Simultaneous determination of four tobacco-specific N-nitrosamines in mainstream smoke for Chinese Virginia cigarettes by liquid chromatography-tandem mass spectrometry and validation under ISO and “Canadian intense” machine smoking regimes

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining four tobacco-specific N-nitrosamines (TSNAs) in mainstream smoke from Chinese Virginia cigarettes was developed. Mainstream cigarette smoke particulate matter was collected on a Cambridge filter pad, further extracted using 100 mM ammonium acetate after 100 μL internal standard addition, and subsequently analyzed with LC-MS/MS. The limit of detection for NNN, NNK, NAT and NAB were 0.006, 0.013, 0.003 and 0.021 ng mL⁻¹ respectively, with a linear calibration range spanning 1-200 ng mL⁻¹. Intra- and inter-day precision for four TSNAs ranged from 3.3% to 8.5% and 2.3% to 10.1%; recovery was between 89.1% and 104.9% for Chinese Virginia cigarettes. The proposed method was applied to evaluate TSNAs yields for 39 commercially available cigarettes in Chinese market under ISO and “Canadian intense” machine smoking regimes, on the ground that it comes closest to representing smoke deliveries from human smoking. Total TSNAs emissions are more than double under the Canadian regime. TSNAs:nicotine ratios were used in our assay to show any differences in yield from different brands. TSNAs:nicotine levels show more than a 10-fold difference across brands and types (Chinese Virginia cigarettes and blended cigarettes) in the Chinese market.     

Monitoring of the smoking process by multicommutation Fourier transform infrared spectroscopy

Nicotine was selected as the target molecule for monitoring of the smoking process by multicommutation Fourier Transform Infrared spectroscopy (FTIR). The method involved the use of CHCl₃ for on-line extraction of nicotine from tobacco, cigarette filters and tobacco ash from NH₄OH alkalinized samples, and absorbance measurement of the characteristic band at 1316 cm⁻¹ in the stopped-flow mode, by obtaining the peak area in the range between 1334 and 1300 cm⁻¹. Under the best operational conditions, the procedure developed provided a detection limit of 0.05 mg mL⁻¹ nicotine, corresponding to 0.5 mg g⁻¹ in the solid sample, a relative standard deviation less than 2.5%, and a sampling frequency of 12 determinations h⁻¹. It can be concluded that nicotine migrates in the smoke mainstream towards the filter during the smoking process. The smoking of cigarettes and cigars is different. Nicotine is retained weakly by both tobacco and filter in the case of cigarettes, and strongly by the unburned tobacco in cigars. The incomplete smoking of cigars and cigarettes reduces nicotine intake and thus reduces the additive effect.     

Optimization and validation of a gas chromatographic method for analysis of (RS,SR)-diastereoisomeric impurity in formoterol fumarate

The levels of 2-amino-9H-pyrido[2,3-b]indole (AalphaC or 2-amino-alpha-carboline), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC or 2-amino-3-methyl-alpha-carboline), 9H-pyrido[3,4-b]indole (norharman), and 1-methyl-pyrido[3,4-b]indole (harman) have been determined in the mainstream smoke condensate from three reference cigarettes, namely Kentucky reference 1R5F, Kentucky reference 2R4F, and CORESTA CM4. The amino-alpha-carbolines, and norharman and harman (beta-carbolines) can be classified as heterocyclic aromatic amines (HAAs) and are listed as biologically active agents in the mainstream smoke of cigarettes. For the analysis, the mainstream smoke condensate from cigarettes was collected on a filter pad, the analytes were isolated using solid-phase extraction (SPE), and quantified without derivatization on a GC-MS. Total amounts of carbolines found in the condensate increased from ultralight 1R5F to full-flavor CM4 cigarettes. The level of harman was about 250 ng/cigarette for the 1R5F cigarette and about 1025 ng/cigarette for the CM4 cigarette. Norharman was typically three times more abundant than harman. The AalphaC was found at about 10 times lower level compared to harman, and MeAalphaC was about 50 times lower than harman. The use of reference cigarettes can provide a common measure for laboratories to assess carboline amounts among cigarette brands.     

