Atmospheric pressure gas chromatography-mass spectrometric profiling of VOCs in fuits

Gas chromatography coupled with mass spectrometry (GCMS) is a well-established analytical approach for volatile compounds analysis and metabolomics. The most widely used ionization technique in GCMS is electron ionization (EI), which produces library searchable spectra dominated by fragments. The molecular ion in EI spectrum is often of very low abundance or absent. Many plant terpenoids have same molecular formula and shows matching fragmentation pattern when using EI, therefore any minor dissimilarity in the relative abundances of the masses in a spectrum lead to a false search result in NIST. Also lack of molecular ion information can give incorrect compound identification, if using spectral matching alone. Alternative approaches, such as the chemical ionization (CI), can be optimized to provide a molecular ion with reduced fragmentation, but with the serious drawback of a major loss of the sensitivity. Atmospheric pressure gas chromatography coupled with mass spectrometry (APGC-MS) is an ionization technique that generates a spectrum conserving the molecular ion species with minimal fragmentation; additionally the system offers high mass accuracy, which is extremely useful in structure elucidation of unknown volatiles. We are establishing an analytical method employing APGC-MS for VOCs in fruits like Grape and Strawberry. In first phase we have done optimization of instrumental parameters using mixture of pure reference standards of fruit aroma compounds, this also aided in understanding ionization patterns of VOCs using APGC-MS since there is no database(like NIST MS) is available for primary identification of the compounds. Using injections of pure reference standards we started developing in-house APGC-MS library of the fruit VOCs. Parallelly, efficiency of thermal desorption system (TDS) for the extraction of VOCs also has been teste

Improving the annotation of unknown metabolites via integration of GCMS and LCMS metabolomics: a close look into the complexity of grapevine glycosides

Every grape cultivar has its own unique genetic characteristics, leading to the production of a different secondary metabolite profile. Volatile compounds in grapes are usually analyzed via GC-MS, while their glycosides are evaluated by indirect methods, after enzymatic or chemical hydrolysis. A common feature of raw data from metabolomics experiments is that they cannot be immediately interpreted as relative concentrations of constituent compounds. A bottleneck here is the signal annotation, which is the process of tentatively associating the pseudospectra found with chemical structure. For the free volatiles, the annotation of large metabolomics dataset can be performed by an automatic pipeline that it is able to focus on hundreds of compounds simultaneously (Wehrens et al., 2014). However, there is scarcity of publications addressing the annotation of the glycosides, which makes the annotation of these compounds a challenging task. In this study we present the molecular profiling of volatile compounds and their precursors in ten selected genotypes, including Vitis vinifera cultivars, American species and interspecific crosses. We tried to “gain from the complexity”, since the difficult task of annotation of the glycosides was achieved through combined use of two orthogonal techniques, GC/MS and LC/HRMS, before and after enzymatic hydrolysis. The results show that both free and glycosidically bound aroma precursors behave differently in each different grape cultivars and species. As many as 66 free aroma volatile molecules (originally existing and released after hydrolysis) were profiled through GC/MS analysis, while 15 glycosides were identified through LC/HRMS and correlation with GC/MS data. Literature Wehrens R., Weingart G., Mattivi F. (2014) MetaMS: An open-source pipeline for GC-MS-based untargeted metabolomics. Journal of Chromatography B, 966, 109–116. Acknowledgements. The project of MG and LN is funded under the International PhD Programme in Genomics and the Molecular Physiology of Fruits (GMPF) by the Fondazione Edmund Mach (FEM), San Michele all’Adige, (TN) Italy. Financial support from the project ADP 2014, by Autonomous Province of Trento, Italy, is greatly acknowledge

Establishing an atmospheric pressure gas chromatography-MS (APGC-MS) based metabolomics platform

