Investigation of number of replicate measurements required to meet cigarette smoke chemistry regulatory requirements measured under Canadian intense smoking conditions.

There has been increased interest in recent years in regulatory reporting of cigarette smoke toxicants. There is a great deal of diversity in current regulatory standards around the world in terms of the identities of regulated toxicants, and the number of replicate analyses stipulated for their measurement. Furthermore, analytical methods developed collaboratively by several organisations and intended for regulatory analysis generally differ in their recommended replicate numbers to those stipulated by regulators. In view of these inconsistencies, we undertook an exercise to examine the most appropriate numbers of replicates required for regulatory analysis of cigarette smoke toxicants. A one-point-in-time sampling exercise was undertaken of the German cigarette market, with 161 brands sampled and analysed in a single laboratory using Canadian Intense smoking conditions. Seven replicate measurements were made for each analyte and product, other than nicotine, CO and nicotine-free dry particulate matter for which eight replicate measurements were made. After confirming the absence of order of analysis effects, a variety of statistical tests (such as group assessment, paired comparisons, linear regression models and ratio analysis) were conducted examining mean values, SDs and CVs to identify the role of numbers of analytical replicates on data quality. The statistical analysis showed no difference in mean values for any of the 18 toxicants irrespective of replicate numbers (between 3 and 7 or 8). The large majority of analytes showed no difference in data variability with replicate number; but some very small differences (much lower than within product variability) were observed for a minority of compounds. Similarly, paired analysis showed no significant differences between mean values obtained using different replicate numbers in most cases, apart from very low differences (<5%) for a small number. Linear regression analysis showed correlations around 96 to 98% (other than crotonaldehyde at 91%) between values obtained with 3 vs 7 replicates. Similarly, per product mean value ratio analysis showed 95% consistency between values obtained with 3 and 7 replicates. We therefore conclude that three replicates is sufficient for precise determination of cigarette mainstream smoke toxicant emissions, and that use of 7 replicates as stipulated in some regulator jurisdictions does not offer any greater accuracy or precision.

Changes in Biomarkers of Exposure on Switching From a Conventional Cigarette to Tobacco Heating Products: A Randomized, Controlled Study in Healthy Japanese Subjects.

BACKGROUND: Smoking is a leading cause of numerous human disorders including pulmonary disease, cardiovascular disease, and cancer. Disease development is primarily caused by exposure to cigarette smoke constituents, many of which are known toxicants. Switching smokers to modified risk tobacco products (MRTPs) has been suggested as a potential means to reduce the risks of tobacco use, by reducing such exposure. METHODS: This randomized, controlled study investigated whether biomarkers of toxicant exposure (BoE) were reduced when smokers switched from smoking combustible cigarettes to using a novel (glo™/THP1.0) or in-market comparator (iQOS/THS) tobacco heating product (THP). One hundred eighty Japanese smokers smoked combustible cigarettes during a 2-day baseline period, followed by randomization to either continue smoking cigarettes, switch to using mentholated or non-mentholated variants of glo™, switch to using a non-mentholated variant of iQOS, or quit nicotine and tobacco product use completely for 5 days. Baseline and post-randomization 24-h urine samples were collected for BoE analysis. Carbon monoxide was measured daily in exhaled breath (eCO). RESULTS: On day 5 after switching, urinary BoE (excluding for nicotine) and eCO levels were significantly (p < .05) reduced by medians between 20.9% and 92.1% compared with baseline in all groups either using glo™ or iQOS or quitting tobacco use. Between-group comparisons revealed that the reductions in the glo™ groups were similar (p > .05) to quitting in many cases. CONCLUSIONS: glo™ or iQOS use for 5 days reduced exposure to smoke toxicants in a manner comparable to quitting tobacco use. THPs are reduced exposure tobacco products with the potential to be MRTPs. IMPLICATIONS: This clinical study demonstrates that when smokers switched from smoking combustible cigarettes to using tobacco heating products their exposure to smoke toxicants was significantly decreased. In many cases, this was to the same extent as that seen when they quit smoking completely. This may indicate that these products have the potential to be reduced exposure and/or reduced risk tobacco products when used by smokers whose cigarette consumption is displaced completely. CLINICAL TRIAL REGISTRATIONS: ISRCTN14301360 and UMIN000024988.

