Introduction: Mass spectrometry has been successfully used for the analysis of metabolites from plant tissues.[1] Modern ambient ionization sources play a pivotal role in the increased applicability of this analytical technique.[2] In this context, tissue spray-mass spectrometry (TSI-MS) provides an easy and rapid way to directly detect metabolites in different organism tissues. In this technique, a high voltage is applied in the analyzed tissue, which is placed in front of the mass spectrometer inlet. Aliquots of solvent can be added above the sample, extracting the metabolites. A spray is generated by electric field, and ionization occurs based on the same principles that governs the electrospray ionization phenomenon.[3] Catharanthus roseus is a widely studied medicinal plant species. Like other plants from Apocynacea family, it produces a vast number of terpenoid indole alkaloids (TIAs) through an intricate arrangement of biosynthetic reactions.[4] Although direct ionization techniques have already been used to analyze metabolites from Catharanthus roseus leaves,[5] an attempt to detect the major products of the TIAs pathway have not yet been studied. In this work, leaves and flowers of Catharanthus roseus were directly analyzed by TSI-MS technique in order to detect the major products of TIAs biosynthetic route. Methodology: TSI-MS experiments with collision induced dissociation (CID) of the detected ions (TSI-MS/MS) were achieved in a Thermo® LCQ-Fleet IT-MS mass spectrometer. ESI ionization source was modified to allow TSI technique applications. Flowers and leaves were cut in triangular pieces and placed in front of the spectrometer inlet. Aliquots of 10,0 μL of a solution of formic acid in methanol, 0,1%(v/v), was used as solvent to generate the spray. A voltage of 3,0 kV was applied to the tissue in order to acquire the spectra. Results and Discussion: TSI-MS and TSI-MS/MS provided the detection of a total of 10 TIAs in leaves and flowers of Catharanthus roseus, including the bisindole alkaloids vincristine, vinblastine and 3,4-anhydrovinblastine, which are characterized by its impressive anticancer activities. These compounds are also some of the final products of the TIAs biosynthetic route. For the confirmation of these metabolites, leaves and flowers extracts were submitted to high-resolution mass spectrometry and LC-MS experiments. Conclusion: Tissue spray – mass spectrometry provided the detection of some of the major products of TIAs biosynthetic route. The developed methodology showed to be simple, rapid and efficient providing qualitative information in few minutes, with minimum sample preparation and low solvent consumption, which is a great advantage in times of sustainable chemistry.

References

1. Ernst, M.; Silva, D. B.; Silva, R. R.; Vêncio, R. Z.; Lopes, N. P., Mass spectrometry in plant metabolomics strategies: from analytical platforms to data acquisition and processing. Nat Prod Rep 2014, 31, 784-806.
2. Clendinen, C. S.; Monge, M. E.; Fernández, F. M., Ambient mass spectrometry in metabolomics. Analyst 2017, 142, 3101-3117.
3. Liu, J.; Wang, H.; Cooks, R. G.; Ouyang, Z., Leaf spray: direct chemical analysis of plant material and living plants by mass spectrometry. Anal Chem 2011, 83, 7608-13.
4. Pan, Q.; Mustafa, N. R.; Tang, K.; Choi, Y. H.; Verpoorte, R., Monoterpenoid indole alkaloids biosynthesis and its regulation in Catharanthus roseus: a literature review from genes to metabolites. Phytochemistry Reviews 2016, 15, 221-250.
5. Hu, B.; Wang, L.; Ye, W. C.; Yao, Z. P., In vivo and real-time monitoring of secondary metabolites of living organisms by mass spectrometry. Sci Rep 2013, 3, 2104.