Supercritical fluid extraction and gas chromatography/mass spectrometry for the analysis of tobacco-specific nitrosamines in cigarettes

A method for measuring four tobacco-specific nitrosamines (TSNAs), an important group of compounds in tobacco products, was developed. These compounds were extracted using supercritical fluid extraction (SFE) and purified by a sodium hydroxide wash of the ethyl acetate eluting solvent and solid-phase extraction. Quantitation was performed using gas chromatography/mass spectrometry (GC/MS). Spiking experiments were carried out to determine the recovery, precision, and limits of detection of this method. The detection limits were 0.04 microgram per sample for N'-nitrosonornicotine and N'-nitrosoanatabine and 0.02 microgram for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosoanabasine. This method was used to measure TSNAs in various brands of cigarette tobacco with excellent reproducibility. The variation of TSNA levels among the cigarettes of different packs and types was significantly smaller than that among different brands. Comparable TSNA levels were obtained with SFE and liquid extraction methods. Signal-to-noise levels were similar for GC/MS and GC/thermal energy analysis when low-level tobacco samples were analyzed.     

Development of a quantitative method for the analysis of tobacco-specific nitrosamines in mainstream cigarette smoke using isotope dilution liquid chromatography/electrospray ionization tandem mass spectrometry

An improved method has been developed for the determination of the four major tobacco-specific nitrosamines (TSNA) in mainstream cigarette smoke. The new method offers decreased sample preparation and analysis time as compared to traditional methodologies. This method uses isotope dilution liquid chromatography coupled to a tandem mass spectrometer with electrospray ionization and is significantly more sensitive than traditional methods. It also shows no evidence of artifactual formation of TSNA. Sample concentrations were determined for four TSNA in mainstream smoke using two isotopically labeled TSNA analogues as internal standards. Mainstream smoke was collected on an industry standard 44-mm Cambridge filter pad, extracted with an aqueous buffer solution, and analyzed without further sample cleanup. This method has been validated through intra- and interlaboratory studies and has shown excellent recoveries, sensitivity, and repeatability. The limits of detection of each TSNA varied from 0.01 to 0.1 ng/mL, and the linear calibration range of the instrument in sample matrix spanned 0.5-200 ng/ mL, which allowed for the determination of the TSNA levels in cigarettes with a wide range of deliveries. Data are also reported from two commercially available industry reference cigarettes and show excellent agreement and reproducibility over a six-month time period (n > 50).     

The detection of nitro pesticides in mainstream and sidestream cigarette smoke using electron monochromator-mass spectrometry

Over the past decade, electron monochromator-mass spectrometry (EM-MS) has been shown to be a selective and sensitive technique for the analysis of a wide variety of electrophilic compounds in complex matrixes. Here, for the first time, three different dinitroaniline pesticides, flumetralin, pendimethalin, and trifluralin, have been shown to be present in both mainstream and sidestream tobacco smoke using an EM-MS system. A number of cigarettes were tested that included three pure-tobacco-type cigarettes, an experimental reference cigarette, and 11 commercial cigarettes. Due to the complexity of the smoke particulate matter, the pesticides were identified only after each sample was subjected to a multistep cleanup process that included phenyl solid-phase extraction; an acid wash; aminopropyl solid-phase extraction; and finally, normal phase LC fractionation. All cigarette types tested showed the presence of the three pesticides in the tobacco smoke, with flumetralin ranging from trace levels up to 37 (+/- 9) ng/cig, pendimethalin ranging from trace levels up to 10.4 (+/- 0.6) ng/cig, and trifluralin ranging from trace levels up to 47 (+/- 17) ng/cig. Acute toxicity information is presented for the three pesticides.     

