The utility of in vitro metabolism studies coupled to liquid chromatography-tandem mass spectrometry for the selection of diagnostic markers of novel psychoactive substances: the case of 4,4’-DMAR

para-Methyl-4-methylaminorex (4,4’-DMAR) is a synthetic, substituted oxazoline derivative and analogue of the scheduled aminorex and 4-methylaminorex (4-MAR), both of which originally designed and synthesized as appetite suppressants. The psychostimulant properties of these substances are due to the interaction with dopamine transporter (DAT), serotonin transporter (SERT) and norepinephrine transporter (NET). Chemically, because of the two chiral centers in the oxazoline ring, 4,4’-DMAR exist as two different racemic (±) –cis and (±) –trans mixture or four distinct enantiomers; recent evidence suggests that only the cis isomers show a biological activity; furthermore, if cis and trans isomers are administered in combination, the effects of the cis isomer are enhanced. A recently validated method was used to detect the main metabolites in rat plasma and brain tissues and it allowed to identify four metabolites, due to hydroxylation, oxidation, hydrolysis and deamination. (Lucchetti, et al., 2017) Here we describe the in vitro metabolism of cis-4,4’-DMAR, investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on samples obtained after incubation with pooled human liver microsomes (HLM) and recombinant isoforms of cytochrome P450 and uridine diphospho glucuronosyl-transferase, with the aim of identifying the most suitable diagnostic marker(s) of intake, to be targeted in human urine in case of voluntary assumption. Following incubation with HLM, no metabolite was detected for the trans isomer, while six metabolites were found for cis-4,4’-DMAR as a result of mono-hydroxylation, di-hydroxylation and oxidation of the hydroxylated derivatives. The main cytochrome P450 isoforms involved in metabolism of this substance seems to be cytochrome P450 2D6 (CYP2D6), which is a polymorphic one. The chromatograms also shows the presence of a small amount of trans-4,4’-DMAR, probably due to the incubation with HLM or the exposure to heat. The same analysis was performed on the racemic mixture and it shows that this association leads to a lower amount of metabolites. This result suggests that the trans isomer could exert an inhibitory effect on the cis metabolism.