Tobacco metabolomics by LC/MS: Geographical variations in leafy components

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  • Published: Jan 3, 2011
  • Author: Steve Down
  • Channels: HPLC
thumbnail image: Tobacco metabolomics by LC/MS: Geographical variations in leafy components

The secrets of the tobacco leaf: GC/MS and LC/MS

Within the tobacco industry, the composition of tobacco leaves is an important factor for health and quality control considerations. The chemical components generate the smoke which has a direct influence on the health of the smoker and those in the near vicinity. The same components are also useful for assessing the quality of the tobacco and identifying the geographical source of the leaves.

There are hundreds, if not thousands, of compounds within tobacco leaves and chromatographic techniques are well placed to smoke them out. In particular, GC/MS has been employed in several studies for chemical fingerprinting of tobacco and brand identification. The volatile and semivolatile compounds are separated by one- or two-dimensional GC before mass spectrometric analysis and comparison, often with the aid of chemometric analysis.

Until now, LC/MS has been used primarily for analysing specific tobacco components, rather than global analysis, but now a group of Chinese scientists has extended the scope to a full metabolomic study. Chunxia Zhao and colleagues from the Dalian Institute of Chemical Physics, the Kunming Cigarette Factory of Hongyun Tobacco (Group) Co., Ltd., and the Yunnan Academy of Tobacco Agricultural Sciences, Yuxi, reported their methodology in the Journal of Separation Science.

Tobacco leaf processing: double extraction to maximise components

Tobacco leaves originating from China and Zimbabwe were ground and extracted with two solvents, to release the hydrophilic and hydrophobic components. Three solvents were tested for each mode and those yielding the greatest number of peaks and the greatest peak areas were selected.

Water proved the best for hydrophilic components. Dichloromethane had the highest extraction efficiency for the hydrophobic components but hexane was selected "in order to reduce the risk of column pollution by strong retention components."

Under optimised extraction conditions with ultrasonication, more than 79 and 84% of the hydrophilic and hydrophobic metabolites, respectively (accounting for 96.3 and 92.2% of the summed responses) had CVs lower than 20%, which was deemed to be good reproducibility.

LC/MS of the tobacco leaf metabolome: geographic variations stand out

The extracts were all analysed by LC/MS using a reversed-phase C18 column and a gradient of acetonitrile in aqueous formic acid. The eluted compounds were analysed by positive-ion electrospray ionisation and the raw data were processed using commercial software which extracted lists of molecular features based on the retention times, m/z values and peak abundances.

A total of 656 components were identified, corresponding to 291 and 365 hydrophilic and hydrophobic compounds, respectively, illustrating the value of the extended extraction procedure.

The two datasets were combined and subjected to chemometric analysis. The score plot from partial least squares-discriminant analysis revealed complete separation of the Chinese and Zimbabwean tobaccos, with no overlap.

A second model based on 90% of the raw data as a training set with the remaining 10% as independent test data confirmed the reliability of the model with excellent geographical discrimination.

Fourteen components were responsible for differentiation and eight of them were identified. Higher levels of caffeic acid, 2-acetylpyrrolidine and lower levels of nicotinic acid and nicotine N-oxide were found in the Chinese tobacco, consistent with published reports. In addition, the Chinese leaf contained lower levels of trigonelline and L-proline and higher levels of delta-valerolactam.

The research team postulated that differences in content of specific components such as these might be responsible for the specific aroma characteristics of particular tobaccos.

The LC/MS technique appears promising for distinguishing between tobacco leaves from different parts of the world. However, this was a small study. Zhao recommended that it should now be extended to leaves from more locations, grown under different conditions and harvested at various times, to confirm its applicability.

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.



A metabolomics comparison by LC/MS has enabled the differentiation of geographically distinct tobacco leaves, say Chinese scientists, with 14 compounds playing key roles in the process

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