Quantitative method for the analysis of tobacco-specific nitrosamines in cigarette tobacco and mainstream cigarette smoke by use of isotope dilution liquid chromatography tandem mass spectrometry

An improved liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for the determination of tobacco specific nitrosamines (TSNA). It utilizes four stable isotope-labeled internal standards instead of two as reported by others. A separate internal standard for each analyte is required to minimize sample matrix effects on each analyte, which can lead to poor analyte recoveries and decreases in method accuracy and precision if only one or two of the internal standards are used, especially for complex sample matrixes and when no sample cleanup steps are performed as in this study. In addition, two ion-transition pairs (instead of one) are used for each analyte for the confirmation and quantification, further enhancing the method's accuracy and robustness. These improvements have led to a new LC-MS/MS method that is faster, more sensitive, and selective than the traditional methods and more accurate and robust than the published LC-MS/MS methods. The linear range of the method was from 0.2 to 250 ng/mL with a limit of detection of each TSNA varied from 0.027 to 0.049 ng/mL. Good correlations between the results obtained by the new method and the traditional method were observed for the samples studied.     

Pollution patterns of polycyclic aromatic hydrocarbons in tobacco smoke

Concentrations of polycyclic aromatic hydrocarbons (PAHs) in tobacco smoke of 12 commercial brand cigarettes were determined in a simulated chamber of 20.25 m3 in size. The total concentrations of 17 PAHs (summation operatorPAHs) in the chamber were 3500 and 1152 ng/m3 in vapor phase and particulate phase, respectively. In vapor phase, the yield of naphthalene (NA) appeared to be the most abundant (2462 ng/cig) followed by fluorene (FLUOR) and acenaphthylene (ACY), while the yield of benzo[ghi]perylene (BP) was the most abundant (259.7 ng/cig) in particulate phase followed by phenanthrene (PHEN) and FLUOR. The proportion of PAHs in particulate phase increased with increasing molecular weight. PAHs with two to six rings accounted for 40.2%, 35.3%, 11.7%, 7.6%, 5.2% of summation operatorPAHs, respectively. There was no obvious correlation between PAHs, benzo[a]pyrene (BaP) concentrations in tobacco smoke and smoking tar contents, nicotine contents. With the source fingerprint of PAHs in tobacco smoke, NA could be regarded as the marker of tobacco smoke source because of its largest contribution to summation operatorPAHs (40.2%), followed by FLUOR (12.7%) and ACY (9.8%). Further study indicated that more than 80% of BaP in indoor air of resident homes in Hangzhou was from tobacco smoke.     

Fluorescent approach to quantitation of reactive oxygen species in mainstream cigarette smoke

A novel approach to monitoring of mainstream smoke reactive oxygen species (ROS) has been developed and applied to the quantitation of smoke oxidants. Redox-active fluorescent probe dihydrorhodamine 6G (DHR-6G) was selected as the molecular probe because it is sensitive to typical smoke ROS. The experimental system couples an automatic cigarette smoke machine fiber-optic fluorometer for real-time monitoring of the reaction progress between cigarette smoke and DHR-6G. Quantitation was achieved based on the amount of rhodamine 6G, which is the sole product from DHR-6G oxidation. With the optimization of the trapping efficiency, we detected 391 nmol of ROS/cigarette in the mainstream CS for a standard cigarette 2R4F under standard Federal Trade Commission smoking protocol. Applying this method, we quantified the ROS of selected cigarettes and found that the cigarettes made of burley tobacco have much ( approximately 10 times) higher ROS content in the smoke than that in the tobacco made of bright tobacco. The smokeless cigarette, Eclipse, has comparable ROS with cigarettes made of bright tobacco.     

