Identification of key volatile flavor compounds in cigar filler tobacco leaves via GC-IMS

Cigar filler leaves are the most important component of cigar because they determine its quality. Therefore, the volatile components of eight cigar filler tobacco leaves were studied and compared using gas chromatography–ion mobility spectrometry (GC– IMS). In this study, 84 compounds with high levels of nitrogenous and ketone compounds were identified. Based on the chemometric principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), the eight cigar samples were significantly distinguished. Meanwhile, we performed a discriminant analysis of volatile organic compounds in the eight cigar samples based on the variable importance in the projection (VIP) scores of the PLS-DA model, and revealed significant differences in the volatile compounds between the different varieties. 11 volatile compounds (VIP > 1) were screened and compared, among which triamine, acetic acid, acetone, and cyclopentanone were the main differential compounds/flavor substances. This study showed that GC–IMS can rapidly identify and compare the volatile compounds of various cigars, providing a theoretical basis for studying the differences in the volatile aroma of cigars, and laying a foundation for the breeding selection of subsequent varieties.


Introduction
Cigar tobacco is an important non-food agricultural crop consumed worldwide because of its unique aroma and taste (Allem et al., 2019;Wen and Huang, 2021). Cigar filler tobacco leaves (CFTLs) are an essential component of cigar tobacco, accounting for 70-85% of the weight of the cigar and determining its smoking quality, commodity value, and industrial prospects (Zheng et al., 2022). Generally, CFTLs must undergo additional fermentation before being used in cigars, which can produce several volatile flavor compounds (VFCs) with characteristic flavors (Cornacchione et al., 2022;Yang et al., 2022). Previous research has shown that VFCs in cigar leaves are complex and diverse and include terpenes, aldehydes, alcohols, ketones, esters, and sulfur-containing substances (Kaneko and Harada, 1972). With the development of chromatography-mass spectrometry, more than 100 characteristic flavors have been detected in cigars, including sulfides, terpenes, and aldehydes (Vu et al., 2021;Zhang et al., 2021a). In China, the raw materials of the cigar are mainly imported from Cuba, Dominica, Brazil, and Indonesia (Ying et al., 2018;). The cultivation of CFTLs in China is still in its infancy, therefore the characteristic VFCs of Chinese CFTLs are still unclear (Gao et al., 2015;Ying et al., 2018;Wang et al., 2022). Due to the diversified introduction channels and scattered preservation, various reasons such as repeated naming lead to the unclear genetic relationship between cigar germplasm in China, which brings inconvenience to the quality identification and the promotion and application of superior lines of cigar. Therefore, it is of great significance to study the key VFCs in cigar tobacco leaves that cultivated in China for the understanding and preservation of domestic cigar tobacco germplasm resources. The current research is mainly focused on volatile compounds in cigar, including the extraction, separation, mass spectrometric analysis of characteristic flavor compounds, and exploration of principal flavor components of raw cigar materials that cultured in China. However, there are no systematically reports on the identification and differential analysis of key VFCs in different strains of cigar raw materials in China. Gas chromatography (GC)-mass spectrometry, GColfactometry, and electronic noses are usually employed to identify volatile aroma compounds in tobacco (Rambla-Alegre et al., 2014;Liu et al., 2018;Qi et al., 2022;Zheng et al., 2022). Among these, gas chromatography-ion mobility spectrometry (GC-IMS), which is more sensitive than previous technologies, has emerged. GC-IMS combines the high separation ability of GC with the high sensitivity of IMS and does not require sample pretreatment (Li et al., 2019;Wang et al., 2020;Gu et al., 2021;. Studies on drug detection , environmental quality monitoring (Zheng et al., 2022), and food flavor analysis (Wang et al., 2020) demonstrated that trace amounts of VFCs can be rapidly detected through processing. In our study, GC-IMS was used to detect and identify VFCs in the tobacco leaf raw materials of eight main cigar varieties. Principal component analysis (PCA) was used to analyze the differences in flavor compounds among the different cigar samples. The key VFCs in cigar tobacco raw materials were studied using the variable importance in the projection (VIP) analysis method (Mao et al., 2018). Finally, fingerprints of the volatile flavors of each cigar material were constructed to identify the differences in the volatile compounds among the different materials. The rapid identification of cigar varieties and the efficient differentiation of the flavors of cigar tobacco raw materials will provide a theoretical basis for the qualitative analysis of cigar tobacco raw materials and the research and development of high-quality domestic cigar products.

Samples and CFTLs preparation
Eight cigar filler tobacco leaves (CFTLs) samples of different varieties were planted and collected from the Chinese Tobacco Hubei Industry Co., Ltd., Wuhan City, Hubei Province (111.34555E, 30.78724N) in 2021. For each sample, 2 kg of the material was collected and dried according to the technical regulations for air-drying high-quality cigars. After drying, the CFTLs were placed in a constanttemperature-and-humidity box at 45 ℃ and 80% humidity for initial fermentation. After fermentation, the CFTLs were removed and immersed in liquid nitrogen for rapid pre-cooling, stored in a Ziplock bag at −80 ℃ until further use.