Evaluation of an online, real-time, soft-photon ionisation time-of-flight mass spectrometer for mainstream tobacco smoke analysis.

Mainstream tobacco smoke is a complex and dynamic aerosol, consisting of particulate and vapour phases. Most approaches to determine mainstream smoke toxicant yields are based on offline techniques that limit the opportunity to observe in real time the processes leading to smoke formation. The recent development of online real-time analytical methods offers many advantages over traditional techniques. Here we report the LM2X-TOFMS (Borgwaldt GmbH, Germany), a commercial instrument that couples a linear smoking engine with a time-of-flight mass spectrometer for real-time per-puff measurement of the vapour phase of mainstream cigarette smoke. Total cigarette and puff-by-puff (µg/puff) yields were evaluated, in line with International Council of Harmonisation recommendations, for seven smoke toxicants: acetaldehyde, acetone, 1,3-butadiene, 2-butanone, benzene, isoprene and toluene. Measurements were unaffected by small system changes including replacing the sampling capillary or time of day (all P > 0.05), indicating that the LM2X-TOFMS is rugged. Control charts showed that the system has good stability and control. Analysis of certified gas mixtures of six concentrations of each analyte showed a highly linear response for all seven analytes (R2 = 0.9922-0.9999). In terms of repeatability, the lowest variation was observed for isoprene with a coefficient of variation (CV) of < 6% for each concentration. Acetaldehyde showed the highest CV, increasing from 8.0 to 26.6% with decreasing gas concentration. Accuracy was analysed in terms of relative error, which was ± 16% for six of the analytes; however, the relative error for acetaldehyde was (- 36.2%), probably due to its low ionisation efficiency under the instrument's vacuum ultraviolet lamp. Three cigarette products (reference and commercial) with different ISO tar levels were analysed by the LM2X-TOFMS puff by puff under ISO regulatory smoking conditions. The relative standard deviation based on average yield per cigarette for each analyte in each product (summed puffs per product, n = 30) ranged from ≤ 9.3 to ≤ 16.2%. Measurements were consistent with published data per cigarette. In conclusion, the LM2X-TOFMS is suitable for determining the vapour-phase yields of seven analytes on a real-time, puff-by-puff basis, and can be utilised for both fast screening (qualitative) and quantitative measurements of mainstream cigarette smoke.

Influence of cigarette filter ventilation on smokers' mouth level exposure to tar and nicotine.

Cigarette filter ventilation allows air to be drawn into the filter, diluting the cigarette smoke. Although machine smoking reveals that toxicant yields are reduced, it does not predict human yields. The objective of this study was to investigate the relationship between cigarette filter ventilation and mouth level exposure (MLE) to tar and nicotine in cigarette smokers. We collated and reviewed data from 11 studies across 9 countries, in studies performed between 2005 and 2013 which contained data on MLE from 156 products with filter ventilation between 0% and 87%. MLE among 7534 participants to tar and nicotine was estimated using the part-filter analysis method from spent filter tips. For each of the countries, MLE to tar and nicotine tended to decrease as filter ventilation increased. Across countries, per-cigarette MLE to tar and nicotine decreased as filter ventilation increased from 0% to 87%. Daily MLE to tar and nicotine also decreased across the range of increasing filter ventilation. These data suggest that on average smokers of highly ventilated cigarettes are exposed to lower amounts of nicotine and tar per cigarette and per day than smokers of cigarettes with lower levels of ventilation.

A randomised, controlled, two-Centre open-label study in healthy Japanese subjects to evaluate the effect on biomarkers of exposure of switching from a conventional cigarette to a tobacco heating product.

