The evaluation of liquid chromatography high-resolution mass spectrometry (LC-HRMS) raw data is a crucial step in untargeted metabolomics studies to minimize false positive findings. A variety of commercial or open source software solutions are available for such data processing. This study aims to compare three different data processing workflows (Compound Discoverer 3.1, XCMS Online combined with MetaboAnalyst 4.0, and a manually programmed tool using R) to investigate LC-HRMS data of an untargeted metabolomics study. Simple but highly standardized datasets for evaluation were prepared by incubating pHLM (pooled human liver microsomes) with the synthetic cannabinoid A-CHMINACA. LC-HRMS analysis was performed using normal- and reversed-phase chromatography followed by full scan MS in positive and negative mode. MS/MS spectra of significant features were subsequently recorded in a separate run. The outcome of each workflow was evaluated by its number of significant features, peak shape quality, and the results of the multivariate statistics. Compound Discoverer as an all-in-one solution is characterized by its ease of use and seems, therefore, suitable for simple and small metabolomic studies. The two open source solutions allowed extensive customization but particularly, in the case of R, made advanced programming skills necessary. Nevertheless, both provided high flexibility and may be suitable for more complex studies and questions.

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.

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Introduction: The availability of toxicokinetic data is critical for detecting and monitoring the intake of psychoactive substances. Timely characterization of novel psychoactive substances (NPS) is particularly important to assess their abuse potential and inform public health responses. Methods: Toxicometabolomics offers a powerful approach to characterize xenobiotic metabolism through high-resolution profiling of biochemical transformations. It thus allows the finding of exogenous biomarkers, such as new drug metabolites, and endogenous biomarkers, which could be indications of acute drug ingestions or sample manipulation, as well as offering information on the mode of action of drugs. In this study, we applied a liquid chromatography–high-resolution mass spectrometry workflow to investigate the toxicometabolomics of two N1-sulfonated N,N-dimethyltryptamine derivatives with potential for both therapeutic use and recreational abuse. Results: Zebrafish (Danio rerio), an increasingly valuable model for preclinical pharmacology and toxicology studies, along with pooled human liver S9 fractions were used to elucidate metabolic pathways and identify key phase I and phase II biotransformations. Furthermore, untargeted metabolomics revealed significant downregulation of L-threonine associated with compound exposure. Conclusions: These findings advance the current understanding of tryptamine metabolism and underscore the utility of toxicometabolomics in the analytical evaluation of NPS.