顶空-气相色谱法测定卷烟主流烟气粒相物中吡啶

为研究气相色谱测定卷烟主流烟气粒相物中吡啶含量,采用碳酸钠水溶液作基质校正剂,通过顶空进样、DB-WAXETR色谱柱分离、火焰离子化检测器检测。结果表明:吡啶在0.20~16.20μg/mL质量浓度范围内线性拟合度为0.9998,加标回收率在98.4%~104.7%之间,定量限为0.08μg/支,RSD小于5%,该方法可以快速、准确地测定卷烟主流烟气粒相物中吡啶。     

Automated in-tube solid-phase microextraction coupled with HPLC for the determination of N-nitrosamines in cell cultures

An automated in-tube solid-phase microextraction (SPME) HPLC analysis method for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and several metabolites has been developed. NNK is one of the tobacco-specific N-nitrosamines (TSNA), which has been linked to cancers associated with the use of or exposure to tobacco products. In-tube SPME is an on-line extraction technique in which analytes are extracted and concentrated from the sample directly into a coated capillary by repeated draw/eject steps. In this study, a tailor-made polypyrrole (PPY)-coated capillary and several commercially available capillaries (capillary GC columns) were used to evaluate their extraction efficiencies for NNK and several metabolites in cell cultures. Compared with commercial capillaries that were currently used for in-tube SPME, the PPY-coated capillary showed better extraction efficiency for all of the compounds studied. After optimization of the extraction conditions, NNK and five metabolite compounds were analyzed in spiked cell cultures, confirming the applicability of the developed method. Excellent linearity was observed for all compounds (av R2 = 0.9942) and detection limits that ranged from 20 to 250 ng/mL. The average within-day and between day variations (% RSD) were 2.9 and 3.6%, respectively. This automated extraction and analysis method simplified the determination of the TSNA, requiring a total sample analysis time of only approximately 30 min.     

Picogram measurement of volatile n-alkanes (n-hexane through n-dodecane) in blood using solid-phase microextraction to assess nonoccupational petroleum-based fuel exposure

We describe here a new method for the analysis of alkanes ( n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-undecane, and n-dodecane) in blood using headspace solid-phase microextraction gas chromatography/mass spectrometry. This method is used to measure picogram per milliliter levels of n-alkanes in blood that may result from nonoccupational exposure to alkanes and other volatile nonpolar compounds from common sources such as petroleum-based fuel. This alkane signature is useful in distinguishing typical fuel biomarkers (e.g., benzene and toluene) from other confounding exposure sources such as cigarette smoke. Development of this method required special attention to sample handling as alkanes are not highly soluble in aqueous matrixes and exist as ubiquitous compounds found in many laboratory materials and the environment. In particular, significant n-hexane contamination ( approximately 0.4 ng/mL) occurred from collecting blood samples in vacutainers. This residue was removed by boiling the vacutainer stoppers in methanol followed by vacuum baking. For all the alkanes, the calculated accuracy demonstrated for the water-based standards ranged from 3.3% to 17% as deduced from the difference of the lowest and middle standards from the curve fit. Quality control data among runs over a 10 month period were found to vary from 14% to -29%, with a few exceptions. The resulting quantification limits for n-hexane through n-decane ranged from 0.069 to 0.132 ng/mL. In the analysis of 1200 blood samples from people with no known occupational exposure, median blood levels for all n-alkanes were below these quantification limits. n-Hexane levels above the method detection limit were, however, found in 1.3% of the samples.     

烟支内烟丝分布对卷烟烟气的影响

通过对不同规格平准器卷烟机卷制的烟支进行试验对比分析,研究了相同条件下烟支内烟丝分布对卷烟烟气指标以及逐口分析的影响。结果表明,使用6槽和3槽平准器卷烟机卷制的烟支,他们的烟支重量和吸阻均较稳定,但6槽与3槽平准器卷烟机卷制的烟支相比,其烟气中的焦油、烟碱和一氧化碳量更低,卷烟逐口间烟气的焦油、烟碱含量变化更小。     

In Situ Burning of Oil Spills

For more than a decade NIST conducted research to understand, measure and predict the important features of burning oil on water. Results of that research have been included in nationally recognized guidelines for approval of intentional burning. NIST measurements and predictions have played a major role in establishing in situ burning as a primary oil spill response method. Data are given for pool fire burning rates, smoke yield, smoke particulate size distribution, smoke aging, and polycyclic aromatic hydrocarbon content of the smoke for crude and fuel oil fires with effective diameters up to 17.2 m. New user-friendly software, ALOFT, was developed to quantify the large-scale features and trajectory of wind blown smoke plumes in the atmosphere and estimate the ground level smoke particulate concentrations. Predictions using the model were tested successfully against data from large-scale tests. ALOFT software is being used by oil spill response teams to help assess the potential impact of intentional burning.     