BACKGROUND: Smoking is a leading cause of numerous human disorders including lung cancer, chronic obstructive pulmonary disease, and atherosclerotic cardiovascular disease. The development of modified risk tobacco products (MRTPs) has been suggested as a possible way to reduce the risks of tobacco smoking by reducing exposure to cigarette smoke toxicants. This study is designed to investigate whether biomarkers of such exposure are reduced when smokers switch from smoking commercial cigarettes to using either a novel or a commercially-available tobacco heating product (THP). DESIGN AND METHODS: This study will assess biomarkers of exposure in current smokers who either remain smoking, switch to THP use, or quit all tobacco use completely, for 5 days. The study is an in-clinic (confinement) two-centre, randomised controlled clinical study with a forced-switching design. Subjects of either gender will be aged 23-55 years (minimum legal smoking age plus 3 years), of Japanese origin and with a verified smoking status (assessed by exhaled breath carbon monoxide and urinary cotinine levels). Subjects will have a usual brand cigarette within the International Organisation for Standardisation (ISO) tar band of 6-8 mg and will be judged to be healthy by medical history, physical examination, vital signs, electrocardiography (ECG), clinical biochemistry and lung function tests. The primary objective of this study is to assess changes within groups in selected biomarkers of exposure (BoE) and of biological effect (BoBE) after a forced switch from a commercial control cigarette to either a menthol or a non-menthol THP. Secondary objectives are to assess between-group differences, to determine nicotine pharmacokinetics for cigarettes and THPs, to assess subject's satisfaction with the study products, and to monitor additional endpoints related to safety and product use. DISCUSSION: Data from this study will advance our scientific understanding of the changes in exposure to cigarette smoke toxicants in smokers who switch to using a THP. TRIAL REGISTRATIONS: UMIN000024988 (25th November 2016); ISRCTN14301360 (14th December 2016).

Characterisation of an aerosol exposure system to evaluate the genotoxicity of whole mainstream cigarette smoke using the in vitro γH2AX assay by high content screening.

BACKGROUND: The genotoxic effect of cigarette smoke is routinely measured by treating cells with cigarette Particulate Matter (PM) at different dose levels in submerged cell cultures. However, PM exposure cannot be considered as a complete exposure as it does not contain the gas phase component of the cigarette smoke. The in vitro γH2AX assay by High Content Screening (HCS) has been suggested as a complementary tool to the standard battery of genotoxicity assays as it detects DNA double strand breaks in a high-throughput fashion. The aim of this study was to further optimise the in vitro γH2AX assay by HCS to enable aerosol exposure of human bronchial epithelial BEAS-2B cells at the air-liquid interface (ALI). METHODS: Whole mainstream cigarette smoke (WMCS) from two reference cigarettes (3R4F and M4A) were assessed for their genotoxic potential. During the study, a further characterisation of the Borgwaldt RM20S® aerosol exposure system to include single dilution assessment with a reference gas was also carried out. RESULTS: The results of the optimisation showed that both reference cigarettes produced a positive genotoxic response at all dilutions tested. However, the correlation between dose and response was low for both 3R4F and M4A (Pearson coefficient, r = -0.53 and -0.44 respectively). During the additional characterisation of the exposure system, it was observed that several pre-programmed dilutions did not perform as expected. CONCLUSIONS: Overall, the in vitro γH2AX assay by HCS could be used to evaluate WMCS in cell cultures at the ALI. Additionally, the extended characterisation of the exposure system indicates that assessing the performance of the dilutions could improve the existing routine QC checks.

Longitudinal study of long-term smoking behaviour by biomarker-supported determination of exposure to smoke.

