Abstract
Metabolome studies in forensic toxicology focus on the search for endogenous biomarkers changed by, e.g., drugs of abuse. However, placebo-controlled studies, the ideal study design, in humans are scarce for ethical reasons. Thus, the idea of using routine samples became popular, although confounding factors cannot be controlled. To systematically evaluate the use of routine samples for metabolomics, a comparison between a placebo-controlled amphetamine study in humans (A, npos=18, nneg=18) to routine samples either positive or negative for amphetamine, prepared and analyzed over six months (re-evaluated, B, npos=28, nneg=35) and prepared and analyzed within a single analytical batch (re-extracted, C) was performed. Samples were analyzed using untargeted liquid chromatography-tandem-mass-spectrometry. Comparison was conducted on feature level and based on significance (p- and fold-change-values). Only 3 features were significant in A, B, and C, and 2 were identified as amphetamine-(fragments). All 31 significant features from A were present in B and C; however, only 11 (36%) and 4 (13%) of them were significant mainly because of higher variation. Still, other significant features were found in routine samples (B/C).
In conclusion, routine samples are generally suitable for detecting differences in the metabