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Alternations in gene methylation and other epigenetic changes regulate normal development as well as drive disease progression. The aim of this study is to investigate global methylation changes in the buccal cells of smokers and smokeless tobacco users.Generally healthy adult male subjects were recruited into smoker (SMK), moist snuff consumer (MSC) and non-tobacco consumer (NTC) cohorts (40 subjects/cohort) (ClinicalTrials.gov Identifier: NCT01923402). Global methylation profiling was performed on Illumina 450 K methylation arrays using buccal cell DNAs.The SMK cohort exhibited larger qualitative and quantitative changes relative to MSC. Approximately half of the differentially methylated 1252 gene loci were grouped as combustible tobacco-related (CTR) signatures and a third of the changes, tobacco-related (TR) signatures, were associated with smoking. Very few (41) differentially methylated gene loci were exclusively associated with moist snuff use and were designated as moist snuff-related (MSR) signature. Pathway enrichment analyses revealed that developmental and immune response pathways, among others, were impacted due to tobacco use.Chronic cigarette smoking causes hyper- and hypo-methylation of genes that could contribute to smoking-related diseases. These results help place combustible and non-combustible tobacco products along a risk continuum and provide additional insights into the effects of tobacco consumption.

Une série d'études cliniques ambulatoires furent menées en vue d'évaluer les variations des biomarqueurs d'exposition au tabac et de l'appréciation subjective des produits lorsque des fumeurs adultes passent à une consommation combinée composée de cigarettes associées à des bandelettes Camel (bandelettes), des bâtonnets Camel (bâtonnets) ou du tabac à sucer Camel (tabac à sucer). Dans toutes les études, les sujets furent invités à fumer ad libitum durant une semaine (période de référence). Ensuite, ils reçurent pour instruction de réduire progressivement, durant les trois semaines suivantes, leur consommation journalière de cigarettes (CJC) d'au-moins 75% et d'inclure la consommation d'un type de produit de tabac sans fumée (PTSF). Les données relatives à la consommation des produits furent consignées chaque jour par les sujets, des questionnaires furent remplis, les filtres des cigarettes fumées ainsi que les pochettes usagées de tabac à sucer furent récoltés chaque semaine et des échantillons d'urine furent prélevés toutes les 24 heures durant la période de référence ainsi qu'à la fin de la période de consommation combinée en vue de mesurer les biomarqueurs d'exposition au tabac. Au total, 100 sujets furent inclus et 88 menèrent les études à terme. A la fin de la période de consommation combinée, des réductions moyennes de la CJC avoisinant 60% furent observées dans toutes les études. Les valeurs médianes des biomarqueurs que sont la cétone de nitrosamine dérivée de la nicotine (4- (méthylnitrosamino)-1-(3-pyridyl)-1-butanone) et l'acroléine ne présentèrent soit aucun changement statistiquement significatif soit des diminutions statistiquement significatives, toutes études confondues. Les biomarqueurs de la nicotine dans le sang et dans l'urine ne présentèrent aucune modification significative. Vingt-cinq biomarqueurs furent exclusivement mesurés lors de l'étude portant sur le tabac à sucer. Parmi ces 25 biomarqueurs, 18 affichèrent une diminution statistiquement significative de 12,4-35,7%. Aucune augmentation statistiquement significative des valeurs des biomarqueurs ne fut observée, quelle que soit l'étude. Les diminutions de certains biomarqueurs confirmèrent certes que des réductions du tabagisme se produisaient bel et bien mais qu'elles s'avéraient inférieures aux réductions déclarées de CJC. Les estimations d'exposition des tissus buccaux laissent à penser que les sujets ne modifièrent pas significativement leur comportement d'aspiration des bouffées pour compenser le recul de la CJC. Les valeurs d'acceptabilité des cigarettes chutèrent de façon significative durant la période de consommation combinée tandis que l'appréciation des bandelettes et du tabac à sucer s'améliora, laissant à penser que la modification du comportement de consommation avait altéré les perceptions des produits chez les sujets. Les résultats de ces études suggèrent que les fumeurs qui basculent vers une consommation combinée associant la cigarette soit à du tabac soluble soit à du tabac à sucer n'augmenteront, selon toute attente, pas leur exposition aux constituants de tabac et sont susceptibles de réduire leur exposition à certaines substances toxiques du tabac, tout particulièrement aux substances associées à la combustion

Cardiovascular disease (CVD) biomarkers of biological effect (BoBE), including hematologic biomarkers, serum lipid-related biomarkers, other serum BoBE, and one physiological biomarker, were evaluated in adult cigarette smokers (SMK), smokeless tobacco consumers (STC), and non-consumers of tobacco (NTC). Data from adult males and females in the US National Health and Nutrition Examination Survey and a single site, cross-sectional study of healthy US males were analyzed and compared. Within normal clinical reference ranges, statistically significant differences were observed consistently for fibrinogen, C-reactive protein (CRP), hematocrit, mean cell volume, mean cell hemoglobin, hemoglobin, white blood cells, monocytes, lymphocytes, and neutrophils in comparisons between SMK and NTC; for CRP, white blood cells, monocytes, and lymphocytes in comparisons between SMK and STC; and for folate in comparisons with STC and NTC. Results provide evidence for differences in CVD BoBE associated with the use of different tobacco products, and provide evidence of a risk continuum among tobacco products and support for the concept of tobacco harm reduction.

An age-stratified, cross-sectional study was conducted in the US among healthy adult male cigarette smokers, moist snuff consumers, and non-tobacco consumers to evaluate cardiovascular biomarkers of biological effect (BoBE). Physiological assessments included flow-mediated dilation, ankle-brachial index, carotid intima-media thickness and expired carbon monoxide. Approximately one-half of the measured serum BoBE showed statistically significant differences; IL-12(p70), sICAM-1 and IL-8 were the BoBE that best differentiated among the three groups. A significant difference in ABI was observed between the cigarette smokers and non-tobacco consumer groups. Significant group and age effect differences in select biomarkers were identified.

INTRODUCTION The objectives of this trial were to investigate short-term changes in product usage, tobacco-related biomarkers of exposure, and subjective effects in smokers who switched to dissolvable tobacco (Camel Orbs) use. METHODS Participants were randomized into 1 of 4 groups (continued smoking, switched to consuming Orbs, switched to dual use of cigarettes and Orbs, and tobacco abstinent) and confined for 6 days with dietary restrictions. Most measurements were at baseline and days 1, 3, and 5 of intervention. Mouth-level tar and nicotine exposures were estimated by filter tip analysis. Twenty biomarkers were quantified in 24-hr urine; 4 were quantified in blood/plasma (carboxyhemoglobin, nicotine, cotinine, and thiocyanate). Ratings for nicotine dependence and withdrawal symptoms were scored. RESULTS After 5 days, substantial and statistically significant reductions (~30%-90%) in all biomarkers were observed in the Orbs and abstinent groups compared to baseline. Numerous smaller reductions (~7%-30%) were also noted in the continued smoking and dual-use groups (generally similar in magnitude for both groups). Subjective questionnaire findings indicated greater withdrawal discomfort levels throughout the intervention period for the nonsmoking groups. For subjects that continued smoking, clinical confinement conditions did not significantly alter product use behavior and toxicant exposure profile compared to baseline. CONCLUSIONS Substantial reductions in toxicant exposure occurred for participants that did not smoke. Cigarette smokers that switched to Orbs use showed reductions in all biomarkers, similar to abstinent group. Changes in toxicant exposure for the dual-use group were similar to the continued-smoking group, consistent with minimal changes observed in that group's product use behavior (small reduction in cigarettes per day and small increase in Orbs use).

