Summary The present study evaluated in vitro extractability of various polycyclic aromatic hydrocarbons (PAHs) from moist snuff, when the extracting agent was water or artificial saliva. The extraction was performed on nine brands of moist snuff samples that are commercially available and were purchased from the market in January 2018. The moist snuff brands were selected to represent brands with different tobacco cut size descriptors and flavors. For the measurement of PAHs, two different analytical methods were used, an HPLC (High Performance Liquid Chromatography) method for measuring only benzo[ a ]pyrene (BaP) and a GC/MS/MS (Gas Chromatography Tandem Mass Spectrometry) method for measuring 21 PAHs (including BaP). These methods were modifications of preexistent methods reported in the literature. The results for BaP indicated that by extracting 500 mg of freeze-dried moist snuff with 6 portions of 20 mL water (120 mL), or with 4 portions of 20 mL artificial saliva, followed by two portions of 20 mL water, the BaP remains close to 100% in the solid material and it is not detected in the extracting solution. PAHs with a molecular weight similar or heavier than BaP also showed no extractability. Lighter PAHs such as fluorene, phenanthrene, anthracene, and 5-methylanthracene showed a relatively good extractability. An intermediate group including fluoranthene, pyrene, and benz[ a ]anthracene showed some extractability in the conditions of this in vitro experiment. This study is not a substitute for clinical studies regarding PAH uptake in human users of moist snuff. However, the results indicate very limited bioavailability of BaP and heavier PAHs from moist snuff. Higher, but variable bioavailability was indicated for lighter PAHs. Important implications of these findings are that: 1) measurably different BaP content of two moist snuff products is unlikely to result in any meaningfully different consumer exposure to BaP; and 2) biomarkers for one PAH cannot necessarily be used as a reliable indicator of exposure to another PAH, particularly if the molecular weights of the precursor PAHs differ since their bioavailabilities can be very different. [Beitr. Tabakforsch. Int. 28 (2019) 214–223]
Summary The width of cut tobacco strands is an important indicator for physical parameters as well as for the smoking quality. In some countries, cut width helps to distinguish fine-cut tobacco and pipe tobacco and thus differentiates taxation rate. A new method for rapid measurement of the width of cut tobacco strands was developed based on digital image processing, because the method described in ISO 20193, though easy to implement in factories, proved time consuming and generated high testing costs. The essence of this method is to determine the statistic width of incisions. The straight-line segments represent the width of strands of cut tobacco, from which the determination of the width for randomly placed tobacco strands could be achieved. Five kinds of samples (‘ISO collaborative study samples 0.4 mm, 1.0 mm, 1.6 mm and 3.0 mm’ and ‘Guangdong baked 0.9 mm’) were used to study the comparability of the measurement results between the method presented in this work and the current ISO method. Results show that accuracy and repeatability are comparable. In addition, the testing efficiency of the method presented in this work appears to be higher than the current ISO method, and it is thus a promising alternative method for measuring the width of strands of cut tobacco.
Summary Conditioning is a very important procedure for tobacco and tobacco products before their corresponding chemical and physical analysis. For cigarettes, forced air flow is generally required during the conditioning procedure. A special wind tunnel was designed to investigate how the forced air flow affects the conditioning of cigarettes in a constant climate laboratory. Two types of cigarettes with blended (including flue-cured and burley tobacco strands) and pure flue-cured tobacco strands were selected as test samples. It was found that the conditioning time to achieve the equilibrium could be shortened from 23 h without forced air flow to 7 h with a forced air flow rate of 2m/s. This is mainly due to the exchange of water molecules between cigarette samples and atmosphere being accelerated by applying the forced air flow. It was concluded that a 48 h conditioning period using the described forced air flow rate setup was unnecessary to attain the equilibrium for cigarettes. [Beitr. Tabakforsch. Int. 28 (2019) 224–229]
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Summary The differences or equivalence of products depend on various sources of variability like analytical methods, manufacturing processes, agricultural practices and environmental conditions. In addition, the capacity to compare and discriminate accurately two products is impacted by the number of characteristics considered for the comparison. Previously, it has been shown that a comparison of two products can be performed using the critical difference (CD), because it takes into consideration both the variability of measurements and laboratories. However, some additional sources of variability need to be added in the comparison when products were not manufactured at the same period of time or in the same factory. Here, an extended critical difference is proposed including manufacturing process variability according to the number of samples and batches collected for each product. The general formula and specific cases corresponding to different situations (one vs two labs, short vs long periods of time, same vs different periods of time, one vs several batches) are given.
