In Vitro and In Vivo Toxicometabolomics of the Synthetic Cathinone PCYP Studied by Means of LC-HRMS/MS

Abstract

Synthetic cathinones are one important group amongst new psychoactive substances (NPS) and limited information is available regarding their toxicokinetics and -dynamics. Over the past few years, nontargeted toxicometabolomics has been increasingly used to study compound-related effects of NPS to identify important exogenous and endogenous biomarkers. In this study, the effects of the synthetic cathinone PCYP (2-cyclohexyl-1-phenyl-2-(1-pyrrolidinyl)-ethanone) on in vitro and in vivo metabolomes were investigated. Pooled human-liver microsomes and blood and urine of male Wistar rats were used to generate in vitro and in vivo data, respectively. Samples were analyzed by liquid chromatography and high-resolution mass spectrometry using an untargeted metabolomics workflow. Statistical evaluation was performed using univariate and multivariate statistics. In total, sixteen phase I and one phase II metabolite of PCYP could be identified as exogenous biomarkers. Five endogenous biomarkers (e.g., adenosine and metabolites of tryptophan metabolism) related to PCYP intake could be identified in rat samples. The present data on the exogenous biomarker of PCYP are crucial for setting up analytical screening procedures. The data on the endogenous biomarker are important for further studies to better understand the physiological changes associated with cathinone abuse but may also serve in the future as additional markers for an intake.

Comparison of reversed-phase, hydrophilic interaction, and porous graphitic carbon chromatography columns for an untargeted toxicometabolomics study in pooled human liver microsomes, rat urine, and rat plasma

“…Since the aim of this study was to evaluate the influence of different analytical LC columns on the resolution of selected endogenous compounds and the results of untargeted metabolomics analyses, only columns were changed and other parameters remained unchanged. However, eluents and gradients had to be adopted and were selected according to the column types and as used in other studies (Hemmer et al, 2022 ; Manier et al, 2019b ; Merck, 2019 , 2020 ; Michely & Maurer, 2018 ). In addition, the choice of mobile phases was evaluated by the detectability of different compound classes using a system suitability test mixture described in the Experimental Section of the Supplementary Information .…”

“…In addition, the choice of mobile phases was evaluated by the detectability of different compound classes using a system suitability test mixture described in the Experimental Section of the Supplementary Information . Sample preparations and all other LC-HRMS parameters such as column oven temperature, and MS settings were identical for all columns according to previously published studies (Hemmer et al, 2022 ; Manier et al, 2019b ).

Fig.

…”

“…Study design, sample collection, sample preparation for pooled human liver microsomes (pHLM), rat blood plasma, and rat urine were as described by Hemmer et al (Hemmer et al, 2022 ).…”

“…Analyses were performed using a Thermo Fisher Scientific (TF, Dreieich, Germany) Dionex UltiMate 3000 RS pump consisting of a degasser, a quaternary pump, and an UlitMate Autosampler, coupled to a TF Q Exactive Plus equipped with a heated electrospray ionization (HESI)-II source according to previous published studies (Hemmer et al, 2022 ; Manier et al, 2019a , 2019b ). Performance of the columns and the mass spectrometer was tested before each batch using a test mixture described in the Experimental Section in the Supplementary Information .…”

“…First, by comparing the chromatographic resolution of selected compounds. Second, their impact on the outcome of an untargeted toxicometabolomics study (Hemmer et al, 2022 ). The (toxico-)metabolome of three different biological matrices should be investigated after exposure to the synthetic cathinone PCYP (2-cyclohexyl-1-phenyl-2-(1-pyrrolidinyl)-ethanone) as model compound.…”