Among the more than 5000 chemicals reported in cigarette smoke condensate (CSC), heterocyclic aromatic amines (HAAs) are considered to be a contributor to observed biological activity. HAAs are non-volatile and are reported at ppb levels in CSC. A new method for HAA analysis at the trace level is reported here. N, O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane was employed to derivatize amino groups by heating the reagent containing a sample of CSC at 80 °C for 30 min followed by analysis employing gas chromatography-mass spectroscopy (GC-MS) in the selected-ion-monitoring (SIM) mode. This derivatization method afforded symmetrical peak shapes on a ZB-50 stationary phase and achieved instrumental limits of quantification (LOQ) at 10:1 S/N from -1 ng/mL for AαC to120 ng/mL for Glu-P-1. The chemical identity of each derivative was confirmed by comparison of retention time and mass spectra of standards. The latter were characterized by the following ions: M·+ or [M-1]+, [M-15]+, and m/z 73 (i.e., trimethylsilyl). CSC and its base sub-fractions were studied using the GC-MS method. Ten HAAs were screened and five were quantified in cigarette smoke condensate, while 2-5 HAAs were quantified in each of three base sub-fractions. Values obtained with the new procedure agree well with values reported in the literature and with results obtained from a commercial laboratory via a different analytical method. The potential contribution of each HAA to the overall mutagenic activity observed for CSC and its base fractions is discussed. When considered together, HAAs account for only a small portion (-7.8%) of the observed mutagenicity of the CSC.

Selected toxicant concentrations and other chemical measures have been determined for 43 U.S. smokeless tobacco products sold in 2006 and 2007. Products evaluated included moist snuff, dry snuff, loose leaf, plug, dissolvable and snus tobacco brands. Reference products available for scientific research purposes and eleven Swedish products were also evaluated and compared to the commercial products studied. Chemical endpoints determined included benzo[a]pyrene (B[a]P), N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-Nitrosodimethylamine (NDMA), nitrite, cadmium, lead, arsenic, nickel, chromium, chloride, water, pH and nicotine. Different toxicant profiles were observed for the products studied, with snus tobacco brands generally containing relatively low concentrations of B[a]P and tobacco specific nitrosamines (TSNAs) compared to other moist snuffs. Smokeless tobacco reference product toxicant profiles were similar to corresponding commercial products, with the exception of the TSNA content of the dry snuff reference material. TSNA concentrations observed for all commercial products were lower than historically reported values, likely reflecting changes in product shelf life, tobacco curing practices and, possibly, product blend formulations during the last 20-30 years. The survey results summarized provide a temporal point of comparison with future data anticipated from FDA "harmful and potentially harmful constituents in tobacco products" reporting.

To investigate whether cadmium has an independent role in diseases associated with tobacco consumption, epidemiology data were reviewed, biomonitoring data were analyzed, and probabilistic risk assessment (PRA) was performed. Results from previous epidemiology studies have indicated that there are adverse health effects potentially in common between cadmium exposure and tobacco consumption. Analysis of publically available biomonitoring data showed that blood (B-Cd) and urine (U-Cd) cadmium were higher in cigarette smokers compared with smokeless tobacco (SLT) consumers, and B-Cd and U-Cd in SLT consumers were not significantly different than in non-consumers of tobacco. Comparison with previously established biomonitoring equivalent (BE) values indicated that B-Cd and U-Cd in the majority of these cigarette smokers and SLT consumers did not exceed the blood and urine BEs. Results of the PRA showed that the mean hazard estimate was below a generally accepted regulatory threshold for SLT consumers, but not for cigarette smokers. In total, this evaluation indicated that cadmium exposures in tobacco consumers differed by product category consumed; cadmium in tobacco may not be associated with tobacco consumption related diseases; if cadmium in tobacco contributes to tobacco consumption related diseases, differences in hazard and/or risk may exist by product category.

L'ampleur de la degradation du melange de glycerol dans une cigarette en train de se consumer pour former de l'acroleine et de l'acetone a ete quantifiee par l'addition de glycerol- 13 C 3 aux trois sortes de cigarettes d'une marque leader sur le marche. Plusieurs tampons Cambridge imbibes d'une solution de 2,4-dinitrophenylhydrazine (DNPH) ont ete employes pour pieger des derives d'hydrazone de composes de carbonyle de faible poids moleculaire dans la fumee principale et la fumee laterale. La chromatographie liquide a haute performance en tandem avec la spectrometrie de masse des ions negatifs a ete employee pour isoler les derives DNPH des produits carbonyles volatiles de combustion et pour etablir leur concentration. Acroleine, acetone et propionaldehyde ont ete les principaux composes etudies. Les derives DNPH de l'acroleine- 13 C 3 et de l'acetone- 13 C 3 ont ete synthetises independamment et ils ont servi de normes externes pour la quantification absolue. Le cout du propionaldehyde entierement marque exclut son utilisation dans cette etude. Les sortes de cigarettes de marque selectionnees pour l'etude presentent les caracteristiques de conception des cigarettes actuellement les plus courantes sur le marche americain et offrent une gamme de taux de «goudron » standardisee representative (14, 10 et 5 mg/cig respectivement selon la methode de filtre Cambridge). Les sortes de cigarettes de la marque etudiees ici font partie d'une famille de cigarettes de marque commercialisees qui ne contiennent pas d'additifs au melange de tabac, y compris le glycerol. La fumee principale a ete generee par une machine a fumer automatique utilisant un regime de fumage standard pour le filtre Cambridge et un regime plus intense exigeant des bouffees plus grandes et plus frequentes et obstruction des orifices a 100%, ce qui est specifie par le gouvernement federal canadien a des fins de regulation. La recherche a indique que seule une faible fraction du glycerol ajoute (∼0,25% a 0,30%, p/p) a ete convertie dans les deux composes etudies, la majeur partie ayant generalement ete observee dans la fumee laterale. Moins de 0,1 % du glycerol ajoute a ete converti en acroleine dans la fumee principale pour toutes les conceptions de cigarettes et les regimes de tumage etudies.

