The phenomenon of new psychoactive substances is constantly evolving, with arylalkylamines being one of the largest groups among them. 5-Methylmethiopropamine (5-MMPA, mephedrene), a derivative of methiopropamine (MPA), is available from online vendors selling “research chemicals” and was first seized by German authorities in 2020. No scientific data about its pharmacological effects and its fate in the human body are available so far. Therefore, we investigated the in vivo and in vitro metabolism of 5-MMPA after detection of this compound in a fatal intoxication.

For tentative identification of phase-I metabolites 5-MMPA was incubated with pooled human liver microsomes (pHLM). Additionally, an authentic urine sample was worked up using protein precipitation with and without prior glucuronide cleavage. Analyses for metabolite identification were carried out using high resolution LC-ESI(+)-QTOF-MS. Chromatographic separation was achieved on a Kinetex® Biphenyl column (100×2.1mm, 2.6μm). For the post-mortem investigation of the fatal intoxication, a standard addition approach using automated SPE for sample workup was performed to quantify 5-MMPA in femoral blood with LC-MS/MS.

In the pHLM assay three phase-I metabolites were identified, of which only two where confirmed in the available authentic urine sample. In addition, six further phase-I and two phase-II metabolites were detected in the analyzed urine sample. Predominant metabolic reactions were hydroxylation, oxidation, N-dealkylation, glucuronidation and combinations thereof. In urine an oxo-hydroxy metabolite, that was not generated in the pHLM assay, was detected with the highest abundance. The concentration of 5-MMPA in the femoral blood sample was 7.100±600ng/mL.

The in vitro pHLM assay was not suitable to predict the main metabolites of 5-MMPA as confirmed in the available real case sample. The majority of the metabolic reactions, and in particular the oxidation of 5-MMPA, appear to be catalyzed by enzymes that are not present in an active state in the pHLM assay. Analyses of an authentic urine sample suggest oxo-hydroxy-5-MMPA (main metabolite) and N-demethyl-5-MMPA as suitable biomarkers for 5-MMPA consumption. To confirm the exaxt chemical strucuture of these markers, isolation and structure elucidation, e.g. by NMR, or the synthesis of reference compounds would be needed. In the fatal intoxication case, the elevated blood concentration of 5-MMPA was considered likely to have contributed to toxicity/death (equivalent to a Toxicological Significance Score according to Elliott et al. (2017) of 3). This represents the first documented fatal case involving 5-MMPA.