Flavor Characteristics of Different Varieties of Flue-Cured Tobacco Based on Headspace Gas Chromatography–Ion Mobility Spectrometry and Headspace Solid-Phase Microextraction–Gas Chromatography–Mass Spectrometry

This study analyzed volatile components in six flue-cured tobacco varieties using HS-GC-IMS and HS-SPME-GC-MS. In total, 93 and 80 compounds including esters, alcohols, and aldehydes were detected. Based on relative odor activity values and statistical modeling, 19 key compounds such as benzaldehyde, acetic acid, and methyl acetate were identified as major contributors to flavor differentiation.

The synergistic use of the two analytical techniques provided a comprehensive overview of the volatile flavor profile of flue-cured tobacco. These results clarify the composition and differences in aroma among six varieties, offering a valuable foundation for future studies and flavor control in tobacco processing.

The original article

Flavor Characteristics of Different Varieties of Flue-Cured Tobacco Based on Headspace Gas Chromatography–Ion Mobility Spectrometry and Headspace Solid-Phase Microextraction–Gas Chromatography–Mass Spectrometry

Xiaoyu Chen, Cong Chen, Yutong Liu, Kun Tang, Mengqi Chen, Jianhua Qiu, Lin Yuan, Hongtao Shen*, and Zhiyong Wu*

ACS Omega 2025, 10, 20, 20382–20401

https://doi.org/10.1021/acsomega.5c00343

licensed under CC-BY 4.0

Selected sections from the article follow. Formats and hyperlinks were adapted from the original.

Flue-cured tobacco, also known as fire-tube flue-cured tobacco, originated in Virginia, USA, so it is also known as Virginia-type tobacco. This curing process involves placing mature tobacco leaves in a temperature- and humidity-controlled chamber with hot gas pipes. By precisely regulating thermal conditions, the leaves undergo sequential yellowing and drying stages, culminating in the development of their signature flavor profile. (1,2) Flue-cured tobacco is an important agricultural product in China’s cash crops, which is the main raw material for production of cigarettes and one of the bulk agricultural products exported by China. (3,4) Meanwhile, flue-cured tobacco is a kind of tobacco that has been specially treated and fermented, has a unique aroma and taste, and is the main raw material for tobacco products, such as cigarettes and cigars. (5,6) Yunnan, Guizhou, Sichuan, and Henan are the main producing areas in China, with different flue-cured tobacco varieties. For example, Xuchang is an important tobacco-producing area in Henan Province, having its flue-cured tobacco varieties with diversity and characteristics, such as Yunongxiang 201, Zhongyan 100, Zhongyan Texiang 301, Xuchang 101, Y2002, Y2008, Xiangcheng flue-cured tobacco, long neck Yellow, etc. (7) These special flue-cured tobacco varieties have their own unique quality characteristics and flavor substances.

The chemical composition is the internal factor that determines the quality of tobacco leaves, and the aroma substance is the important material basis for the formation of tobacco leaf aroma quality. Jing et al. have analyzed the conventional chemical components and differences of different varieties of flue-cured tobacco in seven tobacco-producing areas in Henan Province, so as to clarify the suitable varieties and flavor characteristics of tobacco leaves for the producing area. Among them, the flavor substance content of varieties of tobacco leaves in different producing areas was detected by GC-MS. (8) Li et al. have tested the conventional chemical components, smoking quality, and aroma components content of flue-cured tobacco by correlation analysis in Henan tobacco areas. The results showed that balancing the content of sugar components, nitrogen compounds, and potassium chlorine was beneficial to improve the smoking quality of flue-cured tobacco, and the aromatic components in flue-cured tobacco were extracted by simultaneous distillation extraction and 33 neutral odors were identified by GC-MS. (9) Guo et al. have compared and analyzed the conventional chemical components, aroma substances, and sensory quality of seven varieties of flue-cured tobacco, including K326, NC82, G28, Honghuadajinyuan, Gexin3, Xiaohuangjin1025, and Dabaijin599, in the Linyi tobacco-growing area of Shandong Province. The results showed that the chlorine content, the ratio of two sugars, the ratio of potassium to chlorine, and the nitrogen/alkali ratio were different among different varieties of flue-cured tobacco. Furthermore, 72 kinds of aroma components, mainly including carotenoid degradation products, chlorophyll degradation products, and brown reaction products, were detected by HS-SPME-GC-MS, and their contents were significantly different. At the same time, 17 differential metabolites were identified by the analysis of aroma substances. (10)