Selective determination of tobacco‐specific nitrosamines in mainstream cigarette smoke by GC coupled to positive chemical ionization triple quadrupole MS

A rapid method for the selective determination of four kinds of tobacco‐specific nitrosamines, N‐nitrosonornicotine, N‐nitrosoanatabine, N‐nitrosoanabasine and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone, in mainstream cigarette smoke was developed by GC coupled to positive chemical ionization triple‐quadrupole MS. After mainstream cigarette smoke was collected on a cambridge filter pad, the particulate matter was extracted with 0.1 M HCL aqueous solution, cleaned by positive cation‐exchange solid extraction, and finally injected into GC–MS/MS using isotopically labeled analogues as internal standards. Excellent linearity was obtained over the concentration range of 0.5–200.0 ng mL^?1 for all tobacco‐specific nitrosamines with values for correlation coefficient between 0.9996–0.9999. Limits of detection of each tobacco specific nitrosamine varied from 0.023–0.028 ng cig^?1, and lower limits of quantification varied from 0.077–0.093 ng cig^?1. The recovery of each tobacco specific nitrosamine was from 90.0–109.0%. The relative standard deviations of the intra‐day and inter‐day precisions were 3.1–5.8 and 3.9–6.6, respectively. This method was applied to reference and domestic cigarettes. The result showed that the method was consistent with traditional methods and can be used as an effective approach for the routine analysis of tobacco‐specific nitrosamines.     

Analysis of Vinyl Chloride in Vapor Phase of Mainstream Cigarette Smoke by GC–MS Under ISO and Health Canada Intensive Machine Smoking Regimens

Vinyl chloride (VC) in the vapor phase of mainstream cigarette smoke was determined under both International Organization for Standardization (ISO) and Health Canada intensive (HCI) machine smoking regimens, which was suspected to be carcinogenic compound. VC was collected by passing the mainstream cigarette smoke through a Cambridge Filter Pad (CFP) into cryogenic traps containing methanol. The impinger solutions were fortified with VC-d ₃ and analyzed by GC–MS. Limits of detection for vinyl chloride was 0.9 ng mL^?1 with the recovery in the range of 93.2–98.4 %. Moreover, the intra-day and inter-day precision was 7.39 and 9.77 %, respectively. Under HCI machine smoking regimen, the vinyl chloride yields in vapor phase of mainstream cigarette smoke were much higher and the average increase was greater than 100 % compared with those under ISO smoking condition.     

GC–MS Analysis of Hydrogen Sulfide,Carbonyl Sulfide,Methanethiol, Carbon Disulfide,Methyl Thiocyanate and Methyl Disulfide in Mainstream Vapor Phase Cigarette Smoke

A method is described for the simultaneous analysis of hydrogen sulfide, carbonyl sulfide, methanethiol, carbon disulfide, methyl thiocyanate and methyl disulfide in mainstream vapor phase (MVP) cigarette smoke by gas chromatography–mass spectrometry. The fresh MVP smoke was collected in a gas bag, followed by injection of a 50 μL gas sample into the GC inlet via an automatic six-port valve. The separation was on a CP-PoraPLOT Q column and MS was operated in SIM mode. It was found that while carbonyl sulfide and carbon disulfide are very stable in the gas bag, hydrogen sulfide, methanethiol, methyl disulfide and methyl thiocyanate are extremely reactive and their levels increase or decrease drastically with the storage time in the gas bag. These results suggest that there is an absolute need to analyze the smoke sample as quickly as possible. Maintaining a precise time after the smoke collection is a key factor in order to obtain reproducible results. In this study, all the samples are injected within 2 min after MVP smoke was collected in the bag. Under smoke conditions of 60 mL puff of 2 s duration every 30 s, 12 brands of commercial cigarettes and Kentucky Reference 2R4F cigarettes were analyzed. Average values of three replicates of the 2R4F cigarettes were 31.6 μg cigt^?1 hydrogen sulfide, 40.7 μg cigt^?1 carbonyl sulfide, 25.6 μg cigt^?1 methanethiol, 2.2 μg cigt^?1 carbon disulfide, 23.7 μg cigt^?1 methyl thiocyanate and 17.6 μg cigt^?1 methyl disulfide. All other types of analyzed cigarettes show a similar quantitative distribution for these analytes.     

