipyrido[1,2-α:3’,2-D]imidazole; IQ, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline; and PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.
Initial research on HAAs in cigarette smoke began in the 1960s when harman and norharman were identified from cigarette smoke by paper chromatography and ultraviolet, infrared, and fluorescence spectroscopy (Poindexter & Carpenter, 1962). In the 1980s, AC and MeAC were separated from cigarette smoke condensate by column chromatography and high-performance liquid chromatography (HPLC) with fluorescence detection (Matsumoto, Yoshida, & Tomita, 1981; Yoshida & Matsumoto, 1980). IQ was identified from the smoke of 20 cigarettes by HPLC/electrochemical detection after purification and Blue-Cotton extraction (Yamashita et al., 1986). The level of IQ was estimated to be 0.26 ng delivered per cigarette. In the 1990s, Trp-P-1, Trp-P-2, PhIP, Glu-P-1, and Glu-P-2 were detected in mainstream cigarette smoke using HPLC with fluorescence detection, acid–base partitioning with dichloromethane, solid-phase extraction (SPE), and preparative HPLC fractionations (Kanai, Wada, & Manabe, 1990; Manabe, Tohyama, Wada, & Aramaki, 1991; Manabe & Wada, 1990; Manabe, Wada, & Kanai, 1990). Trp-p-1, AC, IQ, and MeIQ were detected in mainstream cigarette smoke by gas chromatography (GC)-nitrogen phosphorus detection using two connected fused-silica capillary columns, but Trp-P-2, PhIP, and Glu-P-1 were not detectable by this method (Kataoka, Kijima, & Maruo, 1998). All the studies on HAAs in cigarette smoke condensate conducted before 2000 involved several laborious steps for sample cleanup due to limitations of the specificity of the detection methodology. In 2004, Smith, Qian, Zha, and Moldoveanu (2004) published methods for determining four HAAs (harman, norharman, AC, and MeAC) using SPE and GC–mass spectrometry (MS) without derivatization, a significant improvement in reducing the complexity of the analysis. All these studies demonstrated that mainstream cigarette smoke contains many chemical substances that potentially can interfere with the accurate quantification of HAAs.
Our study describes the development and application of a simple, sensitive, and accurate method using isotope dilution HPLC–electrospray ionization tandem mass spectrometric detection (ESI-MS/MS) to quantify six HAAs (harman, norharman, AC, MeAC, Trp-P-1, and Trp-P-2) in mainstream cigarette smoke condensate. Tandem mass spectral detection helped reduce burdensome sample cleanup procedures by offering the advantages of high sensitivity and selectivity through reduction in interferences from other sample components. Additionally, isotopically labeled internal standards helped compensate for sample loss and instrument response variation providing improved accuracy and precision. Six individual isotope-labeled internal standards—norharman-D7, harman-13C2,15N, AC-15N3, MeAC-D3, Trp-P-1-13C2,15N, and Trp-P-2-13C2,15N—were used. Sample preparation consisted of a one-step analyte extraction using 0.1% aqueous formic acid solution. This method was applied to the analysis of HAAs in mainstream smoke particulate matter, generated under two different machine-smoking regimens, from Kentucky research cigarettes, selected domestic cigarettes, and custom-blended cigarettes.
Methods
Reagents and Materials
Native HAA standards and their corresponding isotope-labeled analogs were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). Other HPLC-grade chemicals and solvents were obtained from Fisher Scientific (Pittsburgh, PA). The Kentucky research cigarettes were purchased from the University of Kentucky (Lexington, KY), and the domestic cigarettes were purchased from commercial retail outlets in the metropolitan Atlanta, Georgia, area. The custom-blended cigarettes were unfiltered varieties purchased from Murty Pharmaceuticals, Inc. (Lexington, KY) and contained a single blend of different tobacco types.
Standard Preparation
The individual standard stock solutions and mixed standard stock solutions were prepared in methanol, stored at −20 °C, and protected from light. Calibration standard solutions were prepared by spiking known amounts of mixed standard stock and internal standard solution onto 44-mm Cambridge glass fiber filter pads, followed by extracting in 13 ml of 0.1% aqueous formic acid on a Lab-Line shaker at 250 rpm for 1 hr. These calibration standard solutions were injected to LC/MS/MS to generate calibration curves for Trp-P-1, Trp-P-2, AC, and MeAC. To establish curves of norharman and harman, the prepared calibration standard solutions were then diluted 200-fold with 0.1% aqueous formic acid and injected to LC/MS/MS due to high concentrations of norharman and harman in cigarette smoke.