The types and contents of flavor substances in tobacco are the key factors that affect its quality. Although the differences of chemical components and aroma substances in different genotypes of flue-cured tobacco have been reported, the comprehensive study of volatile aroma substances for different varieties of flue-cured tobacco is still relatively limited. (11−14) To date, a single technology is generally used to detect and analyze the volatile aroma chemical components of different varieties of flue-cured tobacco, such as gas chromatography–mass spectrometry (GC-MS) or headspace solid-phase microextraction–gas chromatography–mass spectrometry (HS-SPME-GC-MS). (15−18) It is noteworthy that although GC-MS can determine a variety of compounds at the same time and has been widely applied in the detection of volatile flavor components, it is easy to miss trace-level, small molecular weight compounds, with which the volatile aroma substances in the sample cannot be fully analyzed. (19−23)

Compared with GC-MS detection technology, headspace gas chromatography–ion mobility spectrometry (HS-GC-IMS) has more efficient separation ability and sensitive response speed, without sample pretreatment, and solid/liquid samples can be directly placed in the headspace for detection, which can characterize chemical ions based on the difference of gas phase migration speed of different ions in the electric field. (24,25) HS-GC-IMS is another effective and emerging detection technology for the characterization of volatile compounds and has been widely used to analyze the difference of volatile components in different samples. (26−29) For instance, Kong et al. have analyzed volatile compositions and their relative content of different Yunyan 87 tobacco leaves with or without typical “glutinous rice”-like characteristics from Yunnan Province by HS-GC-IMS detection technology. The results showed that a total of 57 common odorants (including monomers and dimers) were identified from the tested tobacco leaves, and the different characteristic flavor compounds between the two flue-cured tobacco leaves were identified by statistical methods. Among them, 2-acetyl-1-pyrroline is a key contributor to “glutinous rice”-like aroma. (30) Despite this elegant progress, GC-IMS has limited spectral library data, and its detection ability for macromolecular compounds is low, which may miss the detection of volatile aroma substances. Therefore, more and more research studies have combined SPME-GC-MS and GC-IMS to fully analyze volatile flavors. (31−35) Zhu et al. have analyzed the flavor profile fermented with/without the peel of kiwi wine by means of a combination of gas chromatography-ion mobility spectrometry (GC-IMS), headspace solid-phase microextraction–gas chromatography–mass spectrometry (HS-SPME-GC-MS), and sensory evaluation. The results showed that a total of 124 volatile compounds were detected in kiwi fruit wine by the combination of HS-SPME-GC-MS and GC-IMS. The number of molecules characterized was higher than that of a single technique, indicating that more comprehensive and systematic flavor characterization of kiwi wine can be provided using distinct analytical instruments. (36) However, there is still a lack of research that identifies more comprehensive volatile flavor information in flue-cured tobacco by comparing the volatile aroma characteristics of different varieties of flue-cured tobacco with two techniques.

Therefore, this study combined HS-SPME-GC-MS and HS-GC-IMS with chemometrics to analyze volatile aroma components and relative contents of six varieties of flue-cured tobacco, including Yunongxiang 201, Zhongyan 100, Zhongyan Texiang 301, Xuchang 101, Y2002, and Y2008 in Xuchang from Henan Province. The concept of relative odor activity value (ROAV) was introduced, and the volatile compounds in different varieties of flue-cured tobacco were accurately screened and identified by fingerprint, principal component analysis (PCA), orthogonal partial least-squares discriminant analysis (OPLS-DA), and other statistical methods, aiming to lay a foundation for aroma comparison and regulation of different varieties of flue-cured tobacco.

2. Materials and Methods

2.3. Experimental Methods

2.3.1. HS-GC-IMS Analysis

The experiment was analyzed using an HS-GC-IMS apparatus (FlavourSpec, G.A.S., Dortmund, Germany) with an autosampler (CTC Analytics AG, Zwingen, Switzerland). A 1.0000 g sample was accurately weighed and placed in a 20 mL headspace glass sampling bottle with 100 ppm internal standard 2-methyl-3-heptanone 10 μL added and incubated at 60 °C for 20 min by rotating at a speed of 500 rpm. Headspace samples (500 μL) were automatically injected into an MXT capillary column using an injector (85 °C) with a splitless mode at 45 °C under isothermal conditions in a nitrogen of 99.99% purity. Gas chromatographic gradient program: 0–2 min, carrier gas flow rate of 2 mL/min; 2–10 min, 2–10 mL/min; 10–20 min, 10–100 mL/min; 20–60 min, 100 mL/min. The drift gas flow rate is 75 mL/min. The length of the drift tube is 53 mm; the linear voltage in the tube is 500 V/cm; the temperature of the drift tube is 45 °C; carrier gas/drift gas is N₂; radiation source: β-ray (tritium, 3H); ionization mode: positive ions. It is noteworthy that 3 parallel groups of each sample were measured.

