Investigation of exposure biomarkers in human plasma following differing levels of tobacco-specific N-nitrosamines and nicotine in cigarette smoke.

Tobacco-specific N-nitrosamines (TSNAs) are strong carcinogens widely found in tobacco products, environmental tobacco smoke, lake, and wastewater. The main objective of this study was to investigate the effects of cigarette smoke with different yields of TSNAs (NNK, NNN, NAT, NAB) and nicotine on the levels of biomarkers of exposure in smokers' plasma. Three hundred healthy volunteers were recruited comprising 60 smokers of each of 3 mg, 8 mg and 10 mg ISO tar yield cigarettes and 60 smokers who smoked 10 mg, 8 mg, and 3 mg for 14 days sequentially and 60 non-smokers. All study participants were male, aged from 21 to 45 years old, and were recruited from a same unit in Hebei, China. We measured the levels of NNAL, NAT, NNN, NAB and cotinine in plasma from 240 smokers and 60 non-smokers using a novel method established by online two-dimensional solid phase extraction-liquid chromatography-tandem mass spectrometry. The results showed that NNAL, NAT, NNN, NAB and cotinine in the plasma of smokers smoking cigarette with low TSNAs and nicotine were lower than that with high TSNAs and nicotine. When smokers switched from higher to lower TSNA yields of cigarettes, their plasma NNAL, NAT, NNN, NAB levels significantly decreased. The plasma concentrations of NNAL were significantly correlated with those of cotinine, NNN, NAT and NAB for smokers (p < 0.001). Similarly, the plasma concentrations of cotinine were significantly correlated with those of NNN, NAT and NAB for smokers (p < 0.001). The plasma NNAL, NAT, NNN, NAB and cotinine levels for smokers were significantly higher than those for non-smokers. These findings suggested that the total NNAL, NNN, NAT, NAB and cotinine in plasma were valid and reliable biomarkers for human exposure to TSNAs and nicotine.

Harmful chemicals of heat not burn product and its induced oxidative stress of macrophages at air-liquid interface: Comparison with ultra-light cigarette.

BACKGROUND: Harmful and potential harmful chemicals (HPHCs) and oxidative stress of macrophages are major factors responsible for smoking-caused chronic respiratory diseases. However, comparisons of HPHCs among heat not burn (HnB) product and ultra-light cigarette and their induced oxidative stress of macrophages have not been investigated. AIM: The study detected HPHCs deliveries from HnB and ultra-light and measured their induced oxidative stress of macrophages cultured at air-liquid interface (ALI). METHODS: Total particulate matter, tar and 28 chemicals delivered from HnB, ultra-light and 3R4F cigarettes were determined. Mouse mononuclear macrophages at ALI were exposed to the aerosol of three tobacco products. Cell viability was measured by MTT assay. Reduced glutathione was detected by colorimetry method. Reactive oxygen species (ROS) was determined by fluorescence method. RESULTS: The results showed levels of 26 common HPHCs from both HnB product and ultra-light cigarette were less than that from 3R4F cigarette. HnB product delivered formaldehyde, acetaldehyde, propanal, butyraldehyde and crotonaldehyde more than ultra-light cigarette. The levels of 21 HPHCs were lower in the HnB product compared to the ultra-light cigarette. At the same exposure dose and time, the order of cell viability induced by aerosol of that was HnB > ultra-light > 3R4F, the order of content of intracellular reduced glutathione induced by aerosol of that was HnB > ultra-light > 3R4F. It showed no significant difference of ROS level between ultra-light and HnB in each designed exposure dose. HnB induced more ROS than ultra-light cigarette in each designed exposure time. CONCLUSION: Conclusively, most HPHCs from HnB were lower than that from ultra-light, while certain harmful chemicals were higher than ultra-light, e.g., carbonyl compounds. HnB-induced oxidative stress of macrophages is less than ultra-light cigarette.

Simultaneous analysis of nine aromatic amines in mainstream cigarette smoke using online solid-phase extraction combined with liquid chromatography-tandem mass spectrometry.

A fully automated analytical method was developed and validated by this present study. The method was based on two-dimensional (2D) online solid-phase extraction liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) to determine nine aromatic amines (AAs) in mainstream smoke (MSS) simultaneously. As a part of validation process, AAs yields for 16 top-selling commercial cigarettes from China market were evaluated by the developed method under both Health Canada Intensive (HCI) and ISO machine smoking regimes. The gas phase of MSS was trapped by 25 mL 0.6 M hydrochloric acid solution, while the particulate phase was collected on a glass fiber filter. Then, the glass fiber pad was extracted with hydrochloric acid solution in an ultrasonic bath. The extract was analyzed with 2D online SPE-LC-MS/MS. In order to minimize the matrix effects of sample on each analyte, two cartridges with different extraction mechanisms were utilized to cleanup disturbances of different polarity, which were performed by the 2D SPE. A phenyl-hexyl analytical column was used to achieve a chromatographic separation. Under the optimized conditions, the isomers of p-toluidine, m-toluidine and o-toluidine, 3-aminobiphenyl and 4-aminobiphenyl, and 1-naphthylamine and 2-naphthylamine were baseline separated with good peak shapes for the first time. The limits of detection for nine AAs ranged from 0.03 to 0.24 ng cig-1. The recovery of the measurement of nine AAs was from 84.82 to 118.47%. The intra-day and inter-day precisions of nine AAs were less than 10 and 16%, respectively. Compared with ISO machine smoking regime, the AAs yields in MSS were 1.17 to 3.41 times higher under HCI machine smoking regime. Graphical abstract New method using online SPE-LC/MS/MS for analysis of aromatic amines in mainstream cigarette smoke.