A new direct method using liquid chromatography-tandem mass spectrometry has been developed and validated for quantitation of 3-hydroxypropylmercapturic acid (3-HPMA) in urine. The method is fast, simple, and does not require extraction from urine. Analyte was separated on a hydrophilic interaction liquid chromatography column. Severe ion suppression was circumvented by a fast gradient after separation. Assay specificity, linearity, precision, and accuracy met the required FDA/CDER bioanalytical method criteria. Matrix effect and carryover of the assay were assessed. Urine sample storage stability and standard solution stability were also tested. The limit of quantitation was 22.0 ng/mL. The results for 3-HPMA obtained by our method were significantly correlated with results obtained by a contract lab.

Objective Determine human smoked (HS) cigarette yields of tar and nicotine for smokers using their own brand in their everyday environment. Method A robust, filter analysis method was used to estimate the tar and nicotine yields for 784 subjects. Seventeen brands were chosen to represent a wide range of styles: 85 and 100 mm lengths; menthol and non-menthol; 17, 23, and 25 mm circumference; with tar yields [Federal Trade Commission (FTC) method] ranging from 1 to 18 mg. Tar bands chosen corresponded to yields of 1–3 mg, 4–6 mg, 7–12 mg, and 13+ mg. Results A significant difference (p

This report evaluates the formation of nitrosonornicotine (NNN) and of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) from nicotine, and of NNN from nornicotine in the mainstream smoke of a burning cigarette. The cigarettes analyzed in the study were Kentucky reference cigarettes 1R4F and 2R4F, and five other cigarettes, three of them having tobaccos with low levels of tobacco specific nitrosamines (TSNAs). All cigarettes had ‘tar’ levels around 10 mg [where ‘tar’ is defined as the weight of total wet particulate matter (TPM) minus the weight of nicotine and water]. Cigarettes were smoked according to the U.S. Federal Trade Commission (FTC) puffing regimen, using a 35 mL puff volume, 2 sec puff duration and 60 sec puff intervals. Three separate experiments were performed in this study to evaluate the proportion of TSNAs transferred from preexistent tobacco TSNAs and the proportion formed during smoking (pyrosynthesized). In one experiment, the results were obtained by GC/MS analysis of 13C3-TSNAs formed in smoke when 13C3-nicotine was intentionally added to cigarettes. Another experiment used GC analysis with chemiluminescence detection of TSNAs from smoke before and after an excess of nornicotine was intentionally added to cigarettes, and another experiment consisted of LC/MS/MS analysis of 2H4-TSNAs formed in the smoke when 2H4-nicotine and when 2H4-nornicotine were intentionally added to cigarettes. The use of different analytical methods for the study of TSNA formation conveyed an additional level of confidence regarding the reliability of the results obtained. It was found that NNK was generated during smoking from nicotine with 3 × 10-5% to 8 × 10-5% conversion (0.3 ppm to 0.8 ppm conversion of the nicotine) while the result for NNN generation was not conclusive. One experiment demonstrated the formation of NNN from nicotine between 4 × 10-5% and 1.5 × 10-4% (0.4 ppm to 1.5 ppm reported to nicotine), but another experiment did not provide proof of NNN formation, with a limit of quantitation LOQ for NNN corresponding to 5 × 10-5% (or 0.5 ppm). Nornicotine was proven to generate NNN, and the results for the 2R4F cigarette showed 3.3 × 10-3% yield (33 ppm reported to nornicotine) in one experiment and 4.6 × 10-3% (46 ppm reported to nornicotine) in a different experiment, the agreement being very good. Using the results from this study, it was concluded that pyrosynthesis may account for about 5% to 10% of the NNK in mainstream smoke for a filter cigarette with the FTC ‘tar’ level around 10 mg. Pyrosynthesis may account for higher proportions of smoke TSNAs when the cigarette tobacco is low in TSNAs, since the mainstream smoke TSNAs yield from direct transfer from tobacco is small in this case. The contribution of pyrosynthesis may account for 5% to 25% of NNN in mainstream cigarette smoke, or potentially an even higher proportion when the tobacco blend is both low in TSNAs and high in nornicotine. Anabasine is typically present at low levels in tobacco and therefore the formation of nitrosoanabasine(NAB) is of less interest. Anatabine is present in different tobaccos in a range similar to that of nornicotine and being a secondary amine has the potential to act similarly to nornicotine. However, the pyrosynthesis of nitrosoanatabine (NAT) from anatabine was not evaluated in the present study. The study indicated that complete elimination of TSNAs from tobacco is unlikely to completely eliminate the TSNAs from cigarette smoke, and that high nornicotine tobaccos should be avoided in order to minimize the levels of NNN in cigarette smoke.

A tiered testing strategy has been employed to evaluate the potential of tobacco processes, ingredients, or technological developments to change the biological activity resulting from burning cigarette tobacco. The strategy is based on comparative chemical and biological testing. The introduction of banded cigarette papers in cigarettes to meet New York state "Fire Safety Standards for Cigarettes" constitutes an example of a technological development evaluated utilizing this tiered testing strategy that included a comparison of the chemical and biological effects of cigarettes with and without the banded cigarette paper technologies (BCPT) (representative of current marketed technologies). Specific testing included mainstream cigarette smoke chemistry studies; in vitro studies included genotoxicity (Ames and sister chromatid exchange) and cytotoxicity studies (neutral red); in vivo studies included a 13-week inhalation study in Sprague-Dawley rats and a 30-week dermal tumor promotion study in SENCAR mice. Collectively, data indicated that cigarettes with and without BCPT had a similar toxicological profile in this test battery.

A study was conducted to evaluate biomarkers of biological effect and physiological assessments related to cardiovascular disease (CVD) among adult male cigarette smokers (SMK), moist snuff consumers (MSC) and non-consumers of tobacco (NTC). Additionally, biomarkers of tobacco and tobacco smoke exposure (BoE) were measured in spot urines and are reported here. Except for the BoE to nicotine and NNK, BoE were generally greater in SMK compared with MSC, and BoE were generally not different in comparisons of MSC and NTC. Results demonstrated that MSC had lower systemic exposures to many harmful and potentially harmful constituents than SMK, which is consistent with epidemiological data that indicate a differential in CVD risk between these groups.