Sample Preparation
Cigarettes were conditioned in an environmental chamber held at 22 ± 2 °C and 60% ± 3% humidity for at least 24 hr prior to analysis. A Cerulean (Milton Keynes, UK) ASM 16-port smoking machine was used to smoke the cigarettes, and mainstream smoke condensate was collected on 44-mm Cambridge glass fiber filter pads. Cigarettes were smoked under International Organization for Standardization (ISO; 60s interval, 35 ml puff volume, and 2s puff duration) and Canadian Intense (30s interval, 55 ml puff volume, 2s puff duration, and blocked filter ventilation holes) conditions. ISO smoking was used to provide historically comparative data and more intense smoking deliveries (Canadian Intense) to evaluate the change in delivery that could occur when smokers employed various compensation tactics, such as taking larger puff volumes, more frequent puffs, or blocking vent holes while smoking. Under each smoking regimen, individual filter pads collected the mainstream smoke particulate from one cigarette. Each pad was spiked with the isotope-labeled internal standard solution and then extracted identically to the spiked standards as described earlier. An aliquot of each extract was placed in an autosampler vial and underwent LC/MS/MS analysis for AC, MeAC, Trip-P-1, and Trip-P-2. Each extract was then diluted 200-fold with 0.1% formic acid solution for the analysis of norharman and harman.
HPLC Analysis
A Waters Xterra C18 MS column, 2.1 × 100 mm and 3.5-μm particle size (Waters Corporation, Milford, MA), was used to separate the HAA compounds at a column oven temperature of 45 °C. The injection volume was 20 μl. Mobile phase A was 5 mM aqueous ammonium formate solution adjusted to pH 3.4, and mobile phase B was acetonitrile. The separation was achieved by using a gradient method at a flow rate of 450 μl/min. The total run time, including equilibration, for each sample was 13 min.
Mass Spectrometer
An API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) coupled to an Agilent 1100 HPLC (Agilent Technologies, Inc., Santa Clara, CA) was used to identify and quantify the HAAs. Analyst 1.4 from Applied Biosystems controlled the instrumentation. The mass spectrometer was operated with electrospray ionization, and analyte precursor/fragment ion pairs were monitored in multiple reaction–monitoring mode. The optimal instrument control parameters were as follows: positive ionization mode, collision gas 6 psi, curtain gas 35 psi, nebulizer and heater gas 45 psi, ion spray voltage 3500 V, and source temperature 650 °C. Compound dependent parameters were optimized for each analyte (Table 1).
Table 1.
Compound-Dependent Parameters
Analytes | Formula | Precursor ions | Product ions | DP | EP | CE | CXP |
---|---|---|---|---|---|---|---|
Norharman | C11H8N2 | 169.2 | 115/89 | 65 | 10 | 45/65 | 8/8 |
Harman | C12H10N2 | 183.2 | 115/89 | 60 | 10 | 45/67 | 6/8 |
Trp-P-1 | C13H13N3 | 212.2 | 195/167 | 45 | 10 | 35/49 | 10/10 |
Trp-P-2 | C12H11N3 | 198.1 | 181/154 | 60 | 10 | 32/40 | 10/10 |
AC | C11H9N3 | 184.2 | 140/167 | 60 | 11 | 45/33 | 8/10 |
MeAC | C12H11N3 | 198.1 | 181/129 | 60 | 10 | 32/38 | 10/10 |
Norharman-D7 | C11HD7N2 | 176.2 | 120 | 65 | 10 | 50 | 11 |