BACKGROUND: Long-term studies of smokers who switch to lower nicotine yield cigarettes have been identified by the World Health Organization Study Group TobReg and the US Food and Drug Administration as one key area where new knowledge is required to guide science based regulation. The limited number of long-term switching studies have concluded that smokers who switch to lower nicotine yield cigarettes show evidence of partial compensation. Since the European Union tobacco product directive of 2001 introduced tar and nicotine yield ceilings, there has been no long-term observational switching study. To address the limitations of previous studies where smokers were forced switched for relatively short durations, we plan to undertake a long-term study of spontaneous switching which is appropriately powered and includes non-switchers as a control group. METHODS/DESIGN: Healthy adult smokers aged 21-64 years will be enrolled into this 5-year non-residential, multicentre study across 10 cities in Germany. They will be assessed at 10 timepoints with 6 month intervals during which inclusion criteria will be reassessed and spent cigarette filter tips, saliva and 24 h urine samples will be collected. These samples will be used to determine average daily cigarette consumption, estimate mouth-level exposure to tar and nicotine and measure selected biomarkers of exposure, respectively. Spontaneous changes in subjects' preferred cigarette products and any consequent change in tar or nicotine yield will be monitored. Subjects will be required to complete questionnaires on quality of life, smoking behaviours, smoking-related sensory attributes and recent life changes. DISCUSSION: The planned study is anticipated to contribute to understanding smokers' behaviours and their consequent exposure to smoke constituents. It will also allow assessment of compensatory changes in their behaviour following spontaneous switching of cigarette product smoked. Data from this study are expected to provide insights into study design and conduct for non-clinical assessment of smokers' exposure as part of post marketing surveillance programmes. TRIAL REGISTRATION: Current Controlled Trials Database reference ISRCTN95019245.

Multi-analyte approach for determining the extraction of tobacco constituents from pouched snus by consumers during use.

BACKGROUND: Snus is a smokeless oral tobacco product with a significant history of use in Sweden, where it is regulated under food legislation. Users place a small porous sachet or a pinch of loose snus between the upper jaw and cheek for approximately one hour, leading to partial intake of tobacco constituents. To understand user exposure to tobacco, a multi-analyte approach based on the extraction of pouches by methanol, ethanol and water was validated and applied to the measurement of various constituents, including nicotine, four tobacco-specific nitrosamines (TSNAs), propylene glycol, water, ammonium, nitrate, sodium, chloride, linalool, citronellol, linalyl acetate and geraniol, extracted from snus pouches during use by human consumers. RESULTS: After validation against established single-analyte methods, the multi-analyte approach was used to determine constituent levels in snus pouches before and after one hour of use. Although the concentrations in the snus pouches varied from nanogram (e.g. TSNAs) to milligram (e.g. nicotine, sodium and propylene glycol) quantities (25.1 ng to 35.3 mg per 1 g pouch), the mean percentage extracted varied only from 19.2% for linalyl acetate to 37.8% for the TSNA 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) among all constituents analyzed. The TSNAs, some of which are known carcinogens, showed the highest percentage extraction (range 34.6%-37.8%). Measurement variability was low for all analytes, ranging from 2.4% (total TSNAs, NAT) to 9.5% (geraniol). By contrast, inter-subject variability ranged from 6.7% (NAB) to 52.2% (linalyl acetate), and was greater than 20% for eight of the constituents analyzed. Intra-subject variability ranged from 3.4% (citronellol) to 29.7% (geraniol). CONCLUSIONS: Generally, less than a third of each constituent tested was extracted during one hour of snus use, independent of constituent concentration. The variable nature of in-use extraction was shown to be driven by inter-subject variability. The results provide insight into possible mechanisms controlling constituent extraction in the mouth during snus use, and provide reference data for the development of in-vitro laboratory systems for estimating extraction of tobacco constituents from snus.

An inter-laboratory comparison of urinary 3-hydroxypropylmercapturic acid measurement demonstrates good reproducibility between laboratories.