2.3.2. HS-SPME-GC-MS Analysis

1.0 g of sample was placed in a 20 mL headspace vial with 100 ppm internal standard 2-methyl-3-heptanone 10 μL as the sample to be tested. The vial was then incubated at 60 °C for 20 min. Subsequently, SPME with a 1 cm DVB/CAR/PDMS fiber was used for extraction. The SPME fiber was aged at 270 °C for 10 min before being transferred to the incubation chamber, where it adsorbed the sample at 60 °C for 30 min. After adsorption, the SPME fiber was transferred to the GC injector and desorbed at 250 °C for 10 min. HS-SPME-GC-MS analysis was implemented by an Agilent 7890B GC autosampler system (Agilent, CA, USA) equipped with an Agilent 5977B mass spectrometry detector. The chromatographic column was a DB-5MS (30 m × 0.25 mm × 0.25 μm) elastic quartz capillary column. The initial temperature was maintained at 40 °C for 4 min, increased to 100 °C at a rate of 6 °C/min, and finally increased to 250 °C at a rate of 4 °C/min and maintained for 5 min; the carrier gas was helium (>99.999%) with a flow rate of 1 mL/min; the injection port temperature is 250 °C. The conditions of MS were set as follows: the ion source temperature was 230 °C, and electron impact (EI) with ionization energy was 70 eV. MS1 full scan mode was adopted with a scan range of m/z 35–400. The retention index (RI) of each volatile component was calculated by using the linear interpolation method of normal alkane (C7–C40) retention time (RT). Finally, the concentration of volatile compound was quantitatively determined by the peak-area ratio of compound to 2-methyl-3-heptanone. Three parallel groups of each sample were measured.

3. Results and Discussion

4. The Comparison and Analysis of the Aroma of Six Varieties of Flue-Cured Tobacco Based on HS-GC-IMS and HS-SPME-GC-MS

Regarding the aroma description of volatile flavor components, the flavor characteristics of 6 varieties of flue-cured tobacco were further analyzed. The flavor radar charts were constructed based on the frequency of occurrence of different types of flavor compounds, ROAV value, and sensory evaluation results, as shown in Figure 10. Flavor characteristics were assessed on a maximum 10-point scale, where higher scores indicated stronger flavor characteristics. Figure 10A and Figure 10B are flavor radar charts based on flavor components detected by HS-SPME-GC-MS and HS-GC-IMS, respectively. There are differences between the 8 sensory attributes in Figure 10A,B. The sensory attributes of the compounds detected by HS-SPME-GC-MS are mainly fruity, floral, and green aroma. The sweet, spicy, wine, fatty, nutty aroma composition in 6 samples is weak (Figure 10A). The sensory attributes of volatile flavor compounds detected by HS-GC-IMS are mainly fruity, floral, spicy, fatty, and green aroma. Compared with HS-SPME-GC-MS, the sweet, wine, and nutty aroma is enhanced (Figure 10B). To characterize the 8 flavor differences of 6 samples more comprehensively, we combined the aroma components detected by two distinct analytical instruments to draw a radar map (Figure 10C). Based on sensory evaluation results, a radar map in Figure 10D has been drawn. These results showed that there are some differences in the styles of the six varieties of flue-cured tobacco. K1 stood out in fruity, sweet, and fatty aroma; the fruity flavor is related to compounds including methyl valerate, methyl 2-methylbutyrate, methyl decanoate, 6-methyl-5-hepten-2-one, methyl hexanoate, acetaldehyde, ethyl propanoate, hexanal-M, ethyl isobutyrate, acetal, phenylacetaldehyde, 1-butanol-M, etc.; ethyl isobutyrate, phenylacetaldehyde, butyl propanoate-D, geranyl acetate, and butyl propanoate-M have sweet flavors; the fruity aroma may be related to aromatic amino acid degradation products. It is worth noting that esters and aldehydes have a fruity or sweet aroma at low concentrations. The material basis of the fatty aroma is hexanal-M, 1-penten-3-one-M, (Z)-4-heptenal, pentanal, and propanoic acid; K2 stood out in spicy, nutty and fruity aroma; among these, furfural, 2-acetyl pyrrole, 2-acetylthiazole, benzaldehyde-M, and 2-methylpropanal have a nutty aroma, which may be because high-temperature baking promotes the Maillard reaction, and the protein and sugar are degraded, resulting in Maillard degradation products. K3 stood out in floral aroma, which may be suitable for light-scented cigarettes; the floral aroma is related to compounds including methyl heptanoate, methyl decanoate, beta-damascenone, nonanal, decanal, acetaldehyde, 3-methyl-1-butanol-D, nonanal, propanal, etc.; the aldehydes and ketones have pleasant odors such as floral aroma at low concentrations. K4 stood out in green and wine aroma; the material basis of the green flavor is methyl heptanoate, nonanal, diphenyl ether, acetaldehyde, hexanal-M, 1-penten-3-one-M, (Z)-3-hexenol, 2-pentylfuran, etc. The reason for the green flavor may be the slow degradation of chlorophyll and the retention of more neophytadiene. Methyl decanoate, phenylethyl alcohol, methyl caprylate, ethyl propanoate, ethyl isobutyrate, ethyl butanoate, and ethyl hexanoate-M have wine aroma. The reason for the wine aroma of the K4 sample may be due to the extended fermentation process and sufficient esterification reaction; the spicy and floral aroma of K5 is slightly accentuated; 1-penten-3-one-D, 1-penten-3-one-M, acetophenone, acetone, propanal, and pentanal have spicy flavors; it is worth noting that aldehydes and ketones have a spicy or irritating sensation at higher concentrations. The sweet and fruity aroma of K6 is slightly accentuated; overall, 6 varieties of flue-cured tobacco have differences in aroma components. These results are generally consistent with previous studies. (46,47) Additionally, the influencing factors of these differences may be related to the planting environment, preparation technology, and variety genetics, resulting in differences in the genotypes of 6 flue-cured tobacco varieties. (48) It is noteworthy that there are also some differences between Figure 10C and Figure 10D, which may be because the suction temperature during sensory evaluation is higher than the detection temperature of the two instruments, and some compounds may be thermally decomposed into smaller aroma molecules, making certain differences in aroma style. The VOC profile variations explain sensory flavor typologies among cultivars; the combination of analysis results of HS-SPME-GC-MS and HS-GC-IMS can obtain more comprehensive and accurate information on flavor substances, which is conducive to the comparison and analysis of the aroma of different varieties of flue-cured tobacco.