Harman-13C2,15N | C1013C2H10N15N | 186.2 | 117 | 60 | 10 | 47 | 10 |
Trp-P-1-13C2,15N | C1113C2H13N215N | 215.2 | 197.1 | 50 | 10 | 35 | 9 |
Trp-P-2-13C2,15N | C1013C2H11N215N | 201.2 | 155 | 60 | 8 | 43 | 10 |
AC-15N3 | C11H915N3 | 187.1 | 169.1 | 60 | 10 | 35 | 10 |
MeAC-D3 | C12H8D3N3 | 201.1 | 184.1 | 40 | 9 | 32 | 9 |
Analytes | Formula | Precursor ions | Product ions | DP | EP | CE | CXP |
---|---|---|---|---|---|---|---|
Norharman | C11H8N2 | 169.2 | 115/89 | 65 | 10 | 45/65 | 8/8 |
Harman | C12H10N2 | 183.2 | 115/89 | 60 | 10 | 45/67 | 6/8 |
Trp-P-1 | C13H13N3 | 212.2 | 195/167 | 45 | 10 | 35/49 | 10/10 |
Trp-P-2 | C12H11N3 | 198.1 | 181/154 | 60 | 10 | 32/40 | 10/10 |
AC | C11H9N3 | 184.2 | 140/167 | 60 | 11 | 45/33 | 8/10 |
MeAC | C12H11N3 | 198.1 | 181/129 | 60 | 10 | 32/38 | 10/10 |
Norharman-D7 | C11HD7N2 | 176.2 | 120 | 65 | 10 | 50 | 11 |
Harman-13C2,15N | C1013C2H10N15N | 186.2 | 117 | 60 | 10 | 47 | 10 |
Trp-P-1-13C2,15N | C1113C2H13N215N | 215.2 | 197.1 | 50 | 10 | 35 | 9 |
Trp-P-2-13C2,15N | C1013C2H11N215N | 201.2 | 155 | 60 | 8 | 43 | 10 |
AC-15N3 | C11H915N3 | 187.1 | 169.1 | 60 | 10 | 35 | 10 |
MeAC-D3 | C12H8D3N3 | 201.1 | 184.1 | 40 | 9 | 32 | 9 |
Note. CE = collision energy; CXP = collision cell exit potential; DP = declustering potential; EP = entrance potential.
Table 1.
Compound-Dependent Parameters
Analytes | Formula | Precursor ions | Product ions | DP | EP | CE | CXP |
---|---|---|---|---|---|---|---|
Norharman | C11H8N2 | 169.2 | 115/89 | 65 | 10 | 45/65 | 8/8 |
Harman | C12H10N2 | 183.2 | 115/89 | 60 | 10 | 45/67 | 6/8 |
Trp-P-1 | C13H13N3 | 212.2 | 195/167 | 45 | 10 | 35/49 | 10/10 |
Trp-P-2 | C12H11N3 | 198.1 | 181/154 | 60 | 10 | 32/40 | 10/10 |
AC | C11H9N3 | 184.2 | 140/167 | 60 | 11 | 45/33 | 8/10 |
MeAC | C12H11N3 | 198.1 | 181/129 | 60 | 10 | 32/38 | 10/10 |
Norharman-D7 | C11HD7N2 | 176.2 | 120 | 65 | 10 | 50 | 11 |
Harman-13C2,15N | C1013C2H10N15N | 186.2 | 117 | 60 | 10 | 47 | 10 |
Trp-P-1-13C2,15N | C1113C2H13N215N | 215.2 | 197.1 | 50 | 10 | 35 | 9 |
Trp-P-2-13C2,15N | C1013C2H11N215N | 201.2 | 155 | 60 | 8 | 43 | 10 |
AC-15N3 | C11H915N3 | 187.1 | 169.1 | 60 | 10 | 35 | 10 |
MeAC-D3 | C12H8D3N3 | 201.1 | 184.1 | 40 | 9 | 32 | 9 |
Analytes | Formula | Precursor ions | Product ions | DP | EP | CE | CXP |
---|---|---|---|---|---|---|---|
Norharman | C11H8N2 | 169.2 | 115/89 | 65 | 10 | 45/65 | 8/8 |
Harman | C12H10N2 | 183.2 | 115/89 | 60 | 10 | 45/67 | 6/8 |
Trp-P-1 | C13H13N3 | 212.2 | 195/167 | 45 | 10 | 35/49 | 10/10 |
Trp-P-2 | C12H11N3 | 198.1 | 181/154 | 60 | 10 | 32/40 | 10/10 |
AC | C11H9N3 | 184.2 | 140/167 | 60 | 11 | 45/33 | 8/10 |
MeAC | C12H11N3 | 198.1 | 181/129 | 60 | 10 | 32/38 | 10/10 |
Norharman-D7 | C11HD7N2 | 176.2 | 120 | 65 | 10 | 50 | 11 |
Harman-13C2,15N | C1013C2H10N15N | 186.2 | 117 | 60 | 10 | 47 | 10 |
Trp-P-1-13C2,15N | C1113C2H13N215N | 215.2 | 197.1 | 50 | 10 | 35 | 9 |
Trp-P-2-13C2,15N | C1013C2H11N215N | 201.2 | 155 | 60 | 8 | 43 | 10 |
AC-15N3 | C11H915N3 | 187.1 | 169.1 | 60 | 10 | 35 | 10 |
MeAC-D3 | C12H8D3N3 | 201.1 | 184.1 | 40 | 9 | 32 | 9 |
Note. CE = collision energy; CXP = collision cell exit potential; DP = declustering potential; EP = entrance potential.