BACKGROUND: Biomarkers have been used extensively in clinical studies to assess toxicant exposure in smokers and non-smokers and have recently been used in the evaluation of novel tobacco products. The urinary metabolite 3-HPMA, a metabolite of the major tobacco smoke toxicity contributor acrolein, is one example of a biomarker used to measure exposure to tobacco smoke. A number of laboratories have developed liquid chromatography with tandem mass spectrometry (LC-MS/MS) based methods to measure urinary 3-HPMA; however, it is unclear to what extent the data obtained by these different laboratories are comparable. FINDINGS: This report describes an inter-laboratory comparison carried out to evaluate the comparability of 3-HPMA measurement between four laboratories. A common set of spiked and authentic smoker and non-smoker urine samples were used. Each laboratory used their in-house LC-MS/MS method and a common internal standard. A comparison of the repeatability ('r'), reproducibility ('R'), and coefficient of variation for 3-HPMA demonstrated that within-laboratory variation was consistently lower than between-laboratory variation. The average inter-laboratory coefficient of variation was 7% for fortified urine samples and 16.2% for authentic urine samples. Together, this represents an inter-laboratory variation of 12.2%. CONCLUSION: The results from this first inter-laboratory comparison for the measurement of 3-HPMA in urine demonstrate a reasonably good consensus between laboratories. However, some consistent measurement biases were still observed between laboratories, suggesting that additional work may be required to further reduce the inter-laboratory coefficient of variation.

Assessment of an in vitro whole cigarette smoke exposure system: The Borgwaldt RM20S 8-syringe smoking machine.

BACKGROUND: There have been many recent developments of in vitro cigarette smoke systems closely replicating in vivo exposures. The Borgwaldt RM20S smoking machine (RM20S) enables the serial dilution and delivery of cigarette smoke to exposure chambers for in vitro analyses. In this study we have demonstrated reliability and robustness testing of the RM20S in delivering smoke to in vitro cultures using an in-house designed whole smoke exposure chamber. RESULTS: The syringe precision and accuracy of smoke dose generated by the RM20S was assessed using a methane gas standard and resulted in a repeatability error of ≤9%. Differential electrical mobility particle spectrometry (DMS) measured smoke particles generated from reference 3R4F cigarettes at points along the RM20S. 53% ± 5.9% of particles by mass reached the chamber, the remainder deposited in the syringe or connecting tubing and ~16% deposited in the chamber. Spectrofluorometric quantification of particle deposition within chambers indicated a positive correlation between smoke concentration and particle deposition. In vitro air-liquid interface (ALI) cultures (H292 lung epithelial cells), exposed to whole smoke (1:60 dilution (smoke:air, equivalent to ~5 µg/cm2)) demonstrated uniform smoke delivery within the chamber. CONCLUSIONS: These results suggest this smoke exposure system is a reliable and repeatable method of generating and exposing ALI in vitro cultures to cigarette smoke. This system will enable the evaluation of future tobacco products and individual components of cigarette smoke and may be used as an alternative in vitro tool for evaluating other aerosols and gaseous mixtures such as air pollutants, inhaled pharmaceuticals and cosmetics.

A study to evaluate the effect on Mouth Level Exposure and biomarkers of exposure estimates of cigarette smoke exposure following a forced switch to a lower ISO tar yield cigarette.

A forced switch to a lower ISO tar yield cigarette was used in a clinical study, conducted in Germany, that compared two methods of estimating exposure to cigarette smoke. Pre- and post-switch estimates of Mouth Level Exposure (MLE) to nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), pyrene and acrolein were obtained by chemical analysis of spent cigarette filters for nicotine content. Similarly, pre- and post-switch estimates of uptake of these smoke constituents were achieved by analysis of corresponding urinary biomarkers of exposure (BoE): total nicotine equivalents; total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL); total 1-hydroxypyrene (1-OHP), and 3-hydroxypropyl-mercapturic acid (3-HPMA), plus the nicotine metabolite cotinine, in plasma and saliva. Three hundred healthy volunteers were recruited comprising 100 smokers of each of 9-10 and 4-6 mg ISO tar yield cigarettes and 50 smokers of 1-2mg ISO tar yield cigarettes and 50 non-smokers. Fifty smokers of each of the 9-10 and 4-6 mg ISO tar yield cigarettes took part in the switching aspects of this study whilst the remaining smokers formed non-switching control groups who smoked their usual ISO tar yield cigarette throughout the study. After 5 days, all subjects were admitted into a clinic where baseline measures of MLE and BoE were obtained. The 10mg switching group was then switched to the 4 mg ISO tar yield cigarette and the 4 mg ISO tar yield switching group switched to the 1mg cigarette. Subjects returned home for 12 days, continuing to smoke the supplied cigarettes before being readmitted into the clinic where samples were collected for MLE and BoE analysis. Changes in daily exposure estimates were determined on a group and individual basis for both methods. The pre- to post-switch directional changes in MLEs and their corresponding BoEs were generally consistent and the MLE/BoE relationship maintained. Switching to a lower yield cigarette generally resulted in reductions in exposure with the resultant exposure level being similar to that seen in regular smokers of the lower yield cigarette.