Actuellement plusieurs pays prescrivent une reglementation des tests sur cigarettes, exigeant des conditions de fumage plus intense que celles definies dans les conditions normalisees de la Federal Trade Commission (FTC) et l'Organisation internationale de normalisation (ISO), combinees a l'obstruction intentionnelle des orifices de ventilation des cigarettes au cours des tests. Recemment une recommandation initiale a ete lancee sous les auspices de la Convention-cadre sur le controle du tabac, exigeant a l'echelle internationale des tests sur cigarettes sous des conditions normalisee de 55 cc/20 s/2 s apres l'obstruction des orifices a 100%. Les modifications des rendements de la fumee en fonction des parametres de fumage par machine sont parfaitement connues, cependant les connaissances sur les changements potentiels de la composition de la fumee sont plus restreintes. Dans la presente etude, l'influence des conditions de fumage sur le devenir de la nicotine dans une cigarette en combustion a ete etudiee par chromatographie en phase gazeuse avec detection par emission atomique (GC-AED) en utilisant de la nicotine-d 4 injectee a coeur. Quatre conditions differentes de fumage et deux conditions differentes d'obstruction des orifices ont ete examinees. La GC avec detection par spectrometrie de masse a ete utilise pour identifier les composants marques au deuterium, produisant une reaction AED accrue. Une comparaison de la distribution dans la fumee du courant principal des composants marques au deuterium, le courant secondaire et les reliquats (megots et cendres) d'une cigarette plein arome fumee sous les quatre conditions de fumage etudiees, indique qu'un pourcentage plus important de la nicotine marquee reste intact au cours du fumage en fonction d'une intensification du regime de fumage. L'intensification du regime de fumage mene a une diminution des produits de pyrolyse et d'oxydation de la nicotine dans la fumee du courant secondaire. Cependant une augmentation marginale de ces composants est observee dans la fumee du courant principal et dans le megot. Le rapport entre courant secondaire et courant principal pour la nicotine diminue significativement en fonction d'une intensification du regime de fumage. Des petites quantites de nicotine intacte ont ete observees dans la phase gazeuse du courant secondaire, mais la nicotine n'a pas ete trouvee dans les echantillons de la phase gazeuse du courant principal.

A tiered testing strategy has been developed to evaluate the potential for new ingredients, tobacco processes, and technological developments to alter the biological activity that results from burning tobacco. A series of studies was initially conducted with cigarettes containing 3% high fructose corn syrup (HFCS) as an alternate tobacco casing material to corn syrup/invert sugar, including determination of selected mainstream cigarette smoke (MS) constituent yields, Ames assay, sister chromatid exchange (SCE) assay in Chinese hamster ovary (CHO) cells, a 30-week dermal tumor-promotion evaluation of cigarette smoke condensate (CSC) in SENCAR mice, and a 13-week subchronic inhalation study of MS in Sprague-Dawley rats. A second series of studies was conducted with cigarettes containing 3%, 4% and 5% HFCS including MS chemistry, Ames assay, SCE assay in CHO cells, and a neutral red cytotoxicity assays. Collectively, mainstream smoke chemistry, genotoxicity, dermal tumor-promotion, and inhalation toxicity studies demonstrated no differences between cigarettes with 3% HFCS and cigarettes with 3% corn syrup/invert sugar. Also, mainstream smoke chemistry and genotoxicity of cigarettes with 4% and 5% HFCS were not different from cigarettes with 3% HFCS. In conclusion, the addition of up to 5% HFCS to cigarette does not alter the mainstream smoke chemistry or biological activity of mainstream smoke or mainstream smoke condensate as compared to cigarettes with 3% corn syrup/invert sugar with regard to the parameters investigated and presented.

Mainstream cigarette smoke is a complex mixture that is inhaled into the respiratory system. The physical characteristics and chemical composition of mainstream smoke are reviewed and briefly compared with that of sidestream smoke. Special attention is paid to ageing effects and artifact formation during the sampling and testing of cigarette smoke, with specific examples of artifact formation during sampling discussed (nitrogen dioxide, methyl nitrite, etc.). Historically, the generation of cigarette smoke for chemical and biological testing has been based on standard smoke generation procedures that are intended for product comparisons. More recently, emerging global regulations have called for alternative smoke generation methods, with emphasis on results relevant to conditions of product use, e.g., estimates of maximum smoke emissions. Strategies for establishing such alternative smoke generation methods are discussed and the potential effects of alternative smoking conditions on analytical accuracy and precision are addressed. Current regulatory requirements that include Hoffmann analyte analysis (i.e., constituents reported to be associated with the risks of cigarette smoking) are also summarized and the potential effect of alternative smoke generation methods on individual constituent yields considered. Finally, a limited critique of emerging regulation that relates to mainstream cigarette smoke measurements, including a discussion of recent WHO recommendations, is offered.

A tiered testing strategy has been developed to evaluate the potential of tobacco processes, ingredients, or technological developments to change the biological activity resulting from burning tobacco. The strategy is based on comparative chemical and biological testing. Expanded shredded tobacco stems (ESS) constitute an example of a common tobacco components expansion process currently used in the manufacture of cigarettes to increase the tobacco blend filling capacity. As part of the toxicological evaluation of ESS, test cigarettes containing 9.5%, 18.5%, and 25% ESS were compared to control cigarettes containing 0% ESS. Testing included mainstream cigarette smoke chemistry studies, genotoxicity studies (Ames and sister chromatid exchange), a 13-week inhalation study in Sprague-Dawley rats, and a 30-week dermal tumor promotion study in SENCAR mice. Collectively, data indicated that cigarettes with and without ESS had a similar toxicological profile in this test battery.

A tiered testing strategy has been developed to evaluate the potential for new ingredients, tobacco processes, and technological developments to increase or reduce the biological activity that results from burning tobacco. In the manufacture of cigarettes, honey is used as a casing ingredient to impart both aroma and taste. The primary objective of this document is to summarize and interpret chemical and toxicological studies that have been conducted to evaluate the potential impact of honey on the biological activity of either mainstream cigarette smoke or cigarette smoke condensate. As part of ongoing stewardship efforts, cigarettes produced with honey (5% wet weight) as an alternative to invert sugar in tobacco casing material were subjected to extensive evaluation. Principal components of this evaluation were a determination of selected mainstream smoke constituent yields, Ames assay, sister chromatid exchange assay in Chinese hamster ovary cells, a 30-wk dermal tumor promotion evaluation of cigarette smoke condensate in SENCAR mice, and a 13-wk inhalation study of cigarette smoke in Sprague-Dawley rats. Comparative analytical evaluations demonstrated that the substitution of honey for invert sugar as a casing material in cigarettes had no significant impact on mainstream smoke chemistry. In addition, in vitro and in vivo studies demonstrated that cigarettes containing tobacco cased with honey had comparable biological activity to cigarettes containing invert sugar. Collectively, these data demonstrate that the use of honey as an alternative casing material in the manufacture of cigarettes does not alter the potential toxicity of cigarette smoke condensate (CSC) or cigarette smoke; therefore the use of honey as an ingredient added to cigarette tobacco is acceptable from a toxicological perspective.