Summary Globally, smokeless tobacco (ST) includes a wide array of chemically diverse products generally used in the oral cavity. Although ST has been widely investigated, this study was undertaken to determine the levels of sugars (mono- and di-saccharides), alditols, and humectants present in major ST categories/subcategories by using high performance liquid chromatography coupled with a triple quadrupole mass spectrometer (HPLC-MS/MS). The products studied included chewing tobacco (loose leaf, plug, twist), US moist snuff, Swedish snus, creamy snuff, dry snuff, dissolvable tobacco products, and tobacco-coated toothpicks. The highest mean sugar level was detected in chewing tobacco (9.3–27.5%, w/w ), followed by dissolvable tobacco (2.1%); all other products were lower than 1%. Creamy snuff had the highest mean alditol levels (22.6%), followed by dissolvable tobacco (15.4%); all others had levels lower than 1%. The detected mean humectant levels ranged from non-detectable to 5.9%. This study demonstrates the broad chemical diversity among ST. This research may aid researchers and public health advocates investigating the exposures and risks of ST. [Beitr. Tabakforsch. Int. 28 (2019) 203–213]
Summary The objective of this work was to compare the nicotine content and yield of new very low nicotine content cigarettes (VLN™) to the top 100 cigarette brand styles in the United States. Nicotine in tobacco filler and nicotine in smoke were measured. On a dry weight basis, VLN™ cigarettes averaged 0.5 mg of nicotine/g tobacco as compared to 19.4 for the top 100 brand styles in the United States. On a cigarette basis, VLN™ cigarettes contained 0.27 mg of nicotine compared to 12 mg for the top brands. On an ISO smoke yield basis, VLN™ produced 0.03 mg nicotine/cig compared to 0.903 mg nicotine/cig for the leading brands. VLN™ cigarettes contained > 95% less nicotine on a per gram basis of tobacco or per cigarette basis than the top 100 brand styles in the U.S. VLN™ cigarettes yielded > 95% less nicotine in smoke than the top brand styles in the United States.
Summary Employment of 1-hydroxy-acetone as a carbon source and NH4OH as a source of base and nitrogen, has enabled arrays of pyrazines to be synthesized. Reaction conditions such as temperature, time, carbon/nitrogen mole ratios and pH were optimized to maximize the quantity of pyrazines, thereby providing the synthesis of at least 19–20 structurally different pyrazines. Addition of amino acids, selected aldehydes, and hydrolyzed tobacco-derived F1 protein has positively impacted the array of pyrazines from both qualitative and quantitative aspects. Results further showed that by changing the carbon source from 1-hydroxy-acetone to 1-hydroxy-2-butanone and/or 2-hydroxy-3-butanone, control of the type of pyrazines being synthesized could be realized in that the qualitative and quantitative distributions of the pyrazine array were shifted to higher molecular weight derivatives. A relatively large scale reaction (1.5 L) employing optimized parameters yielded > 2 g of a diverse array of pyrazines dominated by multiple dimethylpyrazine derivatives. While systematically varying reaction conditions and reagent mole ratios can predictably alter the distribution and yield of pyrazines, the two most overwhelmingly significant factors governing these two pyrazine product characteristics included the structure of the carbon source and the presence or absence of aldehydes and free amino acids.
