Session 8

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The recent rapid innovations made in metabolite profiling and bioassays have begun to drive a change of paradigm in natural products research. Indeed, having at hand full or partial of structure of possibly all metabolites in given natural extract at different quantitative levels open the possibility to perform pharmacognosy studies from a more holistic perspective.
The increasingly amount of accurate metabolome data that can be acquired on massive sample sets, notably through high resolution mass spectrometry data dependent MS/MS analyses (HRMS/MS), allows mapping of natural extracts at an unprecedented precision level [1]. While the acquisition of larger volumes of data is ongoing, contextualizing it is a lagging process and new initiative such as LOTUS contributes to this effort by linking chemical, taxonomical and bibliographical information [2].
In this context we push forward our applications and further development of UHPLC-HRMS/MS Molecular network (MN) approaches [3] to provide enhanced annotation confidence level. We have recently built massive MN on natural extracts libraries, including thousands of plant and microorganism extracts. This allows the creation of virtual libraries of NPs through an advanced automated annotation pipeline. We are developing computational methods and structural-taxonomic libraries to navigate this massive chemical space in order to rationally identify valuable bioactive NPs to focus on. For their full chemical and pharmacological characterization, we then deploy an advanced workflow that allows precise targeted isolation based on metabolite profiling data. Different recent applications will illustrate these aspects and give future prospects in natural products research.

Acknowledgements
The authors are grateful to Green Mission Pierre Fabre for establishing and sharing the unique library of extracts and the Swiss National Science Foundation (SNF N° CRSII5_189921 / 1).

References
[1]  J.L. Wolfender, J.M. Nuzillard, J.J.J. van der Hooft, J.H. Renault, Anal. Chem. 91 (2019) 704-742.
[2]  A. Rutz, M. Sorokina, J. Galgonek, D. Mietchen, E. Willighagen, A. Gaudry, J.G. Graham, R. Stephan, R. Page, J. Vondrášek, C. Steinbeck, G.F. Pauli, J.L. Wolfender, J. Bisson, P.M. Allard, bioRxiv (2021) 2021.2002.2028.433265.
[3]           A. Rutz, M. Dounoue-Kubo, S. Ollivier, J. Bisson, M. Bagheri, T. Saesong, S.N. Ebrahimi, K. Ingkaninan, J.L. Wolfender, P.M. Allard, Front. Plant Sci. 10 (2019) 1329.

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Natural products and vegetable extracts are rich source of bioactive molecules such as flavonoids, alkaloids, saponins, and so on. Although many studies have been carried out on the chemical characterization of these samples, there is still much interest in the detection of novel bioactive compounds. Conventional one-dimensional liquid chromatography (1D-LC) does usually provide sufficient resolving power for their determination. However, in many cases, their complexity is so high that the separation capability offered by 1D-LC is not enough thus requiring more powerful analytical technologies for both their characterization and quantification. Comprehensive two-dimensional LC (LC×LC) involving the coupling of two or more orthogonal or quasi-orthogonal separation systems is an interesting alternative capable, through its selectivity and sensitivity, to detect minor components.
For the analysis of natural products and vegetable extracts, different LC×LC methodologies including NP×RP, RP×RP and HILIC×RP have been successfully investigated. In this contribution, applications of MicroLC×LC in the field of natural products are reported and discussed.

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Short peptides are of extreme interest in clinical and food research fields; nevertheless, they still represent a crucial analytical issue. This lecture aims to provide an overview of analytical solutions developed for enrichment, separation, and untargeted identification of short peptides in biological matrices (i.e.urine) and food matrices. Direct analysis of short peptides is not usually feasible due to the potential association with high abundant components, such as proteins, and the low abundance of peptides compared to other molecules, which can cause extensive ion suppression during electrospray ionization (ESI). A dedicated protocol for enrichment and clean-up of short peptides can overcome some of these issues. In particular, the use of carbonaceous material for the enrichment and separation will be discussed, highlighting the advantages of retaining polar, non-polar, acidic, and positively zwitterionic charged compounds due to strong hydrophobic and ion-exchange interaction [1]. However, sample clean-up is not straightforward either due to the heterogeneous nature of short peptides in polarity, hindering their pre-concentration from complex matrices and chromatographic separation. Alternative separation strategies will be considered to improve the coverage of short polar peptides. Another aspect that will be discussed in detail will be developing a database implemented in Compound Discoverer 3.0, a software dedicated to the analysis of short molecules, to create a data processing workflow specifically dedicated to short peptide tentative identification. Finally, an untargeted metabolomics approach of prostate cancer zwitterionic and positively charged compounds in urine will be presented [2].

Acknowledgements
The work was supported by the PRIN project Prot. 2017Y2PAB8, entitled “Cutting Edge Analytical Chemistry Methodologies and Bio-Tools to Boost Precision Medicine in Hormone-Related Diseases”, provided by the Italian Ministry of Education, Universities and Research.

References
[1]  S. Piovesana, A.L. Capriotti, A. Cerrato, C. Crescenzi, G. La Barbera, A. Laganà, C. M. Montone, C. Cavaliere, Anal Chem. 45 (2019) 11474-11481.
[2]  A. Cerrato, C. Bedia, A.L. Capriotti, C. Cavaliere, V. Gentile, M. Maggi, C. M. Montone, S. Piovesana, A. Sciarra, R. Tauler, A. Laganà, Anal. Chim. Acta, 1158 (2021) 338381.