Integration of GC-MS Based Non-Targeted Metabolic Profiling with Headspace Solid Phase Microextraction Enhances the Understanding of Volatile Differentiation in Tobacco Leaves from North Carolina, India and Brazil

Dong-Ming Ma, Saiprasad V. S. Gandra, Navin Sharma, De-Yu Xie

Department of Plant Biology, North Carolina State University, Raleigh, USA.
ITC R & D Centre, ITC Limited, Peenya, Industrial Area, Bangalore, India.

DOI: 10.4236/ajps.2012.312215                

Abstract

In this report, gas chromatography-mass spectrometry (GC-MS) based non-targeted metabolomics is used to develop appropriate headspace solid phase microextractions (HS-SPME) to enhance the understanding of volatile complexity of flue-cured tobacco leaves. Non-targeted metabolic profiling of GC-MS shows that the extraction condition of HS-SPME at 100?C for 30 min provides a better metabolite profile than other extraction conditions tested. GC-MS and principal component analyses (PCA) show that among five types of fibers tested, 100 μm polydimethylsiloxane (PMDS), 65 μm polydimethylsiloxane/divinylbenzene (PMDS/DVB) and 75 μm carboxen/polydimethylsiloxane (CAR/ PMS) provide a better reproducible metabolite profile. Based on an appropriate PDMS extraction condition optimized, we use GC-MS analysis and PCA to compare metabolite profiles in flue-cured leaves of tobacco plants grown in North Carolina, India and Brazil, respectively. The resulting data of PCA show that the global metabolic profiles in North Carolina samples are separated from those in Brazil and India samples, two groups of which are characterized by a partially overlapped pattern. Several peaks that were differentially accumulated in samples were annotated to known metabolites by deconvolution analysis, such as norsolanadione, solavetivone and rishtin. Norsolanadione is detected only in Brazil samples. Solavetivone is detected in samples of India and Brazil but not in those of North Carolina. Rishtin is detected in samples of North Carolina and India but not in Brazil samples. These data indicate that not only can a non-targeted metabolic profiling approach enhance the understanding of volatile complexity, but also can identify marker volatile metabolites in tobacco leaves produced in different growth regions.

Keywords

Tobacco; Headspace Solid-Phase Microextraction; Gas Chromatography Mass Spectrum;Volatile Compounds

