A pseudotargeted method based on gas chromatography and mass spectrometry with selected-ion monitoring was established to investigate the metabolite differences of flue-cured tobacco from three different growing regions. The mixed solvent of acetonitrile/isopropanol/water (3:3:2, v/v/v) was chosen as the optimal extraction system based on the good repeatability and extraction efficiency. A self-developed software coupled with commercial software was used to establish the pseudotargeted method including 289 peaks and 47 groups. Multivariable statistical analysis indicated that tobacco samples can be obviously separated based on the geographical origins. On the basis of a Mann–Whitney U test, organic acids, phenols, and alkaloids had higher levels in Hunan province. In contrast, a large proportion of amino acids (including l-tyrosine, l-proline, and serine), sucrose, and linoleic acid were the highest in Yunnan province. Meanwhile, multiple metabolic pathways (including carbohydrate metabolism, tricarboxylic acid cycle, and nitrogen metabolism) were influenced by growing regions. Twenty-eight differential metabolites, which had great contributions to the classification of tobacco samples of three growing regions, were further defined. The results demonstrated that the developed pseudotargeted method was a powerful tool to investigate the metabolic profiling of tobacco leaves and discriminate tobacco leaves of different growing regions.

A “ppseudo” targeted method using GC-MS-selected ions monitoring was applied to investigate the chemical characteristics of commercial cigarettes made in China and foreign countries. To identify the components and define the quantative ions for SIM acquisition, a quality control sample was analyzed using GC-MS full scan. Acquired data were treated with a homemade software. A peak table with 312 components and their related quantitation ions was achieved for SIM acquisition. Structure elucidation was performed using library searching, retention index, standard compounds, and fitted retention time. The fitted retention time was calculated by a linear correction curve obtained using measured and library retention time to verify compounds. A total of 90 compounds were elucidated. Chemical characteristics of different cigarette brands were investigated. The data acquisition was carried out in SIM mode. The principal component and the hierarchical clustering analyses showed that the Chinese domestic flue-cured cigarettes were significantly different from the domestic blended, the foreign flue-cured, and blended cigarettes. Sixty-seven differential compounds were defined using the nonparametric Mann–Whitney test and the group blending samples comparison. Chinese domestic flue-cured cigarettes have higher concentration of saccharides and lower concentration of organic acids and amino acids.

Amino acids are one of the most important metabolites of organisms. They play an important role in plant growth, development, and product quality. A method based on RP ultra-performance LC with single quadrupole MS and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate precolumn derivatization was developed for the analysis of free amino acids in flue-cured tobacco leaves. Unlike the corresponding UV detection method, this method avoids matrix interference of complicated tobacco components, and the quantitative accuracy and resolution were improved. Twenty free amino acids were detected in flue-cured tobacco leaves. The method showed a good linearity with correlation coefficients of 0.9966–0.9998. The LODs for derivatized amino acids were 0.2–9.7 fmol/μL. Good repeatability with an RSD of 2.5–8.6% and satisfactory intra- and interday precisions were obtained. The developed method was used to investigate free amino acids in flue-cured tobacco leaves in China. The effects of aroma type, variety, and growing regions on free amino acids were investigated. The results showed that free amino acids in tobacco were affected by growing regions and varieties.