基于字符分割与新型LENET网络的票据识别算法

目的 针对现今烟草物流配送中心条烟分拣工作量大,条烟分拣系统在分拣中难免会有“多烟”“少烟”“错烟”等情况出现,为解决这一情况,提高分拣效率。方法 设计一种基于模板匹配提取目标条烟图像的条烟识别系统,为了提高图像质量其图像拍摄平台采用半封闭隧道装置,拍摄图像经预处理后,采用扫描模板尺寸确定、被扫描图像的扫描起始点固定、粗匹配再匹配的二次匹配方法来准确完整地提取目标条烟轮廓,而后经Harris角点检测算法提取条烟图像特征并建立数据库,在RBM模型算法的基础上实现条烟识别纠错。结果 经配送中心实验试用后表明,在速度为1.5 m/s的传送带上,该条烟识别系统纠错率为99.9%左右,误报率≤1/30000,系统工作稳定。结论 该条烟纠错系统很好地解决了条烟分拣出错情况,提高了条烟配送效率,满足烟草物流配送中心的纠错要求。     

Evaluation of hydrazine reduction by cellulose acetate filters using infrared tunable diode laser spectroscopy

Cellulose acetate (CA) filters have been investigated to determine their hydrazine (N2H4) breakthrough characteristics using a system based on tunable diode laser absorption spectroscopy (TDIAS). The breakthrough mass loading sorption curves for hydrazine were dependent on both the flow rate and the concentration. In experiments using a 4.5 ppmv hydrazine standard, the amounts of hydrazine retained by the CA filter were 4.25 microg at a flow rate of 2.82 L/min and 65 microg at a flow rate of 0.28 L/min. These loadings are much greater than the 31.5 ng/cigarette of hydrazine reported in smoke for unfiltered cigarettes. Further, CA filters exposed to four and eight puffs of smoke actually made the filter more efficient in retaining hydrazine compared to CA filters that had not been exposed to smoke. Therefore, if hydrazine is present in smoke at the levels reported in unfiltered cigarettes, all of the hydrazine would be trapped by the CA filter, and would be unable to break through during smoking. A unique feature of this analytical method is that the instrument does not require calibration after molecular parameters have been determined, in this case from previously acquired quantitative hydrazine FT-IR reference spectra.     

Toxic metals distribution in different components of Pakistani and imported cigarettes by electrothermal atomic absorption spectrometer

It was extensively investigated that a significant flux of toxic metals, along with other toxins, reaches the lungs through smoking. In present study toxic metals (TMs) (Al, Cd, Ni and Pb) were determined in different components of Pakistani local branded and imported cigarettes, including filler tobacco (FT), filter (before and after normal smoking by a single volunteer) and ash by electrothermal atomic absorption spectrometer (ETAAS). Microwave-assisted digestion method was employed. The validity and accuracy of methodology were checked by using certified sample of Virginia tobacco leaves (ICHTJ-cta-VTL-2). The percentages (%) of TMs in different components of cigarette were calculated with respect to their total contents in FT of all branded cigarettes before smoking, while smoke concentration has been calculated by subtracting the filter and ash contents from the filler tobacco content of each branded cigarette. The highest percentage (%) of Al was observed in ash of all cigarettes, with range 97.3-99.0%, while in the case of Cd, a reverse behaviour was observed, as a range of 15.0-31.3% of total contents were left in the ash of all branded cigarettes understudy.     