Metabolomics has become a powerful tool to study biological processes in organisms and to identify metabolites and Gas chromatography coupled with mass spectrometry (GCMS) is a well-established analytical approach in metabolomics. The most widely used ionization technique in GCMS is electron ionization (EI), which produces library searchable spectra dominated by fragments. The molecular ion in EI spectrum is often of very low abundance or absent. Many plant terpenoids have same molecular formula and shows matching fragmentation pattern when using EI, therefore any minor variation in the relative abundances of the masses in a spectrum lead to a false positive hit in NIST. Also lack of molecular ion information can give incorrect compound identification, if using spectral matching alone. Alternative approaches, such as the chemical ionization (CI), can be optimized to provide a molecular ion with reduced fragmentation, but with the serious drawback of a major loss of the sensitivity. Atmospheric pressure gas chromatography coupled with mass spectrometry (APGC-MS) is an ionization technique that generates a spectrum conserving the molecular ion species with minimal fragmentation; additionally the system offers high mass accuracy, which is extremely useful in structure elucidation of unknown volatiles. We are establishing Atmospheric Pressure Gas Chromatography-MS (APGC-MS) Based Metabolomics Platform for volatile plant metabolites; primary results using Grape as a model sample are presente

GCxGC-TOFMS in metabolomics of grape and wine samples

Fondazione Edmund Mach (FEM) is a leading institute in grape and wine research. Within the Department of Food Quality and Nutrition a metabolomics platform has been established during the last years. It offers a large variety of analytical instruments (liquid chromatography-tandem mass spectrometry (LC-MS/MS), and gas chromatography-MS/MS) which provide the possibility to measure a broad range of substance classes in many kinds of matrices with both unit resolution MS and also high resolution MS. Recently, the platform was completed with a LECO GCxGC-TOFMS. This talk presents different projects running on this instrument concerning grape and wine samples. The first example deals with Pinot grapes. A comparison of the volatile fraction of a somatic Pinot mutant with the wildtype was done. Volatiles were extracted and enriched with solid phase microextraction (SPME). The GCxGC-TOFMS chromatogram showed around 800 peaks with a S/N > 500 compared to 250 peaks in 1D GC. By applying the statistical compare function of the ChromaTOF software we could find around 50 compounds which were significantly different between mutant and wildtype. We could identify 15 of these compounds by means of authentic standards. The second example deals with the characterization of volatiles of several wines produced from new crossings. In this work the aim was to identify as many substances as possible in the wines, first by comparing mass spectra with an in-house mass spectra library and second by comparing with a commercial library and retention time indices. In parallel the samples were measured with a GC-olfactory to investigate the flavour impact of the volatile metabolites. In the third example the volatile fraction of Lambrusco sparkling wines was used to investigate its capability to serve as marker for the grape varieties used in the wine production. In parallel, the samples were also measured with 1D GC-high resolution MS in CI mode to support the identification of unknown metabolites. This work was partially supported by the AGER, Agroalimentare e Ricerca, cooperative project between grant-making foundations under the section “wine growing and producing”: project New analytical methodologies for varietal and geographical traceability of oenological products; contract n. 2011-0285

Targeted screening of free and glycosidically conjugated VOCs in wine

Five wine varieties were analysed for fifty selected free and glycosidically conjugated volatile organic compounds (VOCs) contents. These free and conjugated volatile precursors were separated by SPE and then glycosides were enzymatically hydrolysed for releasing free volatiles from their respective precursor. Later a sensitive, full-scan GCMS method was employed for the separation and detection of the volatile compounds. Out of selected fifty compounds, several were found in different wines and some were found commonly in all wines. Compounds belonging to several different classes were identified. Identification and confirmation of the compounds was done by retention time/ mass spectra comparison with reference standards and/or NIST MS spectral library matc

Metabolomics profiling using Atmospheric Pressure Gas Chromatography-MS

Gas chromatography coupled with mass spectrometry is a well-established analytical approach to metabolomics. The most widely used ionization technique is electron ionization (EI), which produces library searchable spectra dominated by fragments. The molecular ion in an EI spectrum is often of very low abundance or absent. The lack of molecular ion information, especially for derivatized compounds, can lead to incorrect identification, if using spectral matching alone. Atmospheric pressure GC (APGC) is a ‘soft‘ chemical ionization technique that generates a spectrum conserving the molecular ion species with minimal fragmentation. Here we explore the use of APGC-MS in metabolomics applications