Summary To study the effects of tobacco rod circumference on cigarette combustion status, cigarettes were made with three different circumferences of 24 mm, 20 mm, 17 mm and otherwise identical construction. Their combustion characteristics, including combustion coal volume, characteristic temperature distribution, heating rate, instantaneous burn rate, and yields of selected mainstream smoke chemicals, were systematically measured. The results indicated that the cigarettes with the lowest circumference of 17 mm showed higher combustion temperatures with a smaller coal volume. The maximum instantaneous burn rate was distinctly different for the three cigarettes, from 1.84 mm/s to 2.48 mm/s, when their circumference was reduced from 24 mm to 17 mm. The tobacco mass consumption per puff showed a negative trend when the circumference decreased. The majority of the chemical compounds (16 of 21) measured in mainstream smoke decreased when the circumference was reduced, except for formaldehyde, while the yields of the chemical compounds produced per weight of cut tobacco, consumed during puffing, showed an obverse trend.
Summary A screening method allowing the quantification of 24 aerosol constituents using gas chromatography-mass spectrometry has been developed to assess the aerosol chemistry of heat-not-burn tobacco products. The aim of this method was to quantify phenol, o-cresol, m-cresol, p-cresol, catechol, resorcinol, hydroquinone, 1,3-butadiene, isoprene, benzene, acrylonitrile, toluene, pyridine, styrene, 1,2-propylene glycol, menthol, 2-furanmethanol, acrylamide, naphthalene, nicotine, acetamide, quinoline, triacetin, and glycerine in the aerosol emitted by heated tobacco products. The aerosol was generated by an electrically heated tobacco system (PMI’s Heated Tobacco System (THS 2.4)) with one single aerosol collection method, using the Health Canada smoking regimen and analyzed with two analytical methods. The method was validated according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use and the Association of Official Analytical Chemists guidelines. A regression model based on a linear relationship between concentration and response ratio with a 1/x weighting factor was selected for phenol, o-cresol, m-cresol, p-cresol, 1,3-butadiene, isoprene, benzene, acrylonitrile, toluene, pyridine, styrene, 2-furanmethanol, acrylamide, naphthalene and acetamide. A quadratic regression model with a 1/x weighting factor was chosen for catechol, resorcinol, hydroquinone, 1,2-propylene glycol, menthol, nicotine, quinoline, triacetin and glycerine. Coefficients of variation for repeatability were determined between 7.9% and 17.8% and for intermediate precision between 8.1% and 19.9%. The matrix effect of the heated tobacco aerosol extract was assessed by performing a recovery study, where the aerosol extracts were spiked at different concentrations for the compounds to be analyzed. In addition, the mainstream smoke from 3R4F reference cigarettes was analyzed, and results were compared with previously published studies. The method was successfully validated, providing data consistent with published data and it was shown to be selective, precise and accurate.
Summary Glucose and selected phosphate buffers have been reacted employing systematic variations in reaction temperature and time (150–160 °C for 60–90 min) to optimize the yield of acetol. This mixture was reacted further with NH4OH, systematically varying reaction conditions and reagent ratios to optimize pyrazine yield. The highest yield of pyrazine was obtained when 1 g of glucose was reacted with 25 mL of buffer at 150–160 °C for 60 min, which in turn was reacted with 1 mL of concentrated aqueous NH4OH at 120–130 °C for 17–18 h. Higher temperatures and higher concentrations of glucose caused a decrease in the yield of pyrazines. The addition of hydrolyzed tobacco-derived F1 protein as a secondary source of nitrogen increased the yield of pyrazines by 2–10% depending on F1 protein concentration. Furthermore, the addition of any α-hydroxyketone, similar in structure to acetol, as a pure reagent to the reaction mixture not only increased the yields of pyrazine by ranging from 25–100 % depending on the reagent concentration, but also significantly altered the qualitative and quantitative distribution of the pyrazines. With all of the reaction parameters examined (reaction time, temperature, reagent ratios, etc.) the most significant impacts on both pyrazine yield and distribution were noted when: 1) glucose was pre-reacted with buffer, 2) hydrolyzed F1 protein was added as a second nitrogen source, and 3) when pure α-hydroxyketones were employed as co-reagents. Use of these reaction parameters was found to dramatically shift the pyrazine distribution toward higher molecular weight resulting in a pyrazine array having more desirable physical and sensory attributes.