The objectives of this clinical study were to evaluate changes in tobacco product use behavior and levels of selected biomarkers of exposure (BOEs) for smokers who switched to one of six conditions during clinical confinement: exclusive use of; Camel Snus, Sticks, Strips or Orbs, controlled Dual use of cigarettes and Camel Snus, or tobacco abstinence. The controlled Dual use (DU) condition mandated a 60% reduction in cigarettes smoked per day (CPD). 167 healthy U.S. male and female smokers were randomized to the six groups (n=25–30/group). Subjects smoked their usual brand of cigarette for 1day prior to switching to their designated intervention condition. Levels of thirty-two BOEs in plasma, whole blood, urine and feces were determined before and after switching. Questionnaires that scored nicotine dependence and withdrawal discomfort were also administered. After 5days, exclusive Snus, Sticks, Strips, or Orbs use averaged 6.1, 5.9, 13.5, and 8.5units/day, respectively. DU subjects smoked 7.6 CPD and used 3.2 Snus pouches/day, on average. After 5days, substantial reductions of most biomarkers, including nicotine, were observed in all groups. Toxicant exposures were similar to being tobacco abstinent after switching exclusively to Camel Snus, Sticks, Strips or Orbs. DU reductions were more modest.

An atomic emission detector (AED) for a gas chromatograph (GC) can be used to selectively detect compounds labeled with stable isotopes, such as D, (13)C, and (15)N. This capability can be used to determine precursor-fate relationships within complex matrixes, using stable isotopes rather than radiolabeled isotopes. Employing stable isotopes removes the safety consideration associated with radiolabeling studies. Two previous reports have employed GC-AED in fate studies for (13)C-labeled precursors. The goal of this study was to evaluate the utility of GC-AED for precursor-fate determinations in tobacco science. In this work, GC-AED was used to determine the fate of nicotine-d(4) in a burning cigarette. GC-MSD was then employed to identify the compounds that the AED indicated contained D. Spectral confirmation of the presence of D was performed on each chromatographic peak of interest with both AED and MSD to ensure that the identification of the products was correct. Nicotine and nicotine-d(4) standards were used to evaluate the effect of coelution of unlabeled compounds with the labeled compounds on the AED response for D. It was shown that the AED response for D at λ = 308 nm decreases with increased concentration of unlabeled compound. Detection at λ = 656 nm, however, is unaffected by the presence of the unlabeled compound. Compound-independent calibration is also possible at this wavelength. GC-AED studies with nicotine-d(4) core injected into cigarettes demonstrated that most of the nicotine (79%) is distilled, unchanged, into the mainstream (MS) and sidestream (SS) smoke. The degradation products that do occur include 3-substituted pyridines and nicotine-oxidation products. These are found only in the SS smoke.

Compound-independent calibration (CIC) is a theoretical concept in which a single compound is used to quantitate other compounds within a sample. Gas chromatography with atomic emission detection (...

A new-technology cigarette has been developed. While the new cigarette burns some tobacco, it does not use tobacco as the fuel to sustain combustion and provide heat to the cigarette. Rather, the new cigarette primarily heats tobacco thereby reducing products of smoke formation mechanisms such as tobacco combustion, tobacco pyrolysis and pyrosynthesis. The mainstream smoke composition from a cigarette based on the new design (TOB-HT) has been characterized in comparative chemical testing with two reference cigarettes using the FTC puffing regimen. Thermal properties, UV absorption characteristics, elemental composition and materials balance studies all suggest a simplified smoke aerosol. Twenty-five smoke constituents ("target compounds") identified by the scientific community as compounds that may contribute to the diseases statistically associated with smoking have also been measured. Mainstream smoke concentrations of most target compounds are significantly lower with the TOB-HT cigarette when compared with reference cigarettes in the ultra-light "tar" and light "tar" categories. Taken together, chemical analysis results suggest simplified TOB-HT smoke chemistry with marked reductions in specific chemicals reported to be biologically active.

Evaluation of the Genotoxic and Cytotoxic Potential of Mainstream Whole Smoke and Smoke Condensate from a Cigarette Containing a Novel Carbon Filter. Bombick, D. W., Bombick, B. Reed, Ayres, P. H., Putnam, K., Avalos, J., Borgerding, M. F., and Doolittle, D. J. (1997). Fundam. AppL Toxicol. 39, 11-17. A novel carbon filter has been developed which primarily reduces the amount of certain vapor phase constituents of tobacco smoke with greater efficiency than the charcoal filters of cigarettes currently in the market In vitro indicators of genotoxic and cytotoxic potential were used to compare the cigarette smoke condensate (particulate phase) or whole cigarette smoke (vapor phase and particulate phase) from cigarettes containing the novel carbon filter with smoke condensate or whole smoke from commercial or prototype cigarettes not containing the novel carbon filter. Ames bacterial mutagenicity, sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells, and neutral red cytotoxicity assays in CHO cells were utilized to assess the genotoxic and cytotoxic potential of the cigarette smoke condensates. SCE and neutral red cytotoxicity assays were utilized to assess the genotoxic and cytotoxic potential of the whole smoke. As expected, the novel carbon filter did not significantly affect the genotoxic or cytotoxic activity of the smoke condensate, although we did observe that the use of low-nitrogen tobacco reduced the mutagenicity of the condensate in Salmonella typhimurium strain TA98. However, the whole smoke from cigarettes containing the novel carbon filter demonstrated significant reductions in genotoxic and cytotoxic potential compared to cigarettes without the novel carbon filter. The toxicity of the smoke was correlated (r = 0.7662 for cytotoxicity and r — 0.7562 for SCE induction) to the aggregate mass of several vapor phase components (acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene, ammonia, NO,, HCN, benzene, isoprene, and formaldehyde) in the smoke of the cigarettes utilized in this study. In conclusion, this novel carbon filter, which significantly reduced the amount of carbonyls and other volatiles in mainstream cigarette smoke, resulted in significant reductions in the genotoxic and cytotoxic activity of the smoke as measured by

Chromatographic profiling, an application in which all peaks in a chromatogram are examined, is useful as a tool for providing comparative information regarding sample composition and chemical complexity. The utility of profiling is demonstrated in two studies that involve complex cigarette smoke matrices. In the first study, profiling reveals that both the vapor phase and particulate phase fractions of mainstream smoke from a cigarette that primarily heats tobacco are reduced in complexity in comparison with control products. Relative to a 1R4F control product, total chromatographic responses (TCRs) for the tobacco-heating cigarette are reduced by 89% in the vapor phase and 70% (93% if major components are excluded) in the particulate phase. The second study focuses on a cigarette that combines two design technologies: an experimental tobacco blend and a new carbon filter. This study shows that the new filter design is primarily responsible for reducing the complexity of the vapor phase fraction of mainstream smoke while having relatively little effect on the particulate phase fraction. Compared with three commercial products, the mainstream smoke vapor phase TCR is reduced by approximately 60%.

Arsenic is measurable in tobacco and cigarette mainstream smoke (MSS). Whether arsenic has an independent role in diseases associated with tobacco consumption is not known. Epidemiology and biomonitoring data and probabilistic risk assessment (PRA) methods were used to investigate this potential association. Analysis of data from the National Health and Nutrition Examination Survey (NHANES) showed that urine arsenic concentrations in tobacco consumers were not different or were lower than levels in non-consumers of tobacco. Additionally, urine arsenic levels from NHANES tobacco consumers were five-times or more lower than levels reported in epidemiology studies to be associated with adverse health effects. Results of PRA indicated that mean non-cancer hazard estimates and mean incremental lifetime cancer risk estimates were within accepted ranges. Taken together, these results suggest that arsenic may not be independently associated with tobacco consumption or diseases related to tobacco consumption.

