Development of a heart-cutting supercritical fluid chromatography-high-performance liquid chromatography coupled to tandem mass spectrometry for the determination of four tobacco-specific nitrosamines in mainstream smoke.

This paper proposed a newly developed heart-cutting two-dimensional supercritical fluid chromatography-high-performance liquid chromatography system coupled to tandem mass spectrometry (SFC-HPLC-MS/MS) for the determination of four tobacco-specific nitrosamines (TSNAs) in cigarette mainstream smoke. The orthogonality of five SFC columns and two HPLC columns was evaluated. The 1-AA column was applied for the first dimensional (1D) SFC separation to isolate the target compounds from the complex cigarette smoke matrices, and a trapping column in conjunction with an isocratic pump was employed to capture the 1D elutes. Then, the trapped 1D elutes were transferred into the C18 column through a two-position/six-port valve for the second dimensional (2D) analysis. The ion suppression was significantly reduced by the established SFC-HPLC system; meanwhile, the matrix interferences were eliminated as the results demonstrated. A dynamic range between 0.1 and 20 ng/mL was achieved with LOQs of 0.72 µg/cig for N-nitrosonornicotine (NNN), 0.66 µg/cig for nicotine-derived nitrosamine ketone (NNK), 0.81 µg/cig for N-nitrosoanatabine (NAT), and 0.39 µg/cig for N-nitrosoanabasine (NAB). All the results revealed that the presented method exhibited good repeatabilities and recoveries and could be used as a rapid and reliable approach for routine analysis of TSNAs in mainstream smoke.

Investigating the proteomic expression profile of tobacco (Nicotiana tabacum) leaves during four growth stages using the iTRAQ method.

Despite the importance of tobacco (Nicotiana tabacum) in agriculture and model organism investigations, the proteomic changes that occur in the tobacco leaf as it matures remain to be explored. In this study, an isobaric tags for relative and absolute quantification (iTRAQ) strategy was applied to investigate the proteomic profiles of K326 and Honghua Dajinyuan (HD) tobacco leaves at four growth stages. The proteomic profile varied with growth stage in both K326 and HD. Gene ontology (GO) classification was used to identify the biological processes that showed the greatest changes in protein expression between growth stages of HD and K326. Moreover, the number of differentially expressed proteins was greater in HD than in K326, especially during the rosette growth stage and the fast-growing stage. The galactose metabolism and glycosphingolipid biosynthesis-globo series pathways appeared only during the rosette growth stage of HD. It therefore appears that these pathways may be correlated with tobacco mosaic disease. The identification of these pathways should prove useful in investigations of the pathogenesis of tobacco mosaic virus. Graphical abstract ᅟ.

Quantitative method for analysis of tobacco-specific N-nitrosamines in mainstream cigarette smoke by using heart-cutting two-dimensional liquid chromatography with tandem mass spectrometry.

A heart-cutting two dimensional liquid chromatography coupled with tandem mass spectrometry method was developed for the analysis of tobacco-specific N-nitrosamines (TSNAs) at low concentration level in Virginia-type cigarette smoke. A strong cation exchange column was utilized for the first dimensional separation, which effectively removed acidic and neutral components in the smoke, followed by a reversed phase liquid chromatography coupled with tandem mass spectrometric analysis. To capture components of the TSNAs in the effluent on the trapping column, a compensating pump was applied for online dilution and pH adjustment during the period of the TSNAs fraction transferring and enrichment. Highly sensitive determination of the TSNAs in mainstream cigarette smoke was achieved by isotope deuterated internal standards under the multiple reaction monitoring mode. Compared with traditional methodologies, the method was almost no matrix interference. Limits of quantity for the TSNAs were within 0.027-0.094 ng/mL, and the results showed good reproducibility and accuracy. Finally, the new method was applied for analysis of the Kentucky reference cigarettes and the results agreed well with joint experiments of Cooperation Centre for Scientific Research Relative to Tobacco.

Preparation and characterization of organic-functionalized mesoporous silica as heavy metal adsorbents.