A micro trapping system coupled with a high performance liquid chromatography procedure for methylamine determination in both tissue and cigarette smoke

Both endogenous and exogenous methylamine have been found to be involved in many human disorders. The quantitative assessment of methylamine has drawn considerable interest in recent years. Although there have been many papers about the determination of methylamine, only a few of them involved cigarette smoke or mammalian tissue analysis. The major hurdles of the determination of methylamine are the collection of methylamine from samples and the differentiation of methylamine from the background compounds, e.g., biogenic amines. We have solved this problem using a micro trapping system coupled with an HPLC procedure. The interference from other biogenic amines has been avoided. The high selectivity of this method was achieved using four techniques: distillation, trapping, HPLC separation and selective detection. The chromatograms of both mouse tissues and cigarette smoke are simple, with only a few peaks. The method is easy and efficient and it has been validated and applied to the determination of methylamine in tissues of normal CD 1 mice and cigarette smoke. The methylamine contents were determined to be approximately 268.3 ng g⁻¹ in the liver, 429.5 ng g⁻¹ in the kidney and 547.4 ng g⁻¹ in the brain respectively. The methylamine in the cigarette smoke was approximately 213 ng to 413 ng per cigarette. These results in tissues and in cigarette smoke were found to be consistent with the data in the previous literature. To the best of our knowledge, this is the first report on a method suitable for methylamine analysis in both mammalian tissue and cigarette smoke.     

Magnetic solid‐phase extraction of tobacco‐specific N‐nitrosamines using magnetic graphene composite as sorbent

Tobacco‐specific N‐nitrosamines are carcinogenic components in mainstream cigarette smoke. To explore tobacco‐specific N‐nitrosamine release levels in cigarettes, a magnetic solid‐phase extraction procedure using magnetic graphene composite as sorbent for fast enrichment of tobacco‐specific N‐nitrosamine was developed. Under optimal conditions, a tobacco‐specific N‐nitrosamine determination method was successfully proposed by combining magnetic solid‐phase extraction procedure and high‐performance liquid chromatography coupled with tandem mass spectrometry. The method's limit of detection for tobacco‐specific N‐nitrosamines in mainstream cigarette smoke ranged from 0.018 to 0.057 ng/cigarette. Good linearities were obtained with correlation coefficients above 0.9992. The accuracies of tobacco‐specific N‐nitrosamines in a spiked mainstream cigarette smoke sample were from 89.3 to 109.4%, with a relative standard deviation of less than 11.2%. The proposed method has the merits of rapidity and high sensitivity. Finally, the method was successfully applied to tobacco‐specific N‐nitrosamine analysis in real samples.     

Simultaneous determination of nicotine and 3-vinylpyridine in single cigarette tobacco smoke and in indoor air using direct extraction to solid phase

The aim of the present study was to develop a new analytical method of chromatographic determination of two important markers of ETS exposure: nicotine and 3-vinylpyridine (3-ethenylpyridine, 3-EP) in mainstream (MS) and sidestream (SS) smoke of one single cigarette and in indoor air using direct solid phase extraction combined with gas chromatography. The method can be utilised for both nicotine and 3-EP determination in SS and MS of one single cigarette as well as it allows for a precise determination of compound distribution in indoor air. The application of the same analytical method for both kinds of samples allows anticipating indoor air distribution of both analysed compounds in a very precise way. The precision of the method (calculated as a relative standard deviation) was 9.78% for nicotine and 2.67% for 3-EP; whereas the accuracy (evaluated by a recovery study conducted at three different levels) was 70.1 and 87.3%, respectively. The limit of detection was 0.06 µg per cigarette for both nicotine and 3-EP. The method was evaluated by determining the compounds of interest in two commercially available brands of cigarettes as well as in the reference cigarettes 3R4F and also in indoor air polluted with tobacco smoke. Determined levels of compounds of interest in MS varied from 586 to 772 (nicotine) µg per cigarette and from 3.5 to 10.7 (3-EP) µg per cigarette. In SS smoke the level varied from 14,370 to 22,590 (nicotine) µg per cigarette and from 185 to 550 (3-EP) µg per cigarette, whereas levels in indoor air polluted with tobacco smoke varied from 50.1 to 157.3 (nicotine) µg m^?3and from 7.7 to 20.8 (3-EP) µg m^?3.     