Acute exposure to cigarette smoke or its components triggers diverse cellular effects, including cytotoxicity. However, available data regarding the potential cytotoxic effects of smokeless tobacco (ST) extracts lack consensus. Here, we investigated the relative biological effects of 2S3 reference ST, and whether ST elicits differential cellular/molecular responses compared to combustible tobacco product preparations (TPPs) prepared from 3R4F cigarettes. Total particulate matter (TPM) and whole smoke conditioned medium (WS-CM) were employed as combustible TPPs, while the ST extract was used as non-combustible TPP. HL60, THP1 cells and human PBMCs were used to examine the effects of TPPs in short-term cell culture. Corresponding EC(50) values, normalized for nicotine content of the TPPs, suggest that combustible TPPs induced higher cytotoxicity as follows: WS-CM TPM ≥ ≫ST extract>nicotine. While all three TPPs induced detectable levels of DNA damage and IL8 secretion, the combustible TPPs were significantly more potent than the ST preparation. The major PBMC subsets showed differential cytotoxicity to combustible TPPs as follows: CD4>CD8>monocytes>NK cells. These findings suggest that, relative cytotoxic and other cell biological effects of TPPs are dose-dependent, and that ST extract is the least cytotoxic TPP tested in this study.

Tobacco toxicant-related exposure reduction is an important tool in harm reduction. Cigarette per day reduction (CPDR) occurs as smokers migrate from smoking cigarettes to using alternative tobacco/nicotine products, or quit smoking. Few reports characterize the dose–response relationships between CPDR and effects on exposure biomarkers, especially at the low end of CPD exposure (e.g., 5 CPD). We present data on CPDR by characterizing magnitudes of biomarker reductions. We present data from a well-controlled, one-week clinical confinement study in healthy smokers who were switched from smoking 19–25 CPD to smoking 20, 10, 5 or 0 CPD. Biomarkers were measured in blood, plasma, urine, and breath, and included smoke-related toxicants, urine mutagenicity, smoked cigarette filter analyses (mouth level exposure), and vital signs. Many of the biomarkers (e.g., plasma nicotine) showed strong CPDR dose–response reductions, while others (e.g., plasma thiocyanate) showed weaker dose–response reductions. Factors that lead to lower biomarker reductions include non-CPD related contributors to the measured response (e.g., other exposure sources from environment, life style, occupation; inter-individual variability). This study confirms CPDR dose-responsive biomarkers and suggests that a one-week design is appropriate for characterizing exposure reductions when smokers switch from cigarettes to new tobacco products.

ALAIN BERTHOD* UniversitP Claude Bernard, Lyon I, Laboraroire des Sciences Anatytiques, UA CNRS 435 (J. M. Met-met). 69622 lfilleurbanne Cedes (France) MICHAEL F. BORGERDING R. J. Reynolds Tobncco Company, Bowman Gray Technical Center, Winston-Salem, NC 27102 (USA) and WILLIE L. HINZE Department of Chemi.rtry. Wake Forest University, P.O. Box 7486, Winston-Salem, NC 27109 (USA) ABSTRACT Reduced chromatographic efficiency is a major drawback of micellar liquid chromatography (MLC). The Knox equation h = AI.“’ + Bjv + Cv. was used to determine the individual contribution of the flow anisotropy (A term), molecular band broadening (B term) and mass transfer processes (C term) to the final band broadening. Knox plots of h, the reduced plate height, versus Y, the reduced linear flow-rate, were determined on the same column (i) with an aqueous-organic mobile phase, (ii) with a micellar mobile phase and (iii) with the same aqueous-organic phase. The changes in the A, B and C terms are discussed. Two stationary phases were used: a classical C,, monomer phase and a densely grafted (3.5 pmol/m2) C,, phase. Two micellar solutions were used: a non-ionic micellar solution of Brij 35 and an anionic solution of sodium dodecyl sulfate (SDS). Test solute diffusion coefficients were measured in each mobile phase used. The increase in A is mainly responsible for reduced MLC efficiency. However, the B and C terms also increased significantly with micellar solutions. It is shown that the observed changes in the Knox param- eters can be explained by the change in the stationary phase produced by surfactant adsorption; 6% of the adsorbed SDS (0.14~mol/m2) was irreversibly adsorbed on the C,, phase whose initial efficiency could not be restored. Such

Eight compounds from a Kentucky 1R4F reference cigarette smoke condensate have been determined by selected ion monitoring-mass spectrometry (SIM-MS) to confirm the validity of multidimensional gas chromatography (MDGC) as a quantitative tool in complex mixture analyses. Four electrostatically precipitated smoke condensate samples of 100 cigarettes each are dissolved individually in 25 mL of 2-propanol. The 2-propanol contains two methyl esters (C8 and C14) and seven deuterium-labeled compounds used as internal standards (IS). Analysis of the compounds of interest, pyridine; acetamide; acrylamide; phenol; o-, m-, and p-cresol; and quinoline, is accomplished by using two heartcuts. Heartcut times of the MDGC analysis are selected such that at least one IS is transferred with each group of compounds being analyzed. This study shows that the MDGC technique previously developed and described can be used for quantitative analyses. A comparison is made between the two types of internal standards. The results obtained for both types of internal standards agree within 20% of each other, on the average, with higher standard deviations for approximately 60% of the compounds where methyl esters are used as internal standards.

Cigarettes can be developed that heat rather than burn tobacco. Such products would be expected to have less "tar" and other combustion products than cigarettes that burn tobacco. With one product of this type, benzo(a)pyrene, N-nitrosamines, phenolic compounds, acetaldehyde, acrolein, hydrogen cyanide, and N-heterocyclic compounds have been reduced 10- to 100-fold compared to the Kentucky reference (1R4F) cigarette, a representative low-tar cigarette. The yields of nicotine and carbon monoxide from this new cigarette are less than the yields of 95% and 75%, respectively, of the cigarettes sold in the United States during 1988. Nicotine absorption from smoking this new cigarette is not significantly different from that of tobacco-burning cigarettes yielding equivalent levels of nicotine. The urine mutagenicity of smokers of new cigarettes is significantly less (P less than .05) than that of smokers of tobacco-burning cigarettes and is not significantly different (P greater than .10) from that of nonsmokers. We conclude that cigarettes which heat rather than burn tobacco can reduce the yield of tobacco combustion products. This simplification of smoke chemistry had no effect on nicotine absorption in smokers and resulted in a reduction of biological activity in smokers as measured by urine mutagenicity.