Results and Discussion
Previous work by other researchers on HAAs used hydrochloric acid or organic solvents for extraction. For improved compatibility with our HPLC's mobile phase and mass spectrometer, we selected formic acid as our extraction solution. Chromatographic conditions were optimized to separate the HAA analytes and achieve short run time and reproducible quantitation from the mainstream smoke particulate from 3R4F research cigarettes (University of Kentucky). A series of aqueous solutions containing 0.08%, 0.1%, and 1% formic acid were evaluated for optimal extraction of the filter pads. The formic acid concentration had little influence on extraction efficiency. The extraction time was also optimized through a series of experiments with differing extraction times. Optimal efficiency was achieved with a 40-min extraction time. Further optimization of the chromatography to maintain peak separation and shorter retention times was achieved via a systematic testing of gradient conditions, mobile phase pH, and buffer concentration. The pH primarily influenced the retention times for AC and MeAC and had less effect on the other four HAAs, whereas the ammonium formate buffer concentration had no influence on AC and MeAC but did shift the other HAAs to longer retention times with increasing buffer concentration.
Calibration curves were established by least-square linear regression analysis of the relative response factors (peak area ratios of native analytes divided by their isotopically labeled internal standards) versus the amount spiked on the pads. A linear response existed over a range of levels (about 24-fold) covering the amounts detected in mainstream cigarette smoke collected under differing smoking conditions. Replicate calibration runs at nine different concentration levels yielded correlation coefficients with _R_2 values higher than 0.99.
Replicate measurements of low-level standards were used to calculate the limits of detection (LOD). The SD of each standard level's response was plotted against standard concentrations. The LOD was estimated as 3S0, where S0 is the SD when the standard concentration is extrapolated to zero concentration. The calculated LODs were 3.0 ng for AC, 0.4 ng for MeAC, 0.1 ng for Trp-p-1, 0.1 ng for Trp-p-2, 36 ng for harman, and 68 ng for norharman. The higher LODs for harman and norharman result from the 200-fold dilution of calibration solutions in the standard preparation.
Method accuracy was determined by spiking known amounts of the HAA standards onto Cambridge filter pads containing mainstream smoke condensate from 3R4F cigarettes. Analytes were spiked at two levels: half of and equal to the level of HAAs in 3R4F mainstream smoke when smoked under ISO machine-smoking conditions. The recovered spike was determined by subtracting the average level of HAAs in 3R4F mainstream smoke from the detected level of the spiked pads. Accuracy was calculated by dividing the recovered spike by the amount actually spiked on the pad. Method accuracy ranged from 94% to 125% at the lower spiking level and from 83% to 100% at the higher spiking level. This procedure was also performed with blank filter pads. Improved accuracy from spiking standard solution onto blank filter pads was observed (97%–105%) compared with the spiking experiments on pads containing the mainstream smoke particulate matrix. Precision was determined as the relative standard deviation (RSD) of the replicate measurements of the samples over 3 consecutive days. RSD was typically 5% and was no worse than 12% for both the low and the high concentrations.
Sample matrix effects were investigated by comparing the response of isotopically labeled internal standards both with and without the presence of the smoke matrix generated from smoking different cigarettes under different smoking conditions. Recoveries were reduced, with loss of analyte up to 75%, as the total particulate matter (TPM) increased. Greater matrix effects were observed for the lower level HAAs than for the high level HAAs. The exact nature of the lower response at higher particulate matter loading is not known, but this finding illustrates the importance of labeled internal standards to help compensate for such behavior.
The fully validated method was applied to a series of select Kentucky research cigarettes and domestic cigarettes (Table 2). Good separation was achieved for the six HAAs in mainstream cigarette smoke. All six HAAs were detected and quantified in the mainstream cigarette smoke from all tested brands. Levels of all HAAs in the cigarettes tested with these smoking regimens were at levels well above the detection limits.