Summary Although 2-nitropropane is a potentially harmful compound present in cigarette smoke, there are few fully-validated, modern methods to quantitate it in mainstream cigarette smoke. We developed an isotope dilution gas chromatography-tandem mass spectrometry (ID-GC-MS/MS) method for the detection of 2-nitropropane in mainstream cigarette smoke. The vapor fraction of mainstream cigarette smoke was collected in inert polyvinyl fluoride gas sampling bags and extracted with hexanes containing isotopically labeled internal standard, then purified and concentrated via solid-phase extraction using a normal phase silica adsorbent and a 100% dichloromethane eluant. This method is sensitive enough to measure vapor phase 2-nitro-propane concentrations in the nanogram range, with a 19 ng per cigarette method limit of detection. Product variability estimated from the analysis of 15 cigarette products yielded relative standard deviations ranging from 5.4% to 15.7%, and estimates of precision from two quality control products yielded relative standard deviations of 9.49% and 14.9%. Under the Health Canada Intense smoking regimen, 2-nitropropane in machine-generated mainstream smoke from 15 cigarette products ranged from 98.3 to 363 ng per cigarette.
Summary A previously established method based on headspace solidphase microextraction (HS-SPME) and comprehensive two-dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry (TOFMS) has been used to evaluate and compare the profiles of semi-volatile compounds present in mainstream tobacco smoke particulate matter trapped on glass fibre filters for two types of cigarettes differing only in filter design. In the first cigarette, the filter cavity contained approximately 60 mg of a weakly basic macroporous polystyrene resin cross-linked with divinyl benzene and with surface amine functionality (CR20), whereas in the second cigarette, it was empty. Relative quantitative analysis, chemical identification, and chemical grouping allowed the use of both parametric and non-parametric analyses to identify differences in the chemical composition of the smokes from these cigarettes. The analysis demonstrated that in addition to the selective partial removal of volatile carbonyls and HCN demonstrated previously, CR20 selectively, but incompletely removed 316 compounds from the particulate phase of cigarette smoke, mainly aryl and aromatic hydrocarbons as well as other more volatile species. In contrast, the relative proportion of amines, hydroxylated aromatic compounds and less volatile species was increased in the smoke from the cigarette containing CR20 in the filter. Our findings show that high resolution GC techniques combined with mass spectrometry and chemometric approaches are powerful tools for deconvoluting the complexity of combustion aerosols, as well as helping to identify changes in chemical composition resulting from modifications to cigarette designs. [Beitr. Tabakforsch. Int. 28 (2019) 231–249]
Summary Free amino acids have been isolated via optimized enzymatic hydrolysis of F1 tobacco protein using two cationic resins (Amberlite IR120 and Dowex MAC-2). Optimized enzymatic conversions of the protein as a result of systematic variations in conditions (e.g., time, temperature, pH, enzyme type, enzyme concentration, anaerobic/aerobic environments, and protein concentration) employing commercially available enzymes, were consistently higher than 50% with qualitative amino acid arrays that were consistent with the known composition of tobacco F1 protein. Amberlite IR120 was shown to have a much higher efficiency and capacity for isolation of amino acids from standard solutions and from hydrolysate when compared with the results using Dowex MAC-2. Two columns packed with conditioned Amberlite IR120 (120 × 10 mm,12–15 g resin) and (200 × 25.4 mm, 60–65 g resin) were used to isolate two batches (2.5–3.0 mg and 13–15 mg) of free amino acids, respectively. A relatively inexpensive analytical methodology was developed for rapid analysis of the free amino acids contained within the enzyme hydrolysate. Commercially available enzymes, when employed in optimized reaction conditions, are very effective for enzymatic conversion of tobacco F1 protein to free amino acids.
