Metabolism study of two phenethylamine - derived new psychoactive substances using in silico, in vivo, and in vitro approaches.

New psychoactive substances (NPS) are substances that are not controlled by international drug control conventions but are abused and pose a threat to public health. Proscaline and methallylescaline are two phenylethylamines with psychoactive and stimulant effects and are also derivatives of the classic hallucinogen mescaline. However, limited toxicity information on proscaline and methallylescaline has hindered the identification of these two NPS. Therefore, data on the metabolic profiles of proscaline and methallylescaline are urgently needed. In this study, high-resolution mass spectrometry was used to establish three complementary metabolism models-computational prediction (in silico), zebrafish (in vivo), and human liver microsomes (in vitro)-to study the in vivo and in vitro metabolic fates of proscaline and methallylescaline. The models provided the first identification of 7 proscaline metabolites and 11 methallylescaline metabolites. In addition, hydroxylated and N-acetylated products were identified as the major metabolites of these two phenylethylamines. This enabled the selection of hydroxylated and N-acetylated metabolites as biomarkers of proscaline and methallylescaline, thereby facilitating the specific detection of the intake of these two NPSs in a relatively wide detection window.