Novel organic-inorganic hybrid mesoporous materials have attracted much attention because the functional organic groups can bring these materials special properties, such as selective absorption and new catalytic properties. In this paper, two kind of bis(triethoxysilyl) organic skeleton (EtO)3Si-(CH2)3-NH-C(O)-X-C(O)-NH-(CH2)3-Si(OEt)3 (X = 1,4-benzene (BSPB), 2,5-thienyl (BSPT)) precursors were synthesized firstly. Then bridged organic-inorganic hybrid mesoporous materials were prepared by direct grafting on MCM-41. The MCM-41 was obtained under basic conditions using cetyltrimethylammonium bromide (CTAB) as a template with tetraethyl orthosilicate. FT-IR spectra showed the acylamino, carbonyl, phenyl and thienyl groups had been introduced into the materials. The structures of these materials were further confirmed by X-ray diffraction (XRD) and the N2 adsorption-desorption. In addition, the adsorption capacity of heavy metal ions such as mercury, cadmium, and lead, was evaluated. The results indicated that the functionalized mesoporous materials with high porosity and specific surface area were successfully prepared, and the new prepared materials exhibited good adsorption capacity for heavy metal ions Hg2+, Cd2+, and Pb2+.

Determination of hazardous volatile organic compounds in the Hoffmann list by ion-molecule reaction mass spectrometry.

RATIONALE: Off-line gas or liquid chromatographic mass spectrometry techniques are the most widely used method for analysis of hazardous, carcinogenic volatile organic compounds (VOCs) in mainstream cigarette smoke. However, these conventional techniques can lead to modification of VOCs during sample preparation due to the high reactivity of VOCs. Thus, the development of on-line mass spectrometric methods for analysis of VOCs is desirable to circumvent this problem. METHODS: The accurate identification of VOCs is a critical step in the analysis of cigarette smoke. Here, we use ion-molecule reaction mass spectrometry (IMR-MS) to study the behavior of standard VOCs in the Hoffmann list during this analytical procedure, and then to profile the VOCs in mainstream cigarette smoke using this on-line mass spectrometric method. RESULTS: We first discuss and summarize the charge transfer (CT) ionization and further fragmentation of 20 standard VOCs in the Hoffmann list with the ion reagents Hg(+), Xe(+), and Kr(+). The IMR-MS instrument was then connected to a Borgwaldt-RM20H rotary smoking machine in order to study VOCs in mainstream cigarette smoke on-line. Using this procedure, more than 20 VOCs were identified by IMR-MS by comparison with experimental results obtained on standard VOCs. CONCLUSIONS: The IMR-MS technique can potentially result in reduced molecular fragmentation during analysis of VOCs. However, significant fragmentation still occurs during IMR-MS when the ionization energy (IE) of the ion reagent is much higher than the IE of the VOC, given that excess energy is stored in the newly formed ion during CT ionization. Given that IMR-MS cannot distinguish between isobaric compounds or isomers, we summarize the possible overlapping mass peaks from these isobaric species that may be present in analyses of VOCs. Selection of the ion reagent for IMR-MS should be based on the need to ensure CT ionization of the analytes, as well as avoiding their severe fragmentation.

Development, validation, and application of a liquid chromatography-tandem mass spectrometry method for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in human hair.

The tobacco-specific nitrosamine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a valuable biomarker for human exposure to the carcinogenic nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in tobacco and tobacco smoke. In this work, an efficient and sensitive method for the analysis of NNAL in human hair was developed and validated. The hair sample was extracted by NaOH solution digestion, purified by C(18) solid-phase extraction (SPE) and molecularly imprinted solid-phase extraction, further enriched by reverse-phase ultrasound-assisted dispersive liquid-liquid microextraction (USA-DLLME) into 1.0 % aqueous formic acid, and finally analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. Good linearity was obtained in the range of 0.24-10.0 pg/mg hair with a correlation coefficient of 0.9982, when 150 mg hair was analyzed. The limit of detection and lower limit of quantification were 0.08 and 0.24 pg/mg hair, respectively. Accuracies determined from hair samples spiked with three different levels of NNAL ranged between 87.3 and 107.7 %. Intra- and inter-day relative standard deviations varied from 4.1 to 8.5 % and from 6.9 to 11.3 %, respectively. Under the optimized conditions, an enrichment factor of 20 was obtained. Finally, the developed method was applied for the analysis of NNAL in smokers' hair. The proposed sample preparation procedure combining selectivity of two-step SPE and enrichment of DLLME significantly improves the purification and enrichment of the analyte and should be useful to analyze NNAL in hair samples for cancer risk evaluation and cancer prevention in relation to exposure to the tobacco-specific carcinogen NNK.

Determination of the volatiles from tobacco by capillary gas chromatography with atomic emission detection and mass spectrometry.

A new gas chromatograph-atomic emission detector (GC-AED) coupled with Deans switching technique for analyzing volatiles from tobaccos were developed. The detector operating parameters (reagent gas pressure and make-up gas flow rate) were optimized. The detection limits for the elements carbon (193 nm), hydrogen (486 nm) and oxygen (171 nm) ranged 0.05-0.2, 0.05-0.3 and 1-11 ng, respectively, depending on the compound. The sensitivity and linearity for the elements carbon (193 nm), hydrogen (486 nm) and oxygen (171 nm) decreased in the order O>H>C. Calibration curves were obtained by plotting peak area versus concentration, and the correlation coefficients relating to linearity were at least 0.9359. Elemental response factors measured on these channels, relative to the carbon 193-nm channel, were hydrogen, 0.38-0.48 (mean %RSD=5.64), and oxygen, 0.085-0.128 (mean %RSD=14.9). The evaluation was also done for the new technique and for an established GC-MS technique for the same real samples. The results of GC-AED and GC-MS showed that there was a relatively good agreement between the two sets of data.

Development of single-drop microextraction and simultaneous derivatization followed by GC-MS for the determination of aliphatic amines in tobacco.

In this work, for the first time, headspace (HS) single-drop microextraction and simultaneous derivatization followed by GC-MS was developed to determine the aliphatic amines in tobacco samples. In the HS extraction procedure, the mixture of derivatization reagent and organic solvent was employed as the extraction solvent for HS single-drop microextraction and in situ derivatization of aliphatic amine in the samples. Fast extraction and simultaneous derivatization of the analytes were performed in a single step, and the obtained derivatives in the microdrop extraction solvent were analyzed by GC-MS. The optimized experiment conditions were: sample preparation temperature of 80 degrees C and time of 30 min, HS extraction solvent (the mixture of benzyl alcohol and 2,3,4,5,6-pentafluorobenzaldehyde) volume of 2.0 microL, extraction time of 90 s. With the optimal conditions, the method validations were also studied. The method has good linearity (R(2) more than 0.99), accepted precision (RSD less than 13%), good recovery (98-104%) and low limit of detection (0.11-0.97 microg/g). Finally, the proposed technique was successfully applied to the analyses of aliphatic amines in tobacco samples of seven different brands. It was further demonstrated that the proposed method offered a simple, low-cost and reliable approach to determine aliphatic amines in tobacco samples.

A facile approach for rapid on-site screening of nicotine in natural tobacco.

Nicotine (Nic) exposed to the environment which comes from tobacco products is the main addictive agent and specific classes of hazardous compound that merit concern. In this study, we have established a fast and reliable method to achieve specific detection of Nic in natural nicotiana tabacum within 30 s through a miniaturized platform based on screen printed gold electrode (SPE). A simple electrochemical pretreatment mean was employed on gold surface that led to the exposure of Au (111) facet and a convenient sample pretreatment method was adopted to realize the extraction of Nic in tobacco. The present electrochemical sensor exhibits an ample range of sensing from 10 µg/g to 200 µg/g, which is able to compliance with tobacco industry testing standards of actual samples. Over 60 sampling points from different origins in China or other countries were performed with direct analysis using this method and satisfactory results have been obtained. The proposed approach was demonstrated to be a very promising platform for significantly improving analytical efficiency in laboratories as well as for monitoring the source reduction control of Nic in the environment.

Development of C18-functionalized magnetic silica nanoparticles as sample preparation technique for the determination of ergosterol in cigarettes by microwave-assisted derivatization and gas chromatography/mass spectrometry.