Qualitative and quantitative analysis of VFCs in different varieties of cigar leaves
The ion migration time and RI were used to qualitatively identify VFCs in the detected samples. Totally, 111 peaks were detected in the tobacco leaf samples, and 93 compounds were identified using the NIST 2014 and IMS databases (Figure 1 and Tables S1 and S2), including 12 esters, 4 alkenes, 17 ketones, 4 acids, 20 aldehydes, 13 alcohols, 3 sulfur compounds, 10 nitrogen-containing compounds, and 10 other classes. The monomeric and dimeric forms of nine compounds (acetic acid, sanyamine, cyclopentanone, hexanal, butanol, glutaraldehyde, ethanol, butanal, and propanal) were included. Therefore, 84 compounds were totally identified in the final determination of cigar tobacco samples. In addition, the quantitative analysis of volatile compounds in these cigars was expressed as the peak volume calculated using the IMS system (Table S1). Among which, nitrogen was the main component in the eight samples, with a content accounting for more than 66%. Chuxue No. 80 exhibited the highest nitrogen content (77.77%) and the main nitrogencontaining compounds were triimine and 2acetylpyridine. The ketone contents of the different varieties were significantly different:

Figure-2. VFCs fingerprint from different cigar leaves;
the signal peaks of the volatile compounds contained in the corresponding sample, and each column represents different volatile compounds. The signal peaks represent intensity of the compound. The brightness of the spot represents the concentration of the corresponding volatile compound. Here, monomers and dimers of the same compound were characterized using different columns with the same compound name. Additionally, a small number of volatile compounds were not accurately characterized and are presented in the form of Arabic numerals.

PCA plot for differentiating the volatile compounds in different varieties of cigar leaves
The Dynamic PCA plug-in was used to conduct PCA based on the content (signal peak volume) of volatile compounds in the different varieties of the detected cigar leaves. Distributions of the first and second principal components were obtained through data visualization ( Figure 3). The variance contribution rates of the first two principal components were 49% and 17%. The distances between the samples within each variety were similar, showing good parallelism, and the distances between the samples of each variety were significantly separated. By combining PC1 and PC2, the eight cigar samples were divided into six groups: Hainan No. 2 and Hainan No. 3 were classified in the same group, and Chuxue No. 14 and Jianheng No. 3 were categorized in another group, indicating that they had similar volatile compound compositions. However, the other four cigar varieties were clustered into one group, and the distance between them was relatively large, indicating the composition of volatile compounds of these four varieties differed greatly from each other.

GC-IMS analysis of VFCs in different varieties of cigar leaves
To further explore the key substances causing differences between the tobacco varieties, the VIP scores of all volatile compounds were calculated based on the PLS-DA model, and "VIP> 1" was used as the standard for screening. In this study, 11 (VIP ˃ 1) marker volatile compounds were screened according to the VIP values (Figure 4), among which triimine-M and acetic acid of VIP ˃ 3 were the most important differential compounds, followed by acetone and cyclopentanone-D (2 < VIP < 3), which contributed significantly to the overall difference. Additionally, hexanal, 2-isovaleraldehyde, ethyl isobutyrate, 2butanone, furfuryl alcohol, methyl 3-methyl butyric acid, amyl, and propionaldehyde (1 < VIP < 2) were important differential compounds. Chuxue No. 80 exhibited the highest triamine-M content, followed by Jianheng No. 3. The highest acetic acid content was found in Hainan No. 3, followed by Dexue No. 4. The highest acetone content was observed in Chuxue No. 81, followed by Jianheng No. 3, and Chuxue No. 80. Furthermore, the cyclopentanone contents in Chuxue No. 81 and Chuxue No. 14 were higher than those in the other varieties. The hexanal contents in Chuxue 81 and Chuxue 14 were the highest among all species. Hainan No. 2 and Hainan No. 3 exhibited a higher content of 2-isovaleraldehyde. Moreover, higher ethyl isobutyrate and furfuryl alcohol contents were observed in Chuxue 81 and Dexue 4. Additionally, the amyl methyl 3-methyl butyrate content was higher in Chuxue No. 81. Chuxue No. 81,Chuxue No. 14,and Dexue No. 4 were rich in propionaldehyde.