ACS Omega 2025, 10, 20, 20382–20401: Figure 10. Sensory flavor feature analysis radar chart (A) based on compounds detected by HS-SPME-GC-MS; (B) based on compounds detected by HS-GC-IMS; (C) based on HS-SPME-GC-MS and HS-GC-IMS; and (D) based on sensory evaluation.

5. Conclusion

In this study, the differences of volatile flavor compounds in various flue-cured tobaccos were compared. 93 and 80 aroma components were identified in six varieties of flue-cured tobacco by HS-GC-IMS and HS-SPME-GC-MS, respectively. Esters accounted for the most aroma components, followed by ketones and alcohols. The important aroma components of different flue-cured tobaccos were identified by ROAV analysis. Among them, pentanal, acetaldehyde, 1-penten-3-one, dimethyl thioether, acetal, 3-methyl-1-butanol, ethyl isobutyrate, 6-methyl-5-hepten-2-one, methyl valerate, methyl heptanoate, methyl decanoate, and beta-damascenone have important contributions to the aroma formation of different varieties of flue-cured tobacco. The fruity, sweet, and fatty aroma of the K1 sample is more prominent, which may be due to there being more aromatic amino acid degradation products in K1 samples, and esters and aldehydes have fruity or sweet aroma at low concentrations; K2 stands out in spicy, nutty, and fruity aroma, which may be due to high-temperature baking, which promotes the Maillard reaction and generates Maillard degradation products; the floral aroma of K3 was prominent, and the nutty flavor was weak, which may be due to low temperature and slow roasting, which affected the degree of Maillard reaction. In addition, the aldehydes and ketones have pleasant odors, such as floral aroma, at low concentrations. The green and wine flavor of the K4 sample is more prominent which may be due to the slow degradation of chlorophyll and the extended fermentation process and sufficient esterification reaction; the spicy and floral aroma of K5 is slightly accentuated due to aldehydes and ketones having a spicy or irritating sensation at higher concentrations. The sweet and fruity aroma of K6 is slightly accentuated; furthermore, 19 volatile flavor compounds were screened as aroma markers to distinguish six varieties of flue-cured tobacco by using OPLS-DA based on HS-GC-IMS and HS-SPME-GC-MS, which can be paid attention to when comparing aroma components of different varieties of flue-cured tobacco. The combined analysis of HS-GC-IMS and HS-SPME-GC-MS took advantage of complementary data and displayed the volatile flavor information on different varieties of flue-cured tobacco more comprehensively. This study lays a foundation for aroma analysis and regulation of various flue-cured tobaccos and provides a theoretical basis for enterprise production and flavor evaluation of flue-cured tobacco.