Summary Safety and quality standards for electronic cigarettes (e-cigarettes) have been introduced regionally. In 2016, the U.S. Food and Drug Administration (FDA) issued a rule to regulate e-cigarettes, requiring to report harmful and potentially harmful constituents (HPHCs). In the United Kingdom, the British Standards Institution (BSI) specified the metals to be monitored for e-cigarettes. In this study, a method was developed and validated for the simultaneous determination of 13 metals (Be, Al, Cr, Fe, Co, Ni, Cu, As, Se, Ag, Cd, Sn and Pb) in e-cigarette aerosol. Furthermore, matrix effects of major constituents in the aerosol were investigated using glycerol or 1,2-propylene glycol solutions. E-cigarette aerosol was generated by a rotary smoking machine according to CORESTA Recommended Method N° 81 and collected by an electrostatic precipitator coupled to an impinger containing nitric acid. The collected aerosol was dissolved in nitric acid and an aliquot of this solution was analyzed by inductively coupled plasma mass spectrometry (ICP-MS) equipped with a collision/reaction cell. The linearity of the calibration curve was observed in the range of 0.2 to 100 ng/mL for each analyte; the correlation coefficients were 0.998 or larger, the mean recovery of each standard level ranged from 92.6 to 104.5% and the relative standard deviation amounted to max. 9.5%. Accuracy, repeatability and specificity were validated by spiking three different amounts of analytes into e-cigarette aerosol; the mean recovery of each spiking level ranged from 88.7 to 110.3% with a relative standard deviation amounting to max. 9.2% for all analytes. Background contamination from aerosol generation and collection system existed for some analytes, especially for Al, Fe, Cu and Sn. The potential sources of contamination should be identified and controlled to reduce the impact of contamination on quantification. In addition, the actual values for samples should be reported with method blank statistics. Increase of the concentrations of glycerol and 1,2-propylene glycol in the prepared sample led to the overestimation of As and Se. The amount of polyols in the collected aerosol should be monitored and controlled for the accurate quantification of As and Se.
Summary The present study describes the analysis of several organic acids in tobacco and smokeless tobacco products using a liquid chromatography (LC) method with mass spectrometric (MS) detection (LC-MS). Prior to the application of the LC-MS method, a qualitative analysis for the identification of the organic acids in tobacco and oral tobacco products was performed. The qualitative method used direct silylation of the plant material followed by GC-MS separation and detection. For the acids’ quantitation, a novel LC-MS method has been developed and validated. The acids of interest for quantitation were the following: acetic, citric, fumaric, glyceric, lactic, maleic, malic, oxalic, pyroglutamic, pyruvic, quinic, and trihydroxybutanoic. The LC separation was performed on a Synergy 4u Hydro-RP column 250 × 4.6 mm, with an aqueous mobile phase containing 5% methanol and 0.15% formic acid. The LCMS method has the advantage versus LC methods with other detection types (refractive index, UV absorption at low UV range, or conductivity) of being capable of positive identification of the analytes based on their specific ions, and of having significantly better sensitivity. Unfortunately, the LC-MS method was not generating good results for oxalic acid and acetic acid also expected to be present in some samples of tobacco or tobacco products. The study describes the advantages and disadvantages of the LC-MS method for the analysis of organic acids in tobacco and smokeless tobacco products.