Ergosterol is one of the important precursors of tumorigenic polynuclear aromatic hydrocarbons. A large amount of ergosterol is present in mildewy cigarettes, which derives from fungal contaminations. In this paper, a novel approach based on C(18)-functionalized magnetic silica nanoparticles (C(18)-f-MS NPs) coupled with microwave-assisted derivatization and gas chromatography/mass spectrometry (GC/MS) was developed for the rapid enrichment and determination of ergosterol in cigarettes. Due to that, microwave-assisted derivatization requires very short time (several minutes), and the extraction and concentration of ergosterol become the key step in the sample preparation process. In this study, the prepared C(18)-f-MS NPs with its unique properties (high surface area and strong magnetism) provided an efficient way for extraction and concentration of ergosterol in the samples. Additionally, the analyte of ergosterol adsorbed with C(18)-f-MS NPs in cigarettes can be simply and rapidly isolated (only about 2s) through placing a strong magnet on the bottom of container. In this work, different parameters such as added amounts of C(18)-f-MS NPs, extraction temperature, and extraction time were optimized to enhance the extraction efficiency. Method validations (linear range, detection limit, precision, and recovery) were also studied. The results obtained by the optimal conditions showed that the proposed method based on C(18)-f-MS NPs was a simple, high efficient, and had a rapid approach for the enrichment of ergosterol in cigarettes and was successfully applied to the analysis of ergosterol in normal and mildewy cigarettes followed by microwave-assisted derivatization and GC/MS.

Enantiomeric analysis of anatabine, nornicotine and anabasine in commercial tobacco by multi-dimensional gas chromatography and mass spectrometry.

A fully automated multi-dimensional gas chromatography (MDGC) system with a megabore precolumn and cyclodextrin-based analytical column was developed to analyze the enantiomeric compositions of anatabine, nornicotine and anabasine in commercial tobacco. The enantiomer abundances of anatabine and nornicotine varied among different tobacco. S-(-)-anatabine, as a proportion of total anatabine, was 86.6% for flue-cured, 86.0% for burley and 77.5% for oriental tobacco. S-(-)-nornicotine, as a proportion of total nornicotine, was 90.8% in oriental tobacco and higher than in burley (69.4%) and flue-cured (58.7%) tobacco. S-(-)-anabasine, as a proportion of total anabasine, was relatively constant for flue-cured (60.1%), burley (65.1%) and oriental (61.7%) tobacco. A simple solvent extraction with dichloromethane followed by derivatisation with trifluoroacetic anhydride gave relative standard deviations of less than 1.5% for the determination of the S-(-)-isomers of all three alkaloids. The study also indicated that, a higher proportion of S-(-)-nornicotine is related to the more active nicotine demethylation in the leaf.

Microwave-assisted silylation followed by gas chromatography/mass spectrometry for rapid determination of ergosterol in cigarettes.

Ergosterol is one of the important precursors of tumorigenic polynuclear aromatic hydrocarbons. To the best of our knowledge, a large amount of ergosterol is present in moldy cigarettes, which derives from fungal contaminations. Thus, the development of a simple, fast, and efficient method for the analysis of ergosterol is in great demand. In this paper, GC/MS following microwave-assisted silylation (MAS) was developed for the rapid quantitative analysis of ergosterol in cigarettes for the first time. In our work, total ergosterol in cigarettes after NaOH saponification was extracted with hexane, and then was fast derivatized with bis(trimethylsilyl)trifluoroacetamide (BSTFA) under microwave irradiation. Finally, the ergosterol trimethylsilyl derivative was analyzed by GC/MS. Derivatization conditions including microwave reaction solvent, irradiation time, and power were investigated. Method validations (linear range, LOD, precision, and recovery) were also studied. The results showed that the proposed method provided a fast, simple, and sensitive approach for the determination of ergosterol in cigarettes. Finally it was successfully applied to the analysis of ergosterol in normal and mildewy cigarettes.

Separation and Analysis of Sucrose Esters in Tobacco by Online Liquid Chromatography-Gas Chromatography/Mass Spectrometry.

In this work, a strategy of in-series combination of ultrasound-assisted extraction and online LC-GC/MS was constructed for effective separation and analysis of sucrose esters in tobacco. Sucrose esters were first extracted by ultrasound-assisted extraction with high efficiency and easyhandling. Online LC-GC/MS was then applied for sucrose ester clean-up and analysis. To better evaluate the effectiveness of this strategy, we limited our focus to five groups of sucrose ester isomers. Each group differed in mass from the next by 14 Da. The obtained coefficient of the calibration curve was 0.9986. Limit of detection (LOD) and limit of quantitation (LOQ) were 0.05 and 0.16 µg/ mL, respectively. The recovery was above 90% and the reproducibility was below 4%. This strategy was subsequently applied to the comparison of relative amounts of five groups of sucrose esters extracted from three different parts of aromatic tobacco. The satisfactory performance indicated that this strategy has great prospect for the rapid and high-throughput analysis of sucrose esters in tobacco.