Adsorptive stripping voltammetry of nickel with 1-nitroso-2-napthol using a bismuth film electrode

A sensitive procedure is presented for the voltammetric determination of nickel. The procedure involves an adsorptive accumulation of nickel 1-nitroso-2-napthol (NN) complex on a bismuth film electrode prepared ex situ by electrodeposition. The most suitable operating conditions and parameters such as pH, ligand concentration (C_NN), adsorptive potential (E_ads), adsorptive time (t_ads), scan rate and others were selected and the determination of nickel in aqueous solutions using the standard addition method was possible. The adsorbed Ni-NN complex gives a well defined cathodic stripping peak current at −0.70 V, which was used for the determination of nickel in the concentration range of 10.0-70.0 μg L⁻¹ (pH 7.5; C_NN 6.5 μmol L⁻¹; E_ads −0.30 V; t_ads 60 s) with a detection limit of 0.1 μg L⁻¹. The relative standard deviation for a solution containing 10.0 μg L⁻¹ of Ni(II) was 3.5% (n = 4). The proposed method was validated determining Ni(II) in certified reference waste water (SPS-WW1) and Certified Reference Water for Trace Elements (TMDA 51.3) with satisfactory results. Then lake water samples were analyzed.     

In vivo quantification and pharmacokinetic studies of cotinine in mice after smoke exposure by LC–MS/MS

A sensitive analytical method was developed and validated for the quantification of cotinine in mouse plasma after exposure to smoke of 0.5, 1.0, and 1.5 commercially available cigarettes, using liquid chromatography tandem mass spectrometry. The method was validated over a linear concentration range of 0.075–20.0 ng/mL with the R² value being higher than 0.99. Both the precision (coefficient of variation; %) and accuracy (relative error; %) were within acceptable criteria of <15%. The lower limit of quantification (LLOQ) for cotinine was 0.075 ng/mL with sufficient specificity, accuracy, and precision. Following exposure to 0.5, 1.0, and 1.5 cigarette smoke, it was observed that the AUC and the C_max increased linearly as the doses increased. The pharmacokinetics of cotinine was found linear for the range of 0.5–1.5 commercial cigarette smoke. The quantification of the concentration of cotinine in mouse plasma after smoke exposure will facilitate future behavioral and toxicological experiments in animals and may prove useful in predicting cotinine levels in humans during smoking.     

Determination of cobalamins (hydroxo-, cyano-, adenosyl- and methyl-cobalamins) in seawater using reversed-phase liquid chromatography with diode-array detection

A high-performance liquid chromatography method was developed for the separation and determination of four cobalamins in seawater. Chromatographic separation was performed on a reversed-phase discovery RP-amide C₁₆ column with buffer potassium dihydrogenphosphate and acetonitrile as the mobile phases in linear gradients elution mode. Cobalamins were previously preconcentrated in C₁₈ resins cartridges. Detection was performed using UV-diode array detector in a range of λ of 200–400 nm. The method showed to be linear over a range of 1–300 ng mL⁻¹ with acceptable precision and accuracy. The detection limits ranged between 0.07 pg mL⁻¹ for 5′-deoxyadenosylcobalamin and 0.5 pg mL⁻¹ for hydroxocobalamin. The mean cobalamins recoveries for direct determination ranged between 76 and 93% for hydroxo-, cyano- and methylcobalamin, while the recovery for 5′-deoxyadenosylcobalamin was only 31% suggesting that the preconcentration method was not valid for this cobalamin. The method was successfully applied to coastal seawater where the concentrations ranged from 4.2 to 7.3 ng L⁻¹ for hydroxo-, 1.4–3.9 ng L⁻¹ for cyano-, 2.1–4.6 ng L⁻¹ for 5′-deoxyadenosyl- and 33–83.5 ng L⁻¹ for methylcobalamin.     