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of phenolic polycyclic aromatic hydrocarbons (OH-PAH) in urine of non-smokers and smokers.

Polycyclic aromatic hydrocarbons (PAH) are products of the incomplete combustion of organic materials and, therefore, occur ubiquitously in the environment and also in tobacco smoke. Since some PAH have been classified as carcinogens, it is important to have access to suitable analytical methods for biomarkers of exposure to this class of compounds. Past experience has shown that measuring a profile of PAH metabolites is more informative than metabolites of a single PAH. Assessment of environmental and smoking-related exposure levels requires analytical methods with high sensitivity and specificity. In addition, these methods should be fast enough to allow high throughput. With these pre-conditions in mind, we developed and validated a high-performance liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of phenolic metabolites of naphthalene, fluorene, phenanthrene and pyrene in urine of smokers and non-smokers. Sample work-up comprised enzymatic hydrolysis of urinary conjugates and solid-phase extraction on C18 cartridges. The method showed good specificity, sensitivity, and accuracy for the intended purpose and was also sufficiently rapid with a sample throughput of about 350 per week. Application to urine samples of 100 smokers and 50 non-smokers showed significant differences between both groups for all measured PAH metabolites, and strong correlations with markers of daily smoke exposure in smoker urine. Urinary levels were in good agreement with previously reported data using different methodologies. In conclusion, the developed LC-MS/MS method is suitable for the quantification of phenolic PAH metabolites of naphthalene, fluorene, phenanthrene, and pyrene in smoker and non-smoker urine.

Urinary excretion of the acrylonitrile metabolite 2-cyanoethylmercapturic acid is correlated with a variety of biomarkers of tobacco smoke exposure and consumption.

Acrylonitrile is an IARC class 2B carcinogen present in cigarette smoke. Urinary 2-cyanoethylmercapturic acid (CEMA) is an acrylonitrile metabolite and a potential biomarker for acrylonitrile exposure. The objective of this work was to study the dose response of CEMA in urine of non-smokers and smokers of different ISO tar yield cigarettes. We observed that smokers excreted >100-fold higher amounts of urinary CEMA than non-smokers. The CEMA levels in smokers were significantly correlated with ISO tar yield, daily cigarette consumption, and urinary biomarkers of smoke exposure. In conclusion, urinary CEMA is a suitable biomarker for assessing smoking-related exposure to acrylonitrile.

Evaluation of precision and accuracy of the Borgwaldt RM20S(®) smoking machine designed for in vitro exposure.

The Borgwaldt RM20S(®) smoking machine enables the generation, dilution, and transfer of fresh cigarette smoke to cell exposure chambers, for in vitro analyses. We present a study confirming the precision (repeatability r, reproducibility R) and accuracy of smoke dose generated by the Borgwaldt RM20S(®) system and delivery to exposure chambers. Due to the aerosol nature of cigarette smoke, the repeatability of the dilution of the vapor phase in air was assessed by quantifying two reference standard gases: methane (CH(4), r between 29.0 and 37.0 and RSD between 2.2% and 4.5%) and carbon monoxide (CO, r between 166.8 and 235.8 and RSD between 0.7% and 3.7%). The accuracy of dilution (percent error) for CH(4) and CO was between 6.4% and 19.5% and between 5.8% and 6.4%, respectively, over a 10-1000-fold dilution range. To corroborate our findings, a small inter-laboratory study was carried out for CH(4) measurements. The combined dilution repeatability had an r between 21.3 and 46.4, R between 52.9 and 88.4, RSD between 6.3% and 17.3%, and error between 4.3% and 13.1%. Based on the particulate component of cigarette smoke (3R4F), the repeatability (RSD = 12%) of the undiluted smoke generated by the Borgwaldt RM20S(®) was assessed by quantifying solanesol using high-performance liquid chromatography with ultraviolet detection (HPLC/UV). Finally, the repeatability (r between 0.98 and 4.53 and RSD between 8.8% and 12%) of the dilution of generated smoke particulate phase was assessed by quantifying solanesol following various dilutions of cigarette smoke. The findings in this study suggest the Borgwaldt RM20S(®) smoking machine is a reliable tool to generate and deliver repeatable and reproducible doses of whole smoke to in vitro cultures.

