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The aim of this study was to identify in vivo phase I metabolites of five psychoactive substances: N-ethylpentylone, ethylone, methylone, α-PVP and 4-CDC, using the in house developed experimental setup zebrafish (Danio rerio) water tank (ZWT). High-resolution mass spectrometry allowed for metabolite identification. A pilot study of reference standard collection of N-ethylpentylone from the water tank was conducted. Methods ZWT consisted in 8 fish placed in a 200 mL recipient-containing water for a single cathinone. Experiments were performed in triplicate. Water tank samples were collected after 8 h and pretreated through solid-phase extraction. Separation and accurate-mass spectra of metabolites were obtained using liquid chromatography-high resolution tandem mass spectrometry. Results Phase I metabolites of α-PVP were identified, which were formed involving ketone reduction, hydroxylation, and 2″-oxo-pyrrolidine formation. The lactam derivative was the major metabolite observed for α-PVP in ZWT. N-Ethylpentylone and ethylone were transformed into phase I metabolites involving reduction, hydroxylation, and dealkylation. 4-CDC was transformed into phase I metabolites, reported for the first time, involving N-dealkylation, N,N-bis-dealkylation and reduction of the ketone group, the last one being the most intense after 8 h of the experiment. Conclusions ZWT model indicated to be very useful to study the metabolism of the synthetic cathinones, such as N-ethylpentylone, ethylone, α-PVP and 4-CDC. Methylone seems to be a potent CYP450 inhibitor in zebrafish. More experiments are needed to better evaluate this issue. Finally, this approach was quite simple, straightforward, extremely low cost, and fast for "human-like" metabolic studies of synthetic cathinones. Keywords Synthetic cathinones • Zebrafish • In vivo metabolism • High-resolution mass spectrometry • Human-like experimental model • Reference standard collection in zebrafish water tank Electronic supplementary material The online version of this article (

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<p>Ultrahigh-resolution mass spectrometry like FT-ICR MS has greatly expanded our view of the complexity and reactivity of complex dissolved organic matter (DOM) in the environment. However, both the structural characterization and (semi)-quantification DOM are methodologically not fully resolved, despite recent advances with coupling liquid chromatography (LC) to FT-ICR-MS that allows isomeric separation. Likewise, matrix effects and the existence of multiple isomers with largely varying – but unknown – ionization efficiencies that are simultaneously ionized and detected in direct infusion (DI)-FT-ICR MS prevents (semi)-quantification of DOM compounds and has limited the comparability of samples. Finally, common normalization strategies applied for non-targeted DOM analysis with DI cannot be easily transferred to LC-type DOM data. Here we present a new method that combines a post-column infusion of internal standard (PCI-IS) with LC-FT-ICR MS in order to reduce and compensate matrix effects as well as to provide a robust and reliable way to normalize MS peak intensities and compare samples measured with LC-FT-ICR MS. To this end, DOM samples were analyzed and the peak intensity data normalized by the internal standard and other frequently used normalization methods (e.g. sum of intensity, base peak), and absolute intensity indicating that PCI-IS normalization provides superior precision and accuracy. The potential of this method to provide semi-quantitative information on polarity fractions of DOM is assessed by testing the precision, accuracy, and linearity of PCI-IS normalization of model compounds spiked into DOM samples. Our results indicate that the use of LC separation reduced matrix effects (as compared to DI) and, in combination with the internal standard, improved to potential to obtain reliable peak intensity information.</p>