Discussion
Cigar tobacco is an important economic crop that is widely grown worldwide because of its special aroma and taste, which is made from dried and fermented cigar leaves (Allem et al., 2019). At present, most of the cultural cigar species in China are imported, leading to relatively scarce germplasm resources of cigar tobacco in China Zheng et al., 2022). Therefore, the cultivation and production of cigar filler tobacco leaves in China are still in their primary stages, and more research should be conducted to explore the germplasm resource presentation, explore the quality differences among different varieties, and identify suitable varieties of cigars in China. As an important flavor substance, the volatile aroma components of cigars are complex and diverse, which determines their smoking quality, commodity value, and industrial prospects. Typically, cigar compounds can be detected using GC-MS (Zheng et al., 2022;Ng et al., 2001). In this study, we used GC-IMS to detect and analyze the key VFCs in eight major CFTLs in China. Totally, 84 volatile compounds were identified: 12 esters, 4 alkenes, 16 ketones, 3 acids, 16 aldehydes, 11 alcohols, 3 sulfurcontaining compounds, 10 nitrogen-containing compounds, and 10 other compounds. The quantitative and qualitative results showed that the CFTLs samples mainly contained nitrogen-containing VFCs, followed by ketones, aldehydes, and alcohols. Among these, aldehydes are considered to contribute more to flavor because of their high concentration, low threshold, and high volatility (Zhang et al., 2021a;Wang et al., 2022). In addition, studies have shown that aldehydes were first hydrolyzed by lipids to form free fatty acids; these saturated and unsaturated fatty acids were then thermally decomposed to form hydroperoxides under normal conditions (Kim et al., 2022). Alcohol compounds generally derive from the degradation of secondary hydroperoxides of fatty acids or the reduction of carbonyl compounds (Lawyer et al., 2019). This study revealed significant differences of VFCs between the tested CFTLs, indicating that these differences are the important factors responsible for the different flavor characteristics and sensory qualities of the cigars (Zhang et al., 2021b). To explore the key VFCs in different CFTLs, all volatile compounds were analyzed using fingerprints, PCA, and PLS-DA. The similarities and differences in the volatile compounds among the different varieties were clarified, and the eight varieties were divided into six groups. Significant differences were observed in the structures of flavor substances. Chuxue No. 14,Jianheng No. 3,Hainan No. 2,and Hainan No. 3 exhibited similar flavor substance structures, which can provide a reference for the blending of tobacco leaves in industrial production. The contribution of all identified volatile compounds to the structural differences in flavor compounds was studied using VIP analysis, and 11 marker compounds with important contributions to the structural differences in flavor compounds were selected. Among these, triamine-M and acetic acid were identified as the most important differential markers, followed by acetone and cyclopentanone D. Acetic acid and acetone are important and common aromatic substances in cigars and are known to impart pungent odors (Li et al., 2023). Other compounds such as hexanal, 2isovaleraldehyde, ethyl isobutyrate, and 2-butanone, which endow CFTLs with typical fruity and ethereal properties, are also important differential flavor compounds (Baker et al., 2004). Our study revealed that each cigar variety exhibits characteristic marker compounds in different contents. For example, signal intensity of esters and alkene in Chuxue No. 81 are significantly higher than that in the other varieties., and so as to the ketones in Chuxue No. 14, the alcohols in Dexue No. 4, and the acetophenone in Yongsheng No. 2, which may be the main reason for the flavor differences between the different varieties. Yu et al. (2021) used GC-MS analysis and revealed that the biomarkers of two volatile compounds screened on cigar raw materials were α-curcumene and cedrol, which differs from our results. The differences in volatile compounds among the different varieties can affect the results, whereas the detection ability of GC-IMS and GC-MS may lead to such differences. The volatile components detected by GC-IMS were mostly small molecules with high volatility and low contents, and the detection time was short. Thus, GC-IMS can rapidly identify volatile compounds in different varieties of cigar leaves, as well as the flavors of different samples (Zeki et al., 2020). However, the correlation between the flavor substances in cigar leaves, style characteristics of cigars, and the mechanism of their influence on the sensory quality of cigars among the different varieties requires further research.

Conclusion
In this study, we used GC-IMS to detect, analyze, and build the fingerprints of volatile compounds contained in eight major Chinese CFTLs. We identified and classified 84 volatile compounds, mainly composed of nitrogen, ketones, aldehydes, alcohols, and esters. The GC-IMS fingerprint of the flavor substances contained in cigar tobacco is an efficient tool for the effective identification of the different varieties of cigar tobacco. Additionally, the exploration of key differential volatile compounds can help in revealing the mechanism of flavor differences in the different cigar varieties. This study provides data support for the application of GC-IMS analysis in the rapid grading of cigar tobacco leaves as well as a theoretical basis for studying the flavor-formation mechanism of cigar tobacco leaves. The correlation between these findings via GC-IMS and via GC-MS should be further studied.
Supporting information: Figure S1. Twodimensional GC-IMS spectra of the different cigar tobacco leaves. The ordinate represents the retention time of the volatile compounds during GC separation. The background of the entire graph is blue, the abscissa represents the ion migration time and the red vertical line at 1.0 of the abscissa is the RIP peak (reactive ion peak, normalized). Each point on the right side of the RIP peak represents a volatile compound and the color indicates the signal intensity of a single compound. Red represents high intensity and blue represents low intensity. Table S1. Analysis of volatile organic compounds in different varieties of cigar leaves. Table S2. Peak position and volume of each compound.