A method Is described for determining major constituents In the smoke of a cigarette that heats, but does not burn, tobacco. Dual, simultaneous separations are performed in a single gas chromatographic oven to determine water, glycerol, nicotine, and propylene glycol in a rapid and cost-effective manner. A materials balance of new cigarette smoke total particulate matter was attempted from both Cambridge filter and electrostatic precipitation smoke collection data. Serious deficiencies were found when Cambridge filter smoke collection was applied for this purpose. Electrostatic precipitation smoke collection eliminated these problems. The data obtained by electrostatic precipitation smoke collection Indicate that water, glycerol, nicotine, and propylene glycol make up about 94% of new cigarette smoke total particulate matter.

The design of a new cigarette that heats rather than burns tobacco calls for modifications to the Federal Trade Commission (FTC) method for analytical smoking. These changes Include eliminating sample conditioning at 75 °F and 60% RH, exercising greater care In lighting cigarettes, and smoking cigarettes to self-extinguishment rather than to a predetermined butt length as a measure of complete consumption. By several gross analytical measures, smoke condensate from the new cigarette differs substantially from that of tobacco-burning cigarettes. This Is Inferred from the lack of coloration of smoke condensate collected on Cambridge filters. Elemental analysis demonstrates reduced carbon and nitrogen content concurrent with Increased hydrogen. Thermogravlmetrlc analysis shows almost quantitative weight loss at Tmax = 220 °C. Ultraviolet (UV) spectrophotometrlc analysis shows greatly reduced levels of tobacco-derived smoke components and qualitative differences In chemical entitles being measured. By design, the heat required for smoke formation Is supplied by a carbon heat source embedded In the cigarette tip. Tobacco contained In the cigarette Is not burned and Is exposed to temperatures less than 300 °C. Thus, It Is apparent (1) that smoke from the new cigarette contains little or no "tar" as tar Is classically defined, and (2) that the FTC method even as modified to account for cigarette design differences Is appropriate only for determination of nicotine and carbon monoxide yielded from this cigarette.

A cryogenic trapping method with isotope dilution gas chromatography-mass spectrometry analysis has been developed for the determination of benzene, toluene, styrene and acrylonitrile in mainstream vapor phase cigarette smoke. The method is simple, direct, and quantitative. Vapor phase samples are collected cryogenically in a series of four traps following removal of the particulate phase with a Cambridge filter pad. For all four analytes, 75-85% of the total amounts recovered were found in the initial trap and less than 1% in the final trap. Assessment of instrumental precision by multiple injections of a sample gave relative standard deviations of less than 2%. Linear calibration for all analytes over the analysis range gave an r2 value greater than 0.99 with average relative standard deviations at the mean ranging from 1.4 to 8.2%. The cigarettes analyzed include a reference cigarette (Kentucky 1R4F), a commercial ultra-low "tar" mentholated cigarette, and two cigarettes that heat but do not burn tobacco. The values determined for the four analytes in the 1R4F samples are comparable to reported values of similar cigarettes. The cigarettes which heat rather than burn tobacco yield less of all four analytes compared to the other cigarettes in the study.

A tiered testing strategy has been employed to evaluate the potential for new ingredients, tobacco processes, and technological developments to alter the mainstream smoke or biological activity that results from burning cigarette tobacco. The foundation of this evaluation strategy is comparative testing, typically including chemical and biological assessments. In the manufacture of cigarettes, diammonium phosphate (DAP) and urea have been historically used as ingredients added to tobacco, to reconstituted tobacco sheet, and to other processed tobaccos. As part of ongoing stewardship efforts, a toxicological assessment of cigarettes with and without DAP and urea was conducted. Chemical and biological analyses were conducted for test cigarettes added 0.5% DAP and 0.2% urea in the final blend and also for those added 1.0% DAP and 0.41% urea in the final blend compared to reference cigarettes without added DAP or urea. Principal components of this evaluation included a determination of selected mainstream smoke constituent yields, an Ames assay in Salmonella typhimurium strains TA98 and TA100, a sister chromatid exchange assay in Chinese hamster ovary cells, a 13-week inhalation study of mainstream cigarette smoke in Sprague-Dawley rats, and a 30-week dermal tumor-promotion evaluation of mainstream cigarette smoke condensate in SENCAR mice. Comparative evaluations demonstrated that the addition of DAP and urea to cigarettes at up to 1% and 0.41%, respectively, does not alter the biological activity compared to reference cigarettes without DAP or urea.

Mainstream cigarette smoke (MSS) from 12 US cigarette brands and two reference cigarettes was evaluated to determine concentrations of dioxins (i.e., polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (PCBs)). The study included three 'tar' ranges based on Federal Trade Commission (FTC) determination: Low Yield (LY)or = 5.5, Medium Yield (MY) 9.6-12.2, and High Yield (HY)or = 14.5 mg/cig. Of the brands studied, the HY cigarettes yielded the greatest mean concentrations of 2005 World Health Organization Toxic Equivalents (WHO-TEQs) on a per cigarette basis. WHO-TEQ levels in LY cigarettes were significantly lower than for HY cigarettes (p=0.039) on a yield per cigarette basis and WHO-TEQ concentrations correlated with 'tar' yield (r=0.73, p=0.007), as did concentration on a WHO-TEQ per body mass per day basis (r=0.73, p=0.007). However, a statistically significant relationship was not observed between 'tar' yield levels and WHO-TEQ concentrations on a per mg Total Particulate Matter (TPM) basis. Concentrations for all brands tested ranged from 0.44 to 3.88 fg WHO-TEQ/mg TPM. Maximum daily exposure estimates calculated from this range (0.004-0.074 pg WHO-TEQ/kg bw/day) are below the current WHO Tolerable Daily Intake range of 1-5 pg/kg bw/day.

A tiered testing strategy has been developed to evaluate the potential of tobacco processes, ingredients, or technological developments to change the biological activity resulting from burning tobacco. The strategy is based on comparative chemical and biological testing. Dry ice expanded tobacco (DIET) is an example of a common tobacco expansion process currently used in the manufacture of cigarettes to increase tobacco filling capacity. As part of the toxicological evaluation of DIET, test cigarettes containing DIET were compared with control cigarettes containing tobacco expanded with a traditional expansion agent (Freon-11, also known as trichlorofluoromethane). Testing included mainstream cigarette smoke chemistry studies, genotoxicity studies (Ames and sister chromatid exchange, SCE), a 13-week inhalation study in Sprague-Dawley rats, and a 30-week dermal tumor promotion study in SENCAR mice. Cigarettes containing DIET or Freon-11 expanded tobacco were similar in biological activity.