Table 2.
Heterocyclic Aromatic Amines in Mainstream Cigarette Smoke
Sample name | Smoking regimen | AC (ng/cigarette) | MeAC (ng/cigarette) | Trp-P-1 (ng/cigarette) | Trp-P-2 (ng/cigarette) | Harman (ng/cigarette) | Norharman (ng/cigarette) |
---|---|---|---|---|---|---|---|
Marlboro-UL-K | ISO | 49 | 3.1 | 0.6 | 1.8 | 630 | 1,400 |
Marlboro-L-K | ISO | 77 | 5.1 | 1.3 | 2.8 | 1,000 | 2,000 |
Marlboro-F-K | ISO | 95 | 6.0 | 1.8 | 3.9 | 1,400 | 2,600 |
Newport-F-K-M | ISO | 90 | 6.1 | 1.9 | 3.8 | 1,800 | 2,900 |
1R5F | ISO | 33 | 2.0 | 0.3 | 1.2 | 390 | 800 |
2R4F | ISO | 75 | 5.5 | 1.3 | 2.8 | 1,100 | 2,000 |
3R4F | ISO | 77 | 5.1 | 1.1 | 2.8 | 950 | 1,800 |
CM 6 | ISO | 60 | 4.2 | 1.1 | 4.6 | 1,800 | 3,300 |
3R4F | CI | 160 | 11 | 1.5 | 5.3 | 2,000 | 4,100 |
Marlboro-F-K | CI | 160 | 9.7 | 2.4 | 9.2 | 2,600 | 5,800 |
Sample name | Smoking regimen | AC (ng/cigarette) | MeAC (ng/cigarette) | Trp-P-1 (ng/cigarette) | Trp-P-2 (ng/cigarette) | Harman (ng/cigarette) | Norharman (ng/cigarette) |
---|---|---|---|---|---|---|---|
Marlboro-UL-K | ISO | 49 | 3.1 | 0.6 | 1.8 | 630 | 1,400 |
Marlboro-L-K | ISO | 77 | 5.1 | 1.3 | 2.8 | 1,000 | 2,000 |
Marlboro-F-K | ISO | 95 | 6.0 | 1.8 | 3.9 | 1,400 | 2,600 |
Newport-F-K-M | ISO | 90 | 6.1 | 1.9 | 3.8 | 1,800 | 2,900 |
1R5F | ISO | 33 | 2.0 | 0.3 | 1.2 | 390 | 800 |
2R4F | ISO | 75 | 5.5 | 1.3 | 2.8 | 1,100 | 2,000 |
3R4F | ISO | 77 | 5.1 | 1.1 | 2.8 | 950 | 1,800 |
CM 6 | ISO | 60 | 4.2 | 1.1 | 4.6 | 1,800 | 3,300 |
3R4F | CI | 160 | 11 | 1.5 | 5.3 | 2,000 | 4,100 |
Marlboro-F-K | CI | 160 | 9.7 | 2.4 | 9.2 | 2,600 | 5,800 |
Sample name | Smoking regimen | AC (ng/g tobacco) | MeAC (ng/g tobacco) | Trp-P-1 (ng/g tobacco) | Trp-P-2 (ng/g tobacco) | Harman (ng/g tobacco) | Norharman (ng/g tobacco) |
---|---|---|---|---|---|---|---|
Flue cured tobaccoa | ISO | 130 | 8.1 | 2.2 | 9.4 | 3,100 | 5,700 |
Burley tobaccoa | ISO | 390 | 22 | 3.1 | 16 | 6,200 | 11,000 |
Sample name | Smoking regimen | AC (ng/g tobacco) | MeAC (ng/g tobacco) | Trp-P-1 (ng/g tobacco) | Trp-P-2 (ng/g tobacco) | Harman (ng/g tobacco) | Norharman (ng/g tobacco) |
---|---|---|---|---|---|---|---|
Flue cured tobaccoa | ISO | 130 | 8.1 | 2.2 | 9.4 | 3,100 | 5,700 |
Burley tobaccoa | ISO | 390 | 22 | 3.1 | 16 | 6,200 | 11,000 |
Note. CI = Canadian Intense; F = Full flavor, ISO = International Organization for Standardization; K = King; L = Light; M = Menthol; UL = Ultralight.
a
Custom-made unfiltered cigarettes.