快速分离柱高效液相色谱法测定卷烟主流烟气中的主要羰基化合物

选择2,4-二硝基苯肼(DNPH)为羰基化合物的衍生化试剂,建立了快速分离柱高效液相色谱测定卷烟主流烟气中8种羰基化合物的方法。采用经2,4-二硝基苯肼酸性溶液处理过的剑桥滤片捕集烟气,再用含2%（体积分数）吡啶的乙腈溶液进行萃取,以ZORBAX Stable Bound色谱柱(50 mm×4.6 mm,1.8 μm)进行快速分离,最后由二极管阵列检测器于365 nm下进行检测。该方法的回收率为89.1%～99.2%,相对标准偏差（RSD)在6.0%以下。该方法分析时间短,流动相消耗少,且操作简便、重复性好、回收率高。     

固相萃取-超高效液相色谱法测定卷烟主流烟气中丙烯酰胺

建立了一种固相萃取-超高效液相色谱法(SPE-UPLC)快速检测主流烟气中丙烯酰胺的方法。使用剑桥滤片和吸收瓶捕集主流烟气后,蒸馏水做萃取溶剂,采用C18固相萃取小柱对样品液进行纯化,用UPLC检测,外标法定量。UPLC方法采用ACQUITY UPLCTMBEH C181.7μm 2.1×50 mm色谱柱,柱温30℃,流动相为V(乙腈)∶V(水)=6∶94,流速为0.15 mL/min,紫外检测器(TUV)检测波长为202 nm,分析时间为6 min。烤烟型香烟主流烟气中丙烯酰胺的含量为4.75μg/cig。方法的线性范围为0.1~10 mg/mL,线性相关系数为0.9999;平均回收率为98.7%;检出限为10 ng/mL(S/N=3);相对标准偏差为2.3%。该方法适合主流烟气中丙烯酰胺的快速检测。     

Chemical Studies on Tobacco Smoke. LXI. Volatile Pyridines: Quantitative Analysis in Mainstream and Sidestream Smoke of Cigarettes and Cigars

Abstract

A quantitative analytical method has been developed for volatile pyridines in tobacco smoke using pyridine- ¹⁴C as an internal standard. Mainstream and sidestream smoke are trapped in 0.05 N sulfuric acid anti partitioned with chloroform. The acidic portion is then concentrated, adjusted to pH 3 and extracted with chloroform. Methanol and a trace of 2N sulfuric acid are added to this chloroform portion and the mixture is carefully concentrated.

A trace of sodium hydroxide is added and the final concentrate is analyzed by gas chromatography. Twenty-one volatile pyridines have been identified. The mainstream stroke of a popular. 85-mm U.S. cigarette without filter tip contained the following major pyridines: 33.4 μg pyridine, 12.3 μg α-picoline, 24.2 μg. β+γ-picoline, 7.6 μg 3-ethylpgridine, 5.9 μg: 4-ethylpyridine, and 23.3 μg 3,4-lutidine + 3-vinylpyridine. The sidestream smoke of this cigarette contained up to 28 times higher concentrations of pyridines.

Volatile pyridines were also determined in the mainstream smoke of other non-filter cigarettes, filter cigarettes, little cigars and, a non-tobacco cigarette. Of special interest were the much greater quantities of volatile pyridines in the mainstream and sidestream of cigars in comparison to those of cigarettes. The ratio of pyridines in sidestream to those in mainstream was 5 to 50 in large cigars. Selective filtration was observed with both cellulose acetate and charcoal filter tips. The analytical method resulted in reproducible values for pyridine, with an average recovery rate of more than 90%. In addition to pyridines, two pyrazines were also isolated from the smoke. Processed tobacco contained minute amounts of some volatile pyridines.     

