Abstract
The purpose of this work was to investigate the in vitro metabolism of nitracaine, a new psychoactive substance, using human liver microsome incubations, to evaluate the cytochrome P450 (CYP) enzyme isoforms responsible for the phase-I metabolism and to compare the information from the in vitro experiments with data resulting from an authentic user’s urine sample. Accurate mass spectra of metabolites were obtained using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and were used in the structural identification of metabolites. Two major and three minor phase-I metabolites were identified from the in vitro experiments. The observed phase-I metabolites were formed through N-deethylation, N,N-deethylation, N-hydroxylation, and de-esterification, with CYP2B6 and CYP2C19 being the main enzymes catalyzing their formation. One glucuronidated product was identified in the phase-II metabolism experiments. All of these metabolites are reported for the first time in this study except the N-deethylation product. All the in vitro metabolites except the minor N,N-deethylation product were also present in the human urine sample, thus demonstrating the reliability of the in vitro experiments in the prediction of the in vivo metabolism of nitracaine. In addition to the metabolites, three transformation products (p-nitrobenzoic acid, p-aminobenzoic acid, and 3-(diethylamino)-2,2-dimethylpropan-1-ol) were identified, as well as several glucuronides and glutamine derived of them.
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Acknowledgments
This study has been financially supported by the EU through the FP7 projects under grant agreement #316665 (A-TEAM) and #317205 (SEWPROF). Noelia Negreira and Alexander L.N. van Nuijs acknowledge the University of Antwerp and Research Foundation Flanders (FWO) for their respective postdoctoral fellowships.
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The human urine sample used in this study was anonymously collected via routine investigations at the Emergency Department of the Universitair Ziekenhuis Brussel for diagnosis purposes, and therefore, it did not involve any ethical issues.
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Noelia Negreira and Juliet Kinyua contributed equally to this work.
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Negreira, N., Kinyua, J., De Brabanter, N. et al. Identification of in vitro and in vivo human metabolites of the new psychoactive substance nitracaine by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Anal Bioanal Chem 408, 5221–5229 (2016). https://doi.org/10.1007/s00216-016-9616-7
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DOI: https://doi.org/10.1007/s00216-016-9616-7