2 RATIONALE: The study of natural products by electrospray ionization tandem mass spectrometry (ESI-MS/MS) is an important strategy for the characterization of the major fragmentation reactions which can then help to determine the composition of complex mixtures. Application of ESI-MS/MS to a series of isopimarane diterpenes from Velloziaceae allowed the rationalization of their fragmentation mechanisms. METHODS:Velloziaceae diterpenes were isolated by silica gel column chromatography and investigated by ESI-MS/MS analysis. The fragmentation studies were performed on a quadrupoletime-of-flight instrument using N2 as the collision gas. To help rationalize the fragmentation pathways observed, the geometry and sites of reactivity of the diterpenes were obtained by theoretical calculations using the B3LYP/6-31 + G(d,p) model. Fragmentation mechanisms were proposed on the basis of the calculated protonation sites and product ions energies using density functional theory (DFT) methods. RESULTS:The presence of hydroxyl and carbonyl groups on the terpene core influences the protonation site observed. One compound showed a radical cation as the base peak. MS/MS spectra exhibit water elimination as the major fragmentation pathway (via two ways), either when protonation takes place on the oxygen atom, or through elimination after activation from hydrogen migration. After the elimination of water, the formation of an endocyclic double bond induces a sequential retro-Diels-Alder (RDA) reaction as the major fragmentation step. CONCLUSIONS:A thorough rational analysis of the fragmentation mechanisms of protonated Velloziaceae diterpenes was used to propose the dissociation mechanisms in ESI-MS/MS. The presence of esters in the side chain also influenced the intensity or occurrence of the observed protonated or cationized molecules in ESI-MS. These results will aid the identification of analogues in sample extracts in future metabolomics studies.Keywords: fragmentation mechanism, Velloziaceae, natural products, terpene 3 IntroductionThe Velloziaceae family comprises about 270 species which are distributed mainly in South America and Africa. These plants are found growing in inhospitable environments -sandy and/or rocky soils, with high solar irradiation and low amounts of water. A typical biome is found above 1000 m altitude -known as rupestrian fields 1 . Despite this apparent weakness, many Velloziaceae species present a striking feature of being desiccation tolerant resurrection plants, such as species of the Vellozia genus and the African Xerophyta and Talbotia The structural characterization of the isopimarane diterpenes presented in this paper was performed by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and compared with previously published data. Although NMR is a most efficient method for the complete structural elucidation of natural products, 6 , 7 a relatively large amount of pure sample is required. This involves timeconsuming and costly isolation procedures or the use of expensive and complex (LC)/NMR ...

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