Liquid Chromatography-High-Resolution Mass Spectrometry-Based In Vitro Toxicometabolomics of the Synthetic Cathinones 4-MPD and 4-MEAP in Pooled Human Liver Microsomes
Synthetic cathinones belong to the most often seized new psychoactive substances on
an international level. This study investigated the toxicometabolomics, particularly the in vitro
metabolism of 2-(methylamino)-1-(4-methylphenyl)-1-pentanone (4-MPD) and 2-(ethylamino)-1-(4-
methylphenyl)-1-pentanone (4-MEAP) in pooled human liver microsomes (pHLM) using untargeted
metabolomics techniques. Incubations were performed with the substrates in concentrations ranging
from 0, 12.5, and 25 µM. Analysis was done by means of high-performance liquid chromatography
coupled to high-resolution mass spectrometry (HPLC-HRMS/MS) in full scan only and the obtained
data was evaluated using XCMS Online and MetaboAnalyst. Significant features were putatively
identified using a separate parallel reaction monitoring method. Statistical analysis was performed
using Kruskal-Wallis test for prefiltering significant features and subsequent hierarchical clustering,
as well as principal component analysis (PCA). Hierarchical clustering or PCA showed a distinct
clustering of all concentrations with most of the features z-scores rising with the concentration of
the investigated substances. Identification of significant features left many of them unidentified
but revealed metabolites of both 4-MPD and 4-MEAP. Both substances formed carboxylic acids,
were hydroxylated at the alkyl chain, and formed metabolites after combined hydroxylation and
reduction of the cathinone oxo group. 4-MPD additionally formed a dihydroxy metabolite and a
hydroxylamine. 4-MEAP was additionally found reduced at the cathinone oxo group, N-dealkylated,
and formed an oxo metabolite. These findings are the first to describe the metabolic pathways
of 4-MPD and to extend our knowledge about the metabolism of 4-MEAP. Findings, particularly
the MS data of the metabolites, are essential for setting up metabolite-based toxicological (urine)
screening procedures