Determination of seven nitrobenzene compounds in mainstream cigarette smoke with heart-cutting two-dimensional gas chromatography

A heart-cutting two-dimensional gas chromatography (GC) method was developed for the determination of nitrobenzene compounds (NBCs) in mainstream cigarette smoke. For the method, the particulate matter of cigarette smoke was extracted with cyclohexane, purified with a silica solid-phase extraction (SPE) cartridge and analyzed by heart-cutting two-dimensional GC equipped with two electron capture detectors. The heart-cutting two-dimensional GC was achieved by a single-column GC oven equipped with a microfluidic pressure balanced device (Deans switch). Two-dimensional GC was compared to single-dimensional GC and found to be clearly better for the separation of seven NBCs from a complex smoke matrix. The limits of detection ranged from 1.28 to 9.83 ng/mL, spiked recoveries were between 88.3 and 106.8% and relative standard deviation ranged from 2.79 to 12.78%. The NBCs yields of six kinds of Chinese and international cigarettes brands, which were all smoked according to two smoking protocols (International Organization for Standardization and Health Canada Intense smoking regimens), were determined and compared.     

Near infrared reflectance spectrometry classification of cigarettes using the successive projections algorithm for variable selection

This paper proposes a methodology for cigarette classification employing Near Infrared Reflectance spectrometry and variable selection. For this purpose, the Successive Projections Algorithm (SPA) is employed to choose an appropriate subset of wavenumbers for a Linear Discriminant Analysis (LDA) model. The proposed methodology is applied to a set of 210 cigarettes of four different brands. For comparison, Soft Independent Modelling of Class Analogy (SIMCA) is also employed for full-spectrum classification. The resulting SPA-LDA model successfully classified all test samples with respect to their brands using only two wavenumbers (5058 and 4903 cm⁻¹). In contrast, the SIMCA models were not able to achieve 100% of classification accuracy, regardless of the significance level adopted for the F-test. The results obtained in this investigation suggest that the proposed methodology is a promising alternative for assessment of cigarette authenticity.     

Analysis of Vinyl Chloride in Vapor Phase of Mainstream Cigarette Smoke by GC–MS Under ISO and Health Canada Intensive Machine Smoking Regimens

Vinyl chloride (VC) in the vapor phase of mainstream cigarette smoke was determined under both International Organization for Standardization (ISO) and Health Canada intensive (HCI) machine smoking regimens, which was suspected to be carcinogenic compound. VC was collected by passing the mainstream cigarette smoke through a Cambridge Filter Pad (CFP) into cryogenic traps containing methanol. The impinger solutions were fortified with VC-d ₃ and analyzed by GC–MS. Limits of detection for vinyl chloride was 0.9 ng mL⁻¹ with the recovery in the range of 93.2–98.4 %. Moreover, the intra-day and inter-day precision was 7.39 and 9.77 %, respectively. Under HCI machine smoking regimen, the vinyl chloride yields in vapor phase of mainstream cigarette smoke were much higher and the average increase was greater than 100 % compared with those under ISO smoking condition.

     

Determination of Polycyclic Aromatic Hydrocarbons in Tobacco Smoke by Ionic Liquid Enrichment and Gas Chromatography – Mass Spectrometry

A rapid, efficient and environmentally friendly method based on the ionic liquid (IL) 1-hexyl-3-methylimidazolium hexafluorophosphate ([C₆MIM][PF₆]) was developed for the determination of 16 polycyclic aromatic hydrocarbons (PAHs) in mainstream tobacco smoke. This technique combined ionic liquid (IL) enrichment with solvent reverse extraction for the replacement of solid phase extraction and rotary evaporation in the traditional method and enriched PAHs in the organic solvent. Several parameters, including the type of ionic liquid, volume of ionic liquid and water, extraction time, vortex time and reverse extraction time, were optimized. After pretreatment, the analytes were analyzed by gas chromatography-mass spectrometry (GC-MS) using selective ion monitoring (SIM). Satisfactory results were achieved when this method was applied to determine PAHs in mainstream tobacco smoke. The calibration curves were linear with correlation coefficients ranging from 0.9955 to 0.9999 at concentration levels of 10–800?µg?L^?1, and the relative standard deviations of the optimized method were between 0.7% and 5.3%. The limits of detection were 0.01–0.6?ng cig^?1, and the recoveries of the compounds were 80.2–118%. A comparison of this protocol with literature methods demonstrated that the proposed procedure provides accurate and reliable sample-treatment for the determination of PAHs in tobacco samples.