Flavor research needs GC/MS analysis
GC/MS methods are widely used in metabolomics research. In the case of tea plants, the sample matrix is highly complex and a huge number of potentially relevant flavor compounds are involved. Literature sources count around 600, according to Professor Robbat. This is the reason that a sequential, multidimensional GC-GC/MS method was used in order to comprehensively determine all detectable volatile metabolites in spring and monsoon teas. The acquired data were used to build a metabolite database that relies on a deconvolution software to solve highly complex analysis tasks very quickly using a standard GC-MSD system. This is even possible when multiple compounds co-elute and with overlapping signals. The deconvolution technology developed by Professor Robbat and his colleagues offers added value in comparison with conventional approaches. This includes the possibility to obtain and store retention times and spectra of neat metabolites that aren’t skewed by matrix influence. It is also possible to identify perfectly co-eluting compounds. Professor Robbat and his group routinely measure the relative concentration of more than 400 compounds from infused tea leaves.
Initially the aim had been to use the spring harvested tea samples to find as many metabolites as possible and build a solid reference database. Al Robbat: “In order to build a comparative database, we instead analyzed tea samples consisting of buds and leaves that had been collected over three day periods in both spring and summer”. In other words, the periods spanned both the dry season (spring) and the rainy season (monsoon). Collected tea samples were briefly microwaved in the field to stop enzymatic activity and subsequently sent to the laboratory in sealed plastic bags. In the laboratory, they were vacuum sealed, wrapped in aluminum and stored in a deep freezer awaiting analysis. GC-GC/MS analysis was performed on extracts of the tea samples. The previously used classical extraction procedure was simultaneous distillation and extraction (SDE) using the Likens-Nickerson process on a tea infusion prepared using deionized water and concentrated under a nitrogen atmosphere over anhydrous sodium sulfate. For the work described here, the Gerstel Twister was used since it provides a simple, clean method to extract organic chemical compounds from the tea infusion requiring no or almost no use of solvent. The Twister is a glass coated magnetic stir bar fitted with a PDMS sorbent phase. While stirring the sample, the Twister efficiently extracts organic compounds from the sample. The Twister was also used to collect organics in the field by placing it directly under the leaf and holding it in place with a magnet . For the tandem GC-GC/MS analysis Al Robbat and his colleagues used two 6890 GCs from Agilent Technologies. The first system was fitted with an FID; analyte separation was performed using a column with polar stationary phase. Sample introduction to the Cooled Injection System (CIS) PTV-type inlet was automated using the Gerstel Multi Purpose Sampler (MPS). The second GC system was configured with a non-polar capillary column and a Cryo-Trapping System (CTS) in order to enable heart-cutting and cryofocusing of fractions separated on the first column and to deliver these to the second column as a narrow band for best possible quality of separation as well as MS determination (Agilent Technologies MSD 5973). The second system employs a GC 6890 and two Low Thermal Mass (LTM) units with individual GC columns (Agilent Technologies), to perform the GC-GC/MS experiments. Professor Robbat’s group analyzed spring and summer, high and low elevation samples from both Yunnan and Fujian Provinces, temperatures during sampling varied by up to 10 °C. The samples were taken to study locational differences over a 3-year period (2014 – 2016) . The data analysis software developed by Robbat and sold through Gerstel provided the means to analyze these samples by GC/MS and track compositional changes in tea under stress conditions .
Analysis results confirm sensory impression
“Our analysis results confirm what tea farmers tell you: The spring tea is of much higher quality and has a sweet floral flavor compared with the monsoon tea, which is described as green or earthy. Using tandem GC-GC/MS analysis we succeeded in identifying hundreds of metabolites in the spring and monsoon teas”, Professor Robbat says. His group found 169 metabolites that were common to both tea types and more than 100 compounds that were unique seasonal compounds. A further 163 compounds were detected, but could not be identified. In future, we will increasingly have to contend with extreme weather conditions. This means that plant research must develop tools that can monitor the influence not only on plant growth and yield, but also on taste, flavor and nutritional value of crops. “Our work has delivered a set of tools to monitor seasonal variations in the metabolism of tea plants”, Al Robbat is convinced, “and I’m certain these tools can be used for similar metabolomics studies of crops and human systems”.
 A. Kowalsick, N. Kfoury, A. Robbat Jr., S. Ahmed, C. Orians, T. Griffin, S. B. Cash, J. R. Stepp, Metabolite profiling of Camellia sinensis by automated sequential, multidimensional gas chromatography/mass spectrometry reveals strong monsoon effects on tea constituents, Journal of Chromatography A 1370 (2014) 230-239
 S. Ahmed, J. R. Stepp, C. Orians, T. Griffin, C. Matyas, A. Robbat, S. Cash, X. Dayuan, L. Chunlin, U. Unachukwu, S. Buckley, D. Small, E. Kennelly, Effects of extreme climate events on tea (Camellia sinensis) functional quality validate indigenous farmer knowledge and sensory preferences in tropical china, PLoS ONE 9 (2014)e109126
 N. Kfoury, E. Scott, C. Orians, A. Robbat, Jr., Direct Contact Sorptive Extraction: A Robust Method for Sampling Plant Volatiles in the Field, Journal of Agricultural and Food Chemistry 65 (2017) 8501-8509
 N. Kfoury, J. Morimoto, A. Kern, E. R. Scott, C. M. Orians, S. Ahmed, T. Griffin, S. B. Cash, J. R. Stepp, D. Xue, C. Long, A. Robbat Jr., Striking changes in tea metabolites due to elevational effects, Food Chemistry 264 (2018) 334-341
 A. Robbat Jr., N. Kfoury, E. Baydakov, Y. Gankin, Optimizing targeted/untargeted metabolomics by automating gas chromatography/mass spectrometry workflows, Journal of Chromatography A 1505 (2017) 96-105
* G. Deußing: Redaktionsbüro Guido Deußing, 41464 Neuss/Germany