Summary This research analysed in detail the performance of the new alternative ignition propensity test prescribed in the standard ASTM E2187-16, which is based on the utilization of a substrate comprising a thin steel plate along with one filter paper. The analysis was performed both experimentally, by means of infrared temperature measurements, and theoretically by using a comprehensive finite element model that was able to predict the temperature of the substrate with errors of only 7.3% and 15.7% in space and time, respectively. While the new alternative test was able to reduce the variability of the heat absorbance from 33% to only 4% with respect to the conventional tests, it showed several downsides that critically reduce its reliability. The heat absorbance of the alternative test did not correctly emulate the conventional procedure as it absorbed as much heat as twice. The gravity effect on the plate increased the air gap thickness more than twice, thereby decreasing potentially the heat absorbance by 13%. In addition, a mechanical analysis showed that compressive stresses due to high temperature gradients could cause irreversible buckling, creep and yielding of the plate. Experiments showed that in fact the concavity of the plate was prone to increase after testing. Assuming the maximum concavity allowed by the standards, the heat absorbance was halved in respect to a perfectly flat plate. In view of these results, the utilization of the conventional test method still appears clearly more appropriate than the alternative one.
Summary An array of pyrazines have been synthesized using sugars derived from tobacco cellulose (CDS), ammonium hydroxide, and hydrolyzed tobacco F1 protein as a source of free amino acids (isolated amino acids from F1 hydrolysate, from filtered F1 hydrolysate and from non-filtered F1 hydrolysate). All reactions were performed at 120 °C for 60 min using a 40-mL Parr reaction vessel. Results showed that the addition of hydrolyzed F1 protein as free amino acid source increased the number of pyrazines with branched alkyl chains (for example, 2-butyl-3-methyl pyrazine) compared to when no amino acids were added. However, using isolated amino acids from hydrolyzed F1 protein versus just hydrolyzed F1 protein (filtered or not filtered) did not make a difference in yield or type of branched pyrazines. When non-filtered hydrolyzed F1 protein was used, the solution was much more viscous and contained suspended solid material when compared to the use of filtered hydrolyzed F1 protein. Addition of threonine (THR) to the reaction mixture did not increase the yield of pyrazines but did slightly shift the distribution of pyrazines toward those with three and four carbons attached. Similar but not identical arrays of pyrazines were obtained when somewhat resembling reaction conditions were applied on a larger reaction scale (~1.5 L). A significant 50%-decrease in pyrazine yield was observed when the reaction temperature was reduced from 120 to 100 °C. No noticeable difference in the array of pyrazines from these two reactions was observed. In the majority of cases, the presence of free amino acids resulted in an increase in pyrazine yield coupled with a change in the qualitative array of pyrazines. These results clearly illustrate that sugar prepared from tobacco cellulose (glucose) can be used just like high fructose corn syrup to prepare flavor compounds via Amadori and Maillard reactions. The evidence highlights that hydrolyzed amino acids from F1 tobacco protein can be used via Maillard reactions to produce complementary arrays of pyrazine flavor compounds.
Summary Although smoking is responsible for a huge variety of diseases which result in ~16% of the fatalities in the United States and Europe respectively, cigarettes are still being sold far and wide. Mentholated cigarettes were introduced in 1920, since then to today social recognition and the use of flavored tobacco products is still increasing especially within young people. The EU adopted as its measure to reduce tobacco use among adolescents the prohibition of tobacco products with a characteristic flavor by means of the directive 2014/40/EU of the European Parliament and the Council. For this reason, we developed a method for the simultaneous determination of 14 tobacco flavors like menthol, menthol-like and other compounds via gas-chromatography coupled with mass-spectrometry (GC/MS) and analyzed 21 different tobacco products (mentholated and non-mentholated cigarettes, as well as electrically heated tobacco products (EHTPs)) of the German market regarding their flavoring compound patterns. The highest amounts of flavoring compounds were determined in menthol cigarettes (~10,000 μg/stick) whereas non-mentholated cigarettes and EHTPs featured only ~10 μg/stick. In total, seven flavoring compounds like menthol, L-menthone, L-linalool, isopulegol, geraniol, camphor and WS-3 (cooling agent) were available within the samples. Mentholated cigarettes could be clearly identified since > 99% of the measured flavoring compounds was represented by menthol. Although flavoring compounds in non-mentholated cigarettes and EHTPs were quite comparable, they could be differentiated due to different flavoring compound patterns. Brandspecific flavoring compound patterns were not recognized.