Table 2.
Heterocyclic Aromatic Amines in Mainstream Cigarette Smoke
Sample name | Smoking regimen | AC (ng/cigarette) | MeAC (ng/cigarette) | Trp-P-1 (ng/cigarette) | Trp-P-2 (ng/cigarette) | Harman (ng/cigarette) | Norharman (ng/cigarette) |
---|---|---|---|---|---|---|---|
Marlboro-UL-K | ISO | 49 | 3.1 | 0.6 | 1.8 | 630 | 1,400 |
Marlboro-L-K | ISO | 77 | 5.1 | 1.3 | 2.8 | 1,000 | 2,000 |
Marlboro-F-K | ISO | 95 | 6.0 | 1.8 | 3.9 | 1,400 | 2,600 |
Newport-F-K-M | ISO | 90 | 6.1 | 1.9 | 3.8 | 1,800 | 2,900 |
1R5F | ISO | 33 | 2.0 | 0.3 | 1.2 | 390 | 800 |
2R4F | ISO | 75 | 5.5 | 1.3 | 2.8 | 1,100 | 2,000 |
3R4F | ISO | 77 | 5.1 | 1.1 | 2.8 | 950 | 1,800 |
CM 6 | ISO | 60 | 4.2 | 1.1 | 4.6 | 1,800 | 3,300 |
3R4F | CI | 160 | 11 | 1.5 | 5.3 | 2,000 | 4,100 |
Marlboro-F-K | CI | 160 | 9.7 | 2.4 | 9.2 | 2,600 | 5,800 |
Sample name | Smoking regimen | AC (ng/cigarette) | MeAC (ng/cigarette) | Trp-P-1 (ng/cigarette) | Trp-P-2 (ng/cigarette) | Harman (ng/cigarette) | Norharman (ng/cigarette) |
---|---|---|---|---|---|---|---|
Marlboro-UL-K | ISO | 49 | 3.1 | 0.6 | 1.8 | 630 | 1,400 |
Marlboro-L-K | ISO | 77 | 5.1 | 1.3 | 2.8 | 1,000 | 2,000 |
Marlboro-F-K | ISO | 95 | 6.0 | 1.8 | 3.9 | 1,400 | 2,600 |
Newport-F-K-M | ISO | 90 | 6.1 | 1.9 | 3.8 | 1,800 | 2,900 |
1R5F | ISO | 33 | 2.0 | 0.3 | 1.2 | 390 | 800 |
2R4F | ISO | 75 | 5.5 | 1.3 | 2.8 | 1,100 | 2,000 |
3R4F | ISO | 77 | 5.1 | 1.1 | 2.8 | 950 | 1,800 |
CM 6 | ISO | 60 | 4.2 | 1.1 | 4.6 | 1,800 | 3,300 |
3R4F | CI | 160 | 11 | 1.5 | 5.3 | 2,000 | 4,100 |
Marlboro-F-K | CI | 160 | 9.7 | 2.4 | 9.2 | 2,600 | 5,800 |
Sample name | Smoking regimen | AC (ng/g tobacco) | MeAC (ng/g tobacco) | Trp-P-1 (ng/g tobacco) | Trp-P-2 (ng/g tobacco) | Harman (ng/g tobacco) | Norharman (ng/g tobacco) |
---|---|---|---|---|---|---|---|
Flue cured tobaccoa | ISO | 130 | 8.1 | 2.2 | 9.4 | 3,100 | 5,700 |
Burley tobaccoa | ISO | 390 | 22 | 3.1 | 16 | 6,200 | 11,000 |
Sample name | Smoking regimen | AC (ng/g tobacco) | MeAC (ng/g tobacco) | Trp-P-1 (ng/g tobacco) | Trp-P-2 (ng/g tobacco) | Harman (ng/g tobacco) | Norharman (ng/g tobacco) |
---|---|---|---|---|---|---|---|
Flue cured tobaccoa | ISO | 130 | 8.1 | 2.2 | 9.4 | 3,100 | 5,700 |
Burley tobaccoa | ISO | 390 | 22 | 3.1 | 16 | 6,200 | 11,000 |
Note. CI = Canadian Intense; F = Full flavor, ISO = International Organization for Standardization; K = King; L = Light; M = Menthol; UL = Ultralight.
a
Custom-made unfiltered cigarettes.
The smoking regimen plays a major influence on HAA deliveries under machine-smoking conditions. The level of HAAs produced under Canadian Intense smoking was considerably higher than that produced by ISO regimen for the same cigarettes. In addition, tip ventilation and type of tobacco affected deliveries of HAAs. Each of the HAA levels increased as the concentrations of TPM in the mainstream cigarette smoke increased. Cigarette brands with higher filter ventilation yielded less TPM and lower HAA levels than brands having lower filter ventilation when smoked using the ISO smoking parameters; however, when cigarettes were smoked using the Canadian Intense regimen, with the filter holes blocked, HAA deliveries between cigarettes were much more comparable (Table 2). Factors other than filter ventilation could influence deliveries of the HAAs. We also examined the mainstream smoke from two custom-made unfiltered cigarettes containing exclusively bright and burley tobacco. The HAA levels associated with the cigarettes made exclusively from burley tobacco were significantly higher than those from the bright tobacco cigarettes (Table 2), demonstrating that tobacco type and blend composition likely influence the levels of HAAs measured in mainstream smoke from commercial cigarettes.
To improve the method accuracy and compensate for the matrix effect and sample loss during the sample preparation, we used isotope-labeled analogues as internal standards for this method, resulting in increased cost. The HAA levels reported here were generated using standard smoking machine regimens. Thus, the mainstream smoke levels do not reflect typical human consumption patterns and the resulting exposures. The two different smoking regimens provide comparison means among different products so should serve as a means of understanding the influence of different smoking intensities on the delivery of HAAs.
Previously reported results on mainstream smoke HAA deliveries (Table 3) span a wide range. These data are not directly comparable in most cases because the earlier literature did not always list the specific cigarette brands tested. The values from Smith et al. (2004) for the 1R5F and 2R4F are generally lower than our values but are of the same order of magnitude. One possible explanation for the lower level in previous studies may have resulted from multiple sample cleanup steps, resulting in analyte loss. Such losses are of particular concern because these methods did not include a chemically representative internal standard prior to sample workup to compensate for losses. We found that using isotopically labeled internal standards is essential to minimize sample losses and lessen any matrix effects.
Table 3.
Comparison of Literature Results
Note. ISO = International Organization for Standardization; TPM = total particulate matter.
Table 3.
Comparison of Literature Results
Note. ISO = International Organization for Standardization; TPM = total particulate matter.
As stated earlier, four carcinogenic HAAs (Glup-P-1, Glup-P-2, IQ, and PHIP), in addition to the six determined in this study, have been identified in cigarette smoke (Kanai et al., 1990; Manabe et al., 1991; Yamashita et al., 1986). These HAAs may also contribute to the toxic or carcinogenic burden from cigarette smoke exposure. We were not able to achieve satisfactory quantification on these compounds because of their relatively low levels and high chemical background precluded analysis with the current method. Work is under way to include more of these important carcinogenic compounds.
Now that the FDA has the authority to regulate tobacco products and to set performance guidelines, analytically robust methods on a wide array of constituents will be used to characterize cigarette products and to evaluate potential tobacco product standards. We believe this method meets these requirements and could be useful for characterizing HAA levels in tobacco smoke generated under a wide range of conditions that typical smokers could be readily expected to achieve. However, care must be taken when analyzing tobacco products and comparing constituent deliveries. It is not known what level of reduction in exposure to HAAs would lead to meaningful reductions in overall toxicity. The only proven means to reduce meaningful risk to these and other harmful constituents is through complete tobacco cessation.
Funding
This project was financial supported by the National Center for Chronic Disease Prevention and Health Promotion and the Office of Smoking and Health of the Centers for Disease Control and Prevention.
Declaration of Interests
None declared.
This information is distributed solely for the purpose of predissemination public comment under applicable information quality guidelines. It has not been formally disseminated by the Centers for Disease Control and Prevention. It does not represent and should not be construed to represent any agency determination or policy. Use of trade names and commercial sources is for identification only and does not imply endorsement by the U.S. Department of Health and Human Services.
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Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco 2010.
Topic:
- smoking
- carcinogens
- liquid chromatography
- isotopes
- smoke
- solvents
- ionization
- cigarette smoke
- aromatic amines
- cigarettes
- fiberglass
- filters
- dilution technique
- dilute (action)
- tandem mass spectrometry
- commercial product
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