Development of Multiple Heart-Cutting Two-Dimensional Liquid Chromatography Coupled to Quadrupole-Orbitrap High Resolution Mass Spectrometry for Simultaneous Determination of Aflatoxin B1, B2, G1, G2, and Ochratoxin A in Snus, a Smokeless Tobacco Product.

The combination of multiple heart-cutting two-dimensional liquid chromatography (MHC-LC/LC) and quadrupole-orbitrap high-resolution mass spectrometry (HRMS) for simultaneous determination of the aflatoxins and ochratoxin A in snus is presented in this work. A C18 capillary column was used as the first dimension (1D) to isolate the aflatoxins and ochratoxin A from the complex matrices; then, a 2-position/10-port high-pressure valve equipped with two 60 µL loops was employed to transfer the heart-cuts of 1D-LC into a pentafluorophenyl (PFP) column for the second dimension (2D) separation. With the better separation of the MHC-LC/LC system, the sensitivity of the method was improved, which is essential for the trace mycotoxins analysis. Furthermore, HRMS performed in parallel reaction monitoring mode was employed to eliminate the interferences, and the sample pretreatment procedure was simplified. A new approach utilizing ethyl acetate with 1% formic acid/water solution was adopted to extract aflatoxins and ochratoxin A in snus, which provided parallel recoveries for aflatoxins and ochratoxin A with higher responses in comparison with the QuEChERS method. A dynamic range between 0.2 and 20 µg/kg was achieved with LOQs of 0.05 µg/kg for aflatoxin B1, 0.1 µg/kg for aflatoxin B2, G1, G2, and 1.0 µg/kg for ochratoxin A in dry mass of product. The results revealed that the established method exhibited good repeatability and recovery and could be used as a rapid and reliable approach for routine analysis of aflatoxins and ochratoxin A in snus.

Sensitive determination of trace urinary 3-hydroxybenzo[a]pyrene using ionic liquids-based dispersive liquid-liquid microextraction followed by chemical derivatization and high performance liquid chromatography-high resolution tandem mass spectrometry.

3-Hydroxybenzo[a]pyrene (3-OHBaP) is widely used as a biomarker for assessing carcinogenic benzo[a]pyrene exposure risks. However, monitoring urinary 3-OHBaP suffers from an insufficient sensitivity due to the pg/mL level in urine excretion. In this study, a sensitive method for determination trace urinary 3-OHBaP was developed, involving enzymatic hydrolysis of the glucuronide and sulfate conjugates, ionic liquids dispersive liquid-liquid microextraction (IL-DLLME) enrichment, derivatization with dansyl chloride and HPLC-HRMS/MS analysis in the positive ion mode. Using IL-DLLME makes the enrichment of trace 3-OHBaP very simple, time-saving, efficiency and environmentally-friendly. To enhanced HPLC-HRMS/MS response, an MS-friendly dansyl group was introduced to increase the ionization and fragmentation efficiency. The optimal IL-DLLME extraction parameters and derivatization reaction conditions were investigated. Good linearity was obtained over a concentration range of 0.6-50.0pg/mL with correlation coefficients (r(2)) of 0.9918. The limit of detection (LOD) and limit of quantification (LOQ) values were 0.2pg/mL and 0.58pg/mL, respectively. The recoveries were 92.0±4.2% with the intra-day and inter-day RSD values ranged from 2.2% to 3.8% and from 3.3% to 6.8%, respectively. The proposed IL-DLLME-Dansylation-HPLC-HRMS/MS method was successfully applied to determine urinary 3-OHBaP of non-occupational exposed smokers and nonsmokers.

Tuning the adsorption behaviors of water, methanol, and ethanol in a porous material by varying the flexibility of substituted groups.

