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Brevetoxins (BTX) are a group of marine neurotoxins produced by the harmful alga Karenia brevis. Numerous studies have shown that BTX are rapidly accumulated and metabolized in shellfish and mammals. However, there are only limited data on BTX metabolism in fish, despite growing evidence that fish serve as vectors for BTX transfer in marine food webs. In this study, we aimed to investigate the in vitro biotransformation of BTX-2, the major constituent of BTX profiles in K. brevis, in several species of northern Gulf of Mexico fish. Metabolism assays were performed using hepatic microsomes prepared in-house as well as commercially available human microsomes for comparison, focusing on phase I reactions mediated by cytochrome P450 monooxygenase (CYP) enzymes. Samples were analyzed by UHPLC-HRMS(/MS) to monitor BTX-2 depletion and characterize BTX metabolites based on MS/MS fragmentation pathways. Our results showed that both fish and human liver microsomes rapidly depleted BTX-2, resulting in a 72-99% reduction within 1 h of incubation. We observed the simultaneous production of 22 metabolites functionalized by reductions, oxidations, and other phase I reactions. We were able to identify the previously described congeners BTX-3 and BTX-B5, and tentatively identified BTX-9, 41,43-dihydro-BTX-2, several A-ring hydrolysis products, as well as several novel metabolites. Our results confirmed that fish are capable of similar BTX biotransformation reactions as reported for shellfish and mammals, but comparison of metabolite formation across the tested species suggested considerable interspecific variation in BTX-2 metabolism potentially leading to divergent BTX profiles. We additionally observed non-enzymatic formation of BTX-2 and BTX-3 glutathione conjugates. Collectively, these findings have important implications for determining the ecotoxicological fate of BTX in marine food webs.

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A potent synthetic α2-adrenergic agonist called PT-31, (3-(2-chloro-6-fluorobenzyl)-imidazolidine-2,4-dione), was recently detected as a potential drug to be used as an adjuvant drug to treat chronic pain. The excellent pharmacological property of PT-31 highlights the importance in elucidating its metabolism, which could provide valuable information about its metabolite profile for further pharmacokinetics studies and additionally to estimate the impact of its metabolites on the efficacy, safety and elimination of PT-31. In this work, the study of the in vitro metabolism of PT-31 was initially carried out by using a liquid chromatography coupled to ion trap multiple-stage mass spectrometer (LC-IT-MSn) and a hybrid triple quadrupole/linear ion trap mass spectrometer (LC-QTrap). The production of at least three unknown oxidative metabolites was observed. Structural identification of the unknown metabolites was carried out by combination of LC-MS experiments, including selected reaction monitoring (SRM) and multi-stage full scan experiments. Further analysis of 1H-NMR led to the structural confirmation of the major metabolite. The results indicated that PT-31 was metabolized by a hydroxylation reaction in the imidazolidine-2,4-dione ring in rat and human liver microsomes, producing the metabolite 3-(2-chloro-6-fluorobenzyl)-5-hydroxyimidazolidine-2,4-dione in rat liver microsomes. A carbon hydroxylation onto the benzyl ring, produced two other minor metabolites of the PT-31 in rat liver microsomes.

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Metabolomics is the study of the metabolome, the collection of small molecules in living organisms, cells, tissues, and biofluids. Technological advances in mass spectrometry, liquid- and gas-phase separations, nuclear magnetic resonance spectroscopy, and big data analytics have now made it possible to study metabolism at an omics or systems level. The significance of this burgeoning scientific field cannot be overstated: It impacts disciplines ranging from biomedicine to plant science. Despite these advances, the central bottleneck in metabolomics remains the identification of key metabolites that play a class-discriminant role. Because metabolites do not follow a molecular alphabet as proteins and nucleic acids do, their identification is much more time consuming, with a high failure rate. In this review, we critically discuss the state-of-the-art in metabolite identification with specific applications in metabolomics and how technologies such as mass spectrometry, ion mobility, chromatography, and nuclear magnetic resonance currently contribute to this challenging task.

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Banana peels are well recognized as a source of important bioactive compounds, such as phenolics, carotenoids, biogenic amines, among others. As such, they have recently started to be used for industrial purposes. However, its composition seems to be strongly affected by biotic or abiotic ecological factors. Thus, this study aimed to investigate banana peels chemical composition, not only to get insights on eventual metabolic changes caused by the seasons, in southern Brazil, but also to identify the most relevant metabolites for these processes. To achieve this, a Nuclear magnetic resonance (NMR)-based metabolic profiling strategy was adopted, followed by chemometrics analysis, using the specmine package for the R environment, and metabolite identification. The results showed that the metabolomic approach adopted allowed identifying a series of primary and secondary metabolites in the aqueous extracts investigated. Besides, over the seasons the metabolic profiles of the banana peels showed to contain biologically active compounds relevant to the skin wound healing process, indicating the biotechnological potential of that raw material.

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(-)-grandisin is a tetrahydrofuran lignan that displays important biological properties, such as trypanocidal, anti-inflammatory, cytotoxic, and antitumor activities, suggesting its utility as a potential drug candidate. One important step in drug development is metabolic characterization and metabolite identification. To perform a biotransformation study of (-)-grandisin and to determine its kinetic properties in humans, a high performance liquid chromatography (HPLC) method was developed and validated. After HPLC method validation, the kinetic properties of (-)-grandisin were determined. (-)-grandisin metabolism obeyed Michaelis-Menten kinetics. The maximal reaction rate (Vmax ) was 3.96 ± 0.18 µmol/mg protein/h, and the Michaelis-Menten constant (Km ) was 8.23 ± 0.99 µM. In addition, the structures of the metabolites derived from (-)-grandisin were characterized via gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analysis. Four metabolites, 4-O-demethylgrandisin, 3-O-demethylgrandisin, 4,4'-di-O-demethylgrandisin, and a metabolite that may correspond to either 3,4-di-O-demethylgrandisin or 3,5-di-O-demethylgrandisin, were detected. CYP2C9 isoform was the main responsible for the formation of the metabolites. These metabolites have not been previously described, demonstrating the necessity of assessing (-)-grandisin metabolism using human-derived materials.

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