Determination of tobacco-specific N-nitrosamines in urine of smokers and non-smokers.

Tobacco-specific N-nitrosamines (TSNA) include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosonornicotine (NNN), N'-nitrosoanabasine (NAB) and N'-nitrosoanatabine (NAT) and are found in tobacco and tobacco smoke. TSNA are of interest for biomonitoring of tobacco-smoke exposure as they are associated with carcinogenesis. Both NNK and NNN are classified by IARC as Group 1 carcinogens. Samples of 24 h urine collections (n = 108) were analysed from smokers and non-smokers, using a newly developed and validated LC-MS/MS method for determining total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the major metabolite of NNK), and total NNN, NAB and NAT. TSNA levels in smokers' urine were significantly higher than in non-smokers. In smokers, urinary excretion of total TSNA correlated significantly (r > 0.5) with markers of smoking dose, such as daily cigarette consumption, salivary cotinine and urinary nicotine equivalents and increased with the ISO tar yield of cigarettes smoked. The correlation between urinary total NNN and the smoking dose was weaker (r = 0.4-0.5). In conclusion, this new method is suitable for assessing tobacco use-related exposure to NNK, NNN, NAB and NAT.

Patterns and behaviors of snus consumption in Sweden.

INTRODUCTION: Snus is an oral snuff consisting of moist finely ground tobacco which is available in a loose form or with portions of the tobacco sealed in small sachets termed "pouches." The product has a long history of use in Sweden. Currently, there is very little published information on levels of consumption and usage behaviors for snus in Sweden. The objective of this study was to obtain data on the frequency and duration of loose and pouched snus consumption in Sweden and investigate usage behaviors. METHODS: Telephone surveys of snus users randomly selected from telephone directories in all regions of Sweden were conducted in 2007 and 2008. In total, 2,914 respondents answered questions on snus usage, including the types of products used and the quantity and frequency of use. RESULTS: The majority of respondents (96%) used either pouched or loose snus alone. A minority (12.6%) reported dual use of smokeless and combustible tobacco products. Average daily consumption was 11-12 g for pouched snus and 29-32 g for loose snus. The typical duration of use of each pouch/portion was 60-70 min. DISCUSSION: This survey has provided new insights into contemporary snus use in Sweden, such as the marked differences in daily consumption between loose and pouched snus, length of time that snus users typically keep pouches in the mouth, differential patterns of use in males and females, and the simultaneous use of multiple pouches in a small proportion of users.

Urinary excretion of phenolic polycyclic aromatic hydrocarbons (OH-PAH) in nonsmokers and in smokers of cigarettes with different ISO tar yields.