Fully automated analysis of four tobacco-specific N-nitrosamines in mainstream cigarette smoke using two-dimensional online solid phase extraction combined with liquid chromatography-tandem mass spectrometry.

A fully automated method for the detection of four tobacco-specific nitrosamines (TSNAs) in mainstream cigarette smoke (MSS) has been developed. The new developed method is based on two-dimensional online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE/LC-MS/MS). The two dimensional SPE was performed in the method utilizing two cartridges with different extraction mechanisms to cleanup disturbances of different polarity to minimize sample matrix effects on each analyte. Chromatographic separation was achieved using a UPLC C18 reversed phase analytical column. Under the optimum online SPE/LC-MS/MS conditions, N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were baseline separated with good peak shapes. This method appears to be the most sensitive method yet reported for determination of TSNAs in mainstream cigarette smoke. The limits of quantification for NNN, NNK, NAT and NAB reached the levels of 6.0, 1.0, 3.0 and 0.6 pg/cig, respectively, which were well below the lowest levels of TSNAs in MSS of current commercial cigarettes. The accuracy of the measurement of four TSNAs was from 92.8 to 107.3%. The relative standard deviations of intra-and inter-day analysis were less than 5.4% and 7.5%, respectively. The main advantages of the method developed are fairly high sensitivity, selectivity and accuracy of results, minimum sample pre-treatment, full automation, and high throughput. As a part of the validation procedure, the developed method was applied to evaluate TSNAs yields for 27 top-selling commercial cigarettes in China.

Changes in TSNA contents during tobacco storage and the effect of temperature and nitrate level on TSNA formation.

Samples of burley, sun-cured, and flue-cured tobacco from the main producing areas of relevant tobacco types in China were collected to study the changes in tobacco-specific nitrosamine (TSNA) contents during storage and to investigate the effect of storage temperature and tobacco nitrate level on TSNA formation of cured tobacco. Contents of TSNAs in burley and sun-cured tobacco increased substantially during 1 year under natural storage environment, with total TSNA content increasing about 215% for both tobacco types. The most rapid increase occurred during the high temperature season. Temperature had a significant promoting effect on TSNA formation during storage. Storage temperature as high as 27 °C for 12 days was enough to induce the increase of TSNA formation, while the most significant effect was shown when the temperature was above 30 °C. The increased rate of accumulation became greater as the temperature increased. Total TSNA content in air-cured burley tobacco after the treatment of 60 °C for 24 days was 772% higher than that in the low temperature control. Different types of tobacco showed different results in terms of the response of TSNA formation to high temperature. TSNA formation in flue-cured tobacco did not increase after high-temperature treatment for 36 days, while burley and sun-cured tobacco saw a dramatic increase of TSNA content. This difference could be explained by the fact that burley tobacco and sun-cured tobacco usually had more than 10 times the nitrate content than flue-cured tobacco. As the nitrate nitrogen increased in cured burley tobacco, TSNA formation during leaf storage at high temperature significantly increased. Addition of nitrate onto flue-cured tobacco to the level equivalent to burley tobacco followed by high-temperature treatment increased the TSNA concentration comparable to burley tobacco. The interaction between high temperature and abundant nitrate content in cured tobacco could be responsible for TSNA formation during storage.

Determination of four tobacco-specific nitrosamines in mainstream cigarette smoke by gas chromatography/ion trap mass spectrometry.

A specific and sensitive method was developed and validated to quantitatively analyze four tobacco-specific nitrosamines (TSNAs) in the particulate phase of mainstream cigarette smoke. Cigarette smoke particulate matter was collected according to ISO 4387. The particulate matter was extracted with acetic ether, cleaned up with a Supelclean ENVI-Carb silod-phase extraction (SPE) cartridge, concentrated under the protection of nitrogen and analyzed by gas chromatography (GC)/ion trap mass spectrometry (MS) with a very-low-flow-loss column (VF-17 MS) in MS(n) mode using N-nitrosopentyl-3-picolylamine (NNPA) as an internal standard. TSNAs were identified by chromatographic retention time, matching the spectra of the standards and the samples and the consistency of the ratio of the abundance of the ions detected in the standards and the samples. Limits of detection of each TSNA varied from 0.01 to 0.06 ng/cig. A linear calibration range for this method is large enough to measure TSNA levels in cigarette smoke. The recovery of each TSNA was from 91.5 to 102.7%. The method achieved excellent reproducibility (RSD: 1.8-4.8% for intra-assay, 3.4-7.1% for inter-assay). It also shows no evidence of artifact formation. This method is very suitable for the routine detection of TSNAs in mainstream cigarette smoke.