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	J. C. Leffingwell, “CAROTENOIDS AS FLAVOR AND fragrance precursors,” Leffingwell Reports, Vol. 2, No. 6, 2002, pp. 1-5.
[2]	J. Cai, B. Liu, P. Ling and Q. Su, “ Analysis of Free and Bound Volatiles by Gas Chromatography and Gas Chromatography-Mass Spectrometry in Uncased and Cased Tobacco,” Journal of Chromatography A, Vol. 947, No. 2, 2002, pp. 267-275. doi:10.1016/S0021-9673(02)00015-8
[3]	B. Rodu, “The Scientific Foundation for Tobacco Harm Reduction, 2006-2011,” Harm Reduction Journal, Vol. 8, No. 1, 2011, p. 19. doi:10.1186/1477-7517-8-19
[4]	L. F. Huang, K. J. Zhong, X. J. Sun, M. J. Wu, K. L. Huang, Y. Z. Liang, F. Q. Guo and Y. W. Li, “Comparative Analysis of the Volatile Components in Cut Tobacco from Different Locations with Gas Chromatography-Mass Spectrometry (GC-MS) and Combined Chemometric Methods,” Analytica Chimica Acta, Vol. 575, No. 2, 2006, pp. 236-245. doi:10.1016/j.aca.2006.05.079
[5]	Y. Li, T. Pang, Z. Guo, X. Wang, J. Deng, K. Zhong, X. Lu and G. Xu, “Accelerated Solvent Extraction for GC-Based Tobacco Fingerprinting and Its Comparison with Simultaneous Distillation and Extraction,” Talanta, Vol. 81, No. 1-2, 2010, pp. 650-656. doi:10.1016/j.talanta.2009.12.054
[6]	X. Zhu, Y. Gao, Z. Chen and Q. Su, “Development of a Chromatographic Fingerprint of Tobacco Flavor by Use of GC and GC-MS,” Chromatographia, Vol. 69, No. 7-8, 2009, pp. 735-742. doi:10.1365/s10337-009-0968-4
[7]	S. Risticevic, H. Lord, T. Gorecki, C. L. Arthur and J. Pawliszyn, “Protocol for Solid-Phase Microextraction Method Development,” Nature Protocols, Vol. 5, No. 1, 2010, pp. 122-139. doi:10.1038/nprot.2009.179
[8]	E. Aprea, H. Gika, S. Carlin, G. Theodoridis, U. Vrhovsek and F. Mattivi, “Metabolite Profiling on Apple Volatile Content Based on Solid Phase Microextraction and Gas-Chromatography Time of Flight Mass Spectrometry,” Journal of Chromatography A, Vol. 1218, No. 28, 2011, pp. 4517-4524. doi:10.1016/j.chroma.2011.05.019
[9]	G. Weingart, B. Kluger, A. Forneck, R. Krska and R. Schuhmacher, “Establishment and Application of a Metabolomics Workflow for Identification and Profiling of Volatiles from Leaves of Vitis vinifera by HS-SPME-GC-MS,” Phytochemical Analysis, Vol. 23, No. 4, 2011, pp. 345-358. doi:10.1002/pca.1364
[10]	N. Li, Y. Mao, C. Deng and X. Zhang, “Separation and Identification of Volatile Constituents in Artemisia Argyi Flowers by GC-MS with SPME and Steam Distillation,” Journal of Chromatographic Science, Vol. 46, No. 5, 2008, pp. 401-405.
[11]	S. Reale, P. Fasciani, L. Pace, F. De Angelis and G. Marcozzi, “Volatile Fingerprints of Artemisinin-Rich Artemisia annua cultivars by Headspace Solid-Phase Microextraction Gas Chromatography/Mass Spectrometry,” Rapid Communications in Mass Spectrometry, Vol. 25, No. 17, 2011, pp. 511-2516. doi:10.1002/rcm.5155
[12]	S. Bijlsma, I. Bobeldijk, E. R. Verheij, R. Ramaker, S. Kochhar, I. A. Macdonald, B. van Ommen and A. K. Smilde, “Large-Scale Human Metabolomics Studies: A Strategy for Data (Pre-) Processing and Validation,” Analytical Chemistry, Vol. 78, No. 2, 2006, pp. 567-574. doi:10.1021/ac051495j
[13]	J. Xia, N. Psychogios, N. Young and D. S. Wishart, “MetaboAnalyst: A Web Server for Metabolomic Data Analysis and Interpretation,” Nucleic Acids Research, Vol. 37, 2009, pp. W652-W660. doi:10.1093/nar/gkp356
[14]	J. Xia and D. S Wishart, “Web-Based Inference of Biological Patterns, Functions and Pathways from Metabolomic Data Using Metabo Analyst,” Nature Protocols, Vol. 6, No. 6, 2011, pp. 743-760. doi:10.1038/nprot.2011.319
[15]	S. S. Yang, C. B. Huang and I. Smetena, “Optimization of Headspace Sampling Using Solid-Phase Microextraction for Volatile Components in Tobacco,” Journal of Chromatography A, Vol. 942, No. 1-2, 2002, pp. 33-39. doi:10.1016/S0021-9673(01)01376-0
[16]	H. Kataoka, H. L. Lord and J. Pawliszyn, “Applications of Solid-Phase Microextraction in Food Analysis,” Journal of Chromatography A, Vol. 880, No. 1-2, 2000, pp. 35-62. doi:10.1016/S0021-9673(00)00309-5
[17]	E. Baltussen and C. A. Cramers, “Sorptive Sample Preparation—A Review,” Analytical and Bioanalytical Chemistry, Vol. 373, No. 1-2, 2002, pp. 3-22. doi:10.1007/s00216-002-1266-2
[18]	E. Demole and P. A. Enggist, “A Chemical Study of Burley Tobacco Flavour (Nicotiana tabacum L.) VI. Identification and Synthesis of Four Irregular Terpenoids Related to Solanone, Including a ‘Prenylsolanone’,” Helvetica Chimica Acta, Vol. 58, No. 6, 1975, pp. 1602-1607. doi:10.1002/hlca.19750580614