Polycyclic aromatic hydrocarbons (PAH) are products of the incomplete combustion of organic materials, and they occur ubiquitously in the environment. They are also present in tobacco smoke. Some PAH have been classified as carcinogens; therefore, it is important to develop and assess suitable biomarkers for PAH exposure. A high-performance liquid chromatographic method with fluorescence detection was developed to determine 1- and 2-hydroxynaphthalene (1- and 2-OH-Nap), 2-hydroxyfluorene (2-OH-Flu), 2-/3-hydroxyphenanthrene (2-/3-OH-Phe), 1-/9-hydroxyphenanthrene (1-/9-OH-Phe), and 1-hydroxypyrene (1-OH-Pyr) in human urine. The method is sensitive (LOQ ranging from 0.01 ng/mL for 1-OH-Pyr to 1 ng/mL for the naphthols), precise (interday precision ranging from 1.4 to 6.9%), and accurate (97-106%). The method was applied to 108 urine samples from 25 nonsmokers and 83 smokers. Smokers excreted significantly higher amounts of 1-OH-Nap (16.1 vs. 2.9 microg/24 h), 2-OH-Nap (20.9 vs. 9.7 microg/24 h), 2-OH-Flu (1.87 vs. 0.75 microg/24 h), 2-/3-OH-Phe (0.73 vs. 0.50 microg/24 h), 1-/9-OH-Phe (0.66 vs. 0.35 microg/24 h), and 1-OH-Pyr (0.36 vs. 0.20 microg/24 h) compared to nonsmokers. In conclusion, the method is suitable for discriminating PAH exposure between different ISO tar yield cigarette smokers, and it may be applicable in evaluating future potential reduced exposure tobacco products.

A study to estimate and correlate cigarette smoke exposure in smokers in Germany as determined by filter analysis and biomarkers of exposure.

A clinical study, conducted in Germany, compared two methods of estimating exposure to cigarette smoke. Estimates of mouth level exposure (MLE) to nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), pyrene and acrolein were obtained by chemical analysis of spent cigarette filters for nicotine content. Estimates of smoke constituent uptake were achieved by analysis of corresponding urinary biomarkers: for nicotine; total nicotine equivalents (nicotine, cotinine, trans-3'-hydroxycotinine plus their glucuronide conjugates), for NNK; (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) plus glucuronide, for pyrene; 1-hydroxy pyrene (1-OHP) plus glucuronide and for acrolein; 3-hydroxylpropyl-mercapturic acid (3-HPMA) plus the nicotine metabolite cotinine in plasma and saliva. Two hundred healthy volunteer subjects were recruited; 50 smokers of each of 1-2 mg, 4-6 mg and 9-10 mg ISO tar yield cigarettes and 50 non-smokers (NS). Smokers underwent two periods of home smoking, each followed by residence in a clinic. Smoking was permitted ad libitum, and spent cigarette filters, cigarette consumption data, 24h urine, as well as plasma and saliva samples were collected. Significant correlations (p<0.001) were found between MLE and the relevant biomarker for each smoke constituent. The Pearson correlation coefficients (r) were 0.83 (nicotine), 0.76 (NNK), 0.82 (acrolein) and 0.63 (pyrene). Mean MLE estimates for nicotine, NNK and pyrene showed a dose response in line with ISO tar yield smoked, with 10 mg > 4 mg >1 mg, and for acrolein 10 mg> 4 mg > *1mg (where * indicates not significant at 95% confidence level). The mean exposure estimates from biomarkers for nicotine, NNK and acrolein also showed a dose response in line with ISO tar yield with 10 mg > 4 mg > 1 mg > NS, and for pyrene 10 mg > *4 mg> 1 mg> NS. This study shows that estimates of exposure obtained by filter analysis and biomarkers of exposure correlate significantly over a wide range of smoke exposures and that filter analysis may provide a simple and effective alternative to biomarkers for estimating smokers' exposure.

Simultaneous determination of four tobacco-specific N-nitrosamines (TSNA) in human urine.

Tobacco-specific N-nitrosamines (TSNA) include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT). TSNA are suggested to play an important role in tobacco smoke carcinogenesis. We have developed and validated an LC-MS/MS method for the determination of total (free and conjugated) TSNA in human urine. The limits of detection (LOD) were 2.0, 0.8, 1.1 and 0.7 pg/ml for NNAL, NNN, NAB and NAT, respectively. Smokers were found to have significantly higher levels of TSNA in their urine than nonsmokers. In conclusion, the newly developed method is suitable for assessing the tobacco use-related exposure to NNK, NNN, NAB and NAT.