Exploring the adsorption and separation of water, methanol, and ethanol is important concerning the use of a sustainable energy source from biofuel. In this paper, the effects of the flexibility of substituted groups have been studied based on three iso-reticular metal-organic frameworks (MOFs), in which the pore surface is decorated with propargyl (-CH2-C[triple bond, length as m-dash]CH), allyl (-CH2-CH[double bond, length as m-dash]CH2), and propyl (-CH2-CH2-CH3) groups respectively. These substituted groups stretch into the channel, acting as gates, and the gate-opening for guests is controlled by the flexibility as well as host-guest interactions. Our study results indicate that (i) the adsorption capacity of water, methanol and ethanol enhances accordingly with the increase of the flexibility of substituted groups; (ii) the adsorptive discrimination of water, methanol, and ethanol on this porous sorbent could be tuned by varying the substituted groups.

Identifying the tobacco related free radicals by UPCC-QTOF-MS with radical trapping method in mainstream cigarette smoke.

Tobacco related free radicals (TFRs) in the cigarette smoke are specific classes of hazardous compounds that merit concern. In this study, we developed a hybrid method to identify TFRs directly based on ultra-performance convergence chromatography with a quadrupole time-of-flight mass spectrometry (UPCC-QTOF MS) combined spin trapping technique. The short-lived TFRs were stabilized successfully in situ through spin trapping procedure and UPCC was applied to facilitate efficient separation of complex derivative products. Coupling of orthogonal partial least squares discriminant analysis (OPLS-DA), UPCC-QTOF MS system enabled us to identify specific potential TFRs with exact chemical formula. Moreover, computational stimulations have been carried out to evaluate the optimized stability of TFRs. This work is a successful demonstration for the application of an advanced hyphenated technique for separation of TFRs with short detection time (less than 7min) and high throughput.

Facile Synthesis of CeO2-LaFeO3 Perovskite Composite and Its Application for 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone (NNK) Degradation.

A facile and environmentally friendly surface-ion adsorption method using CeCO3OH@C as template was demonstrated to synthesize CeO2-LaFeO3 perovskite composite material. The obtained composite was characterized by X-ray diffraction (XRD), fourier transform infrared spectra (FT-IR), field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), thermo-gravimetric analysis and differential scanning calorimetry (TG-DSC), N2 adsorption/desorption isotherms and X-ray photoelectron spectra (XPS) measurements. The catalytic degradation of nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was tested to evaluate catalytic activity of the CeO2-LaFeO3 composite. Much better activity was observed for the CeO2-LaFeO3 composite comparing with CeO2 and LaFeO3. These results suggested that perovskite composite materials are a promising candidate for the degradation of tobacco-specific nitrosamines (TSNAs).

Synthesis of divinylbenzene polymer/Fe3O4 hybrid monolithic column for enrichment and online thermal desorption of methylmercury in real samples.

A novel method of divinylbenzene polymer (DVB)/Fe3O4 hybrid monolithic column solid phase extraction and on line thermal desorption was developed to analyze methylmercury (MeHg) in water and fish samples. The monolithic column was prepared by in situ polymerization in silica tube and its heating characteristic was studied in detail. Special accent was put on the study of parameters influencing adsorption and desorption of MeHg, such as pH, flow rate of sample solution, temperature of desorption and flow rate of carrier. Under optimum conditions, the detection limit and relative standard deviation of MeHg were 0.09 ng L(-1) and 2.6% (10 ng L(-1), N=11), respectively. Recoveries of MeHg were never less than 96%. Standard reference material GBW10029 was analyzed for validation of the methodology. This method was successfully applied to the determination of MeHg in water and fish samples.

Analysis of free and bound volatiles by gas chromatography and gas chromatography-mass spectrometry in uncased and cased tobaccos.

The free and bound volatiles of tobaccos were analyzed by capillary GC and GC-MS. Bound volatiles were isolated by dichloromethane extraction followed by stream distillation continuous extraction (SDE) at pH 2.5 acid hydrolysis. The bound aromatic compounds were hydrolyzed by acid at pH 2.5, and the bound volatiles were liberated and extracted into dichloromethane by SDE simultaneously. In total, 23 volatiles were identified, with neophytadiene, 2-ethyl hexanol, damascenone, benzene ethanol, palmitic acid, stearic acid, linoleic acid, farnesyl acetone, 3-oxo-ionol, and megastigmatrienone being the major components. They consisted mainly of compounds exhibiting aromatic characteristics. The quality and quantity of free and bound volatiles exhibited different distributions in uncased or cased tobaccos. The volatiles existed in higher amounts in bound form than in free form. Compared with uncased tobaccos, free form volatiles showed a decrease after the casing process, while bound volatiles showed an increase.