A new look at radicals in cigarette smoke

Radicals in cigarette smoke have been proposed to contribute to the harm caused by cigarette smoking. For the first time, using HPLC and high-resolution mass spectrometry analysis of stable radical adducts, we have identified specific radical species in cigarette smoke: 7 acyl and 11 alkylaminocarbonyl radicals. Their combined abundance measured in fresh whole smoke from a single 2R4F cigarette is approximately 225 nmol (1.4 x 10(17) radicals). The fiberglass Cambridge filter pad conventionally employed to separate the gas phase from mainstream smoke was found to reduce the apparent yield of these radicals, introducing artifacts of measurement. The long-accepted steady-state mechanism for the formation of carbon-centered radicals in cigarette smoke involving NO2 chemistry cannot account for these newly identified radicals, and it does not in general appear to be a major source of carbon-centered radicals in fresh mainstream cigarette smoke. Consequently, we suggest that the precise nature of radicals in cigarette smoke warrants reexamination.     

Copper Tannic Acid Coordination Nanosheet: A Potent Nanozyme for Scavenging ROS from Cigarette Smoke

The global tobacco epidemic is still a devastating threat to public health. Toxic reactive oxygen species (ROS) in the cigarette smoke cannot be efficiently eliminated by currently available cigarette filters. The resultant oxidative stress causes severe lung injury and further diseases. To tackle this challenge, herein, a novel copper tannic acid coordination (CuTA) nanozyme is reported as a highly active and thermostable ROS scavenger. The CuTA nanozyme exhibits intrinsic superoxide dismutase‐like activity, catalase‐like activity, and hydroxyl radical elimination capacity. These synergistic antioxidant abilities make the CuTA nanozyme a promising candidate for the improvement of commercial cigarette filters. Mouse model results show that commercial cigarettes loaded with CuTA nanozyme efficiently scavenge ROS in the cigarette smoke, reduce oxidative stress–induced lung inflammation, and minimize the resultant acute lung injury. The developed CuTA nanozyme offers an efficient ROS scavenger with multiple antioxidant ability and opens up new opportunities for the modification of cigarette filters to reduce the toxic effects of cigarette smoke.     

大气悬浮颗粒PM10对感烟火灾探测器本底效应研究

针对大气污染引起的悬浮颗粒,尤其是可吸入颗粒（PM10）对感烟火灾探测器产生的本底效应进行了分析，并以离子型感烟探测器为例对本底值进行计算，通过比较，显示不同程度的大气颗粒物污染对感烟探测器本底影响的差异极小，排除了感烟探测器在悬浮颗粒污染严重地区发生灵敏度和误报率升高的可能。确认目前各国感烟火灾探测器检测标准对灵敏度指标的规定可以统一，粒子计数等高分辨率颗粒浓度测量技术的发展将使该本底效应的影响不可忽略。     

Development of nitroxide radicals–containing polymer for scavenging reactive oxygen species from cigarette smoke

We developed a nitroxide radicals–containing polymer (NRP), which is composed of poly(4-methylstyrene) possessing nitroxide radicals as a side chain via amine linkage, to scavenge reactive oxygen species (ROS) from cigarette smoke. In this study, the NRP was coated onto cigarette filters and its ROS-scavenging activity from streaming cigarette smoke was evaluated. The intensity of electron spin resonance signals of the NRP in the filter decreased after exposure to cigarette smoke, indicating consumption of nitroxide radicals. To evaluate the ROS-scavenging activity of the NRP-coated filter, the amount of peroxy radicals in an extract of cigarette smoke was measured using UV–visible spectrophotometry and 1,1-diphenyl-2-picrylhydrazyl (DPPH). The absorbance of DPPH at 517 nm decreased with exposure to cigarette smoke. When NRP-coated filters were used, the decrease in the absorbance of DPPH was prevented. In contrast, both poly[4-(cyclohexylamino)methylstyrene]- and poly(acrylic acid)-coated filters, which have no nitroxide radical, did not show any effect, indicating that the nitroxide radicals in the NRP scavenge the ROS in cigarette smoke. As a result, the extract of cigarette smoke passed through the NRP-coated filter has a lower cellular toxicity than smoke passed through poly[4-(cyclohexylamino)methylstyrene]- and poly(acrylic acid)-coated filters. Accordingly, NRP is a promising material for ROS scavenging from cigarette smoke.     