A tiered testing strategy has been developed to evaluate the potential for tobacco processes, ingredients, and other technological developments to increase or decrease the biological activity resulting from burning tobacco. The strategy is based on comparative chemical and biological testing. Propane expanded tobacco is an example of a processed tobacco used in the modern manufacture of cigarettes. Test cigarettes containing propane expanded tobacco were compared to control cigarettes containing tobacco expanded with a traditional expansion agent (Freon-11). The toxicological evaluation included chemistry studies using mainstream cigarette smoke (determination of selected constituent yields), in vitro studies using cigarette smoke condensate (Ames study in Salmonella typhimurium and sister chromatid exchange study in Chinese hamster ovary cells) and in vivo studies (13-week inhalation study of mainstream cigarette smoke in Sprague-Dawley rats and 30-week dermal tumor promotion study of cigarette smoke condensate in SENCAR mice). Although statistically significant differences in several smoke constituents were observed, most constituents from cigarettes containing 100% propane expanded tobacco were within market survey ranges. Furthermore, biological tests indicated that the cigarettes containing propane or Freon-11 expanded tobacco were not significantly different.

Cigarette-smoke condensate (CSC) is a complex mixture containing over 3800 identified chemicals including nicotine, water, mutagens, antimutagens, cytotoxins and inert chemicals. Although CSC is mutagenic in the Ames test, its effect on the activity of other mutagens has not been characterized. Using the Ames Salmonella bacterial mutagenesis assay, we found CSC exerts a significant inhibitory effect on mutagens requiring bioactivation. Those studied included heterocyclic amines (Glu-P-1, Glu-P-2, IQ, MeIQ, Trp-P-1 and Trp-P-2), benzo[a]pyrene (B[a]P) and aflatoxin B1. However, CSC had no effect on the activity of direct-acting mutagens (2-nitrofluorene, sodium azide, 4-nitro-1,2-phenylenediamine, 4-nitroquinoline N-oxide and methyl methanesulfonate). With indirect-acting mutagens, the reduced number of revertants observed in the presence of CSC was not attributable to cytotoxicity. CSC exhibited a potent inhibitory effect on the cytochrome P-450 dependent monooxygenases, ethoxyresorufin O-deethylase and B[a]P hydroxylase. This suggests inhibition of the cytochrome P-450 isozymes as one possible mechanism for the antimutagenicity of CSC. Fractionation studies of CSC revealed that the neutral, weakly acidic (phenolic) and basic fractions are all effective as antimutagens against Glu-P-1, a representative heterocyclic amine. This indicates that several classes of chemicals contribute to the inhibitory effect of CSC on the mutagenicity of the heterocyclic amines.

A simple method has been developed to separate and quantitate monovalent ionic species in mainstream cigarette smoke aerosols based on ion chromatography (IC) with conductivity detection. The method entails collecting the smoke aerosol particulate phase by electrostatic precipitation, dissolving the smoke condensate in methanol (MeOH), and separating the ionic species on either a cation- or anion-exchange column. The method has been applied to the analysis of smoke aerosols from two cigarettes, 1R4F Kentucky Reference cigarettes and a new cigarette that heats but does not burn tobacco. The predominant cations in smoke aerosols from 1R4F Kentucky Reference and the new cigarettes are sodium (Na+), ammonium (NH4+), and potassium (K+) ions; the predominant anions are acetate (AcO-) and formate (HCOO-). Trace amounts of chloride (Cl-), nitrite (NO2-), and nitrate (NO3-) ions are also present.

Cigarette smoke condensate is a complex chemical matrix and determination of phenolic compounds in it frequently requires extensive and laborious sample preparation. By utilizing derivatization techniques and capillary column gas chromatography with mass spectrometry in the selected-ion mode, separation and quantitation of selected phenolic compounds found in mainstream cigarette smoke can be accomplished with minimal sample preparation. This method has been used to determine concentrations of phenol, o-cresol, m-cresol, p-cresol, catechol, resorcinol and hydroquinone in cigarette smoke condensate from a number of commercially available cigarettes and a new cigarette which heats, but does not burn, tobacco. Unlike tobacco-burning cigarettes, levels of the phenolic compounds in the new cigarette smoke are at or below the detection limits for most of the compounds. This result is attributed to the unique design of the new cigarette.

This paper will summarize several new findings obtained in our laboratory on the use of micellar mobile phases in liquid chromatography. The topics to be addressed include (i) stationary phase modification by the mobile phase surfactant in micellar liquid chromatography, (ii) investigation of the retention mechanism in micellar liquid chromatography (MLC) using an alkyl-benzene homologous series, (iii) evaluation of the effects of organic additives upon retention and efficiency in MLC, and (iv) preliminary characterization of several new classes of surfactant molecules for use in MLC. The information gained from these studies provides new insights into the dynamics of MLC and demonstrates their potential usefulness in several new separation applications including the resolution of optical isomers.

Micellar liquid chromatography (MLC) of a homologous series of alkylbenzenes with two nonionic micellar mobile phases, i.e., poly(oxyethylene(10 or 23))dodecanol (Brij-22 or Brij-35), indicates a linear relationship between the capacity factor, k', and the homologue carbon number, n/sub c/. This is in contrast to the usual log k' vs n/sub c/ relationship found for a conventional methanol-water mobile phase. In order to identify the origin of this difference, the various partition coefficients unique to MLC were determined as a function of carbon homologue number in the usual manner, employing an improved procedure for estimation of the chromatographic phase ratio based on measurement of the cumulative pore volumes of the stationary phase. Examination of the partition coefficient data, together with solute selectivity and water solubility data, leads to the conclusion that separate and distinct retention mechanisms are operative in MLC, depending upon the degree of water solubility or insolubility of the solute. Thus, the retention of sparingly soluble or water-insoluble solutes is largely governed by the partition coefficient associated with the direct transfer of the solute from the mobile phase micelle to the surfactant-modified (hemimicellar) stationary phase. A solubility limit equation, derived from the original Armstrong-Nome MLC theory, is shown tomore » describe the observed linear k' vs n/sub c/ behavior adequately for the homologous series with these micellar mobile phases.« less

Etude des caracteristiques d'adsorption, en chromatographie liquide micellaire, de deux agents de surface non ioniques, le poly(oxyethylene) ether de dodecyle (Brij-22, Brij-35) et un agent de surface anionique, dodecylsulfate de sodium sur une colonne C18

Cette phase presente de bonnes performances comparee a la phase acetonitrile-eau. Exemple de separation: extrait de tabac

Comparaison de trois systemes du commerce: SGE (dans un four de Hewlett-Packard 5880), Valco (avec un CG IBM 9630), et Siemens (Si Chromat 2). Les systemes a deux fours sont a preferer pour des raisons pratiques (piegeage, mise en place de la colonne), sinon les performances sont equivalentes

The major components of an alkaloid-free, flue-cured, tobacco essential oil sample are isolated and identified. This is accomplished by utilizing modern hyphenated analytical methods. The instrumentation developed to accomplish this are an automated multidimensional gas chromatograph/mass spectrometer/flame ionization detector (MDGC/MS/FID) and a multidimensional gas chromatograph/matrix isolation/Fourier transform infrared spectrometer (MDGC/MI/FTIR). A total of 306 compounds is identified in the essential oil, of which 80 are found as tobacco constituents for the first time.