In vitro metabolism studies on new psychoactive substances: N-ethylhexedrone and Buphedrone

Over the last two decades synthetic cathinones (SC), often described as “Bath Salts”, have emerged as one of the most sought for class of Novel Psychoactive Substances (NPS) among the users. These substances are derivatives of cathinone, a naturally occurring beta-ketone amphetamine analogue found in the leaves of the Catha edulis (Khat) plant. Because of their initial legality and stimulating effects, often similar to cocaine or methylenedioxy-methamphetamine (MDMA), they gained a great popularity on the drug markets. As the banning law on cathinones progressed, new substances with different structures distant from the “mother” molecule, and thus more unpredictable in action, have been synthetized. Emerging SC have been commonly appearing in dark web as research chemicals and adulterants of MDMA and other stimulants or even as substitutes of older synthetic cathinones like mephedrone and MDPV. Having the active dose often lower than those substances and inducing higher desire of redosing, the ingestion of these NPS has often led to psychological distress, toxicity and even to fatalities in USA and in EU countries. Moreover, since NPS enter recreational markets every week, as reported by the EMCDDA, there is a constant need to monitor the use/consumption of these newly synthesized substances and obtain sufficient knowledge on their metabolites excretion profiles.

Until present, GC-MS has been used to analyse urine samples to assess the use of this NPS class, but methodologies as LC-MS have also been optimized to detect and quantify drugs and metabolites in biological samples. Notably, parent drugs are usually far less abundant than their metabolites in this type of samples. Hence, this study aims to access the main metabolites of selected SC in order to generate tools in need to help to estimate its use in population.

For that purpose, an emerging cathinone, N-ethylhexedrone (Hexen), was selected to be studied and compared with other cathinone derivative, Buphedrone. To access the main metabolites of these selected SC, in silico prediction of the metabolites of those compounds was carried out in order to map possible routes of metabolism of those compounds. In silico studies were complemented with in vitro metabolism studies (drugs incubated with liver microsomes), in order to detect and confirm the main metabolites present in the reaction mixture at several time points. Standards were synthesized for the analysis of these SC, their metabolites, as well as samples collected from the in vitro study at different time points during incubation, by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The obtained metabolic profiles were evaluated aiming to find metabolites that can be used as markers to estimate the consumption of these drugs. In conclusion, knowledge about the metabolites is of major relevance to allow detection and estimation of the drug consumption patterns and to unveil the metabolic route for assessing their toxicity, including drug-drug interactions.