Determination of nicotine and other minor alkaloids in international cigarettes by solid-phase microextraction and gas chromatography/mass spectrometry

Nicotine, nornicotine, anabasine, and anatabine are the most abundant alkaloids in tobacco. Along with the addictiveness of nicotine, other properties, including their occurrence in tobacco at relatively high concentrations, and as the primary precursors for the highly carcinogenic tobacco-specific nitrosoamines, make these chemicals important from a public health standpoint Therefore, developing a fast and accurate quantitative method to screen large numbers of cigarette samples for these alkaloids was important. This report describes the first use of headspace analysis using solid-phase microextraction combined with gas chromatography/mass spectrometry for the unambiguous detection of tobacco alkaloids. Detection and confirmation of each analyte isestablished by both chromatographic retention times and the ratio of reconstructed ion chromatogram peak areas from characteristic quantitation ion and confirmation ion. Twenty-eight cigarette brands from 14 countries were analyzed. Surprisingly, the minor alkaloids' response factors varied considerably among different styles of cigarettes. Accurate quantification was achieved using a three-point standard addition protocol. The standard addition approach was essential to obtain accurate measurements by minimizing matrix effects that would otherwise have contributed to quantitation bias. Significant differences in the alkaloid profiles were measured in the different cigarette brands. These results strongly suggest that such differences reflect variations associated with blend compositions, tobacco quality, and manufacturing practices.     

Real-time fourier transform-infrared analysis of carbon monoxide and nitric oxide in sidestream cigarette smoke

Fourier transform infrared (FT-IR) spectroscopy was compared directly to independent standard analytical techniques for the routine measurement of carbon monoxide (CO) and nitric oxide (NO) yields from cigarette sidestream smoke. The FT-IR instrument was configured in-line with a nondispersive infrared (NDIR) analyzer for CO analysis and a chemiluminescence (CL) analyzer for NO analysis to monitor the sidestream smoke from a single port of a linear smoking machine. A cold trap was inserted prior to the FT-IR to minimize the levels of vapor phase interferents, such as water. Univariate and multivariate regression analysis were evaluated for the prediction of cigarette yield from time-resolved spectral data at 1, 2, 4 and 8 cm-1 spectral resolution. Regressions were developed using three different spectral ranges including unique rotation-vibration lines, the R-branch, and the entire absorption band. As per standard methods, yields were calculated from the concentration traces generated during the smoke runs for five different cigarettes spanning the expected range of mainstream total particulate matter deliveries. The FT-IR traces for the smoke runs revealed improved temporal resolution yielding analytical information from smoke generated in between puffs. The performance between the validation methods and the FT-IR calibrations was statistically compared. In general, for the determination of CO, the FT-IR calibrations underestimated the yield measured by NDIR by less than 10%. For the NO measurement, the univariate FT-IR calibrations overestimated the NO yield measured by the CL analyzer, whereas the partial least squares (PLS) calibrations showed good agreement. PLS calibrations were developed for both analytes providing no significant difference when compared to the respective standard analytical techniques. Results for sidestream CO and NO yields for Kentucky reference cigarette 1R4F utilizing 8 cm-1 calibrations compared favorably to values reported elsewhere in the literature. Hence, calibration of the FT-IR system at 8 cm-1 spectral resolution clearly revealed the potential of this method, providing enhanced temporal resolution, simultaneous determination of several smoke components, and reduced complexity of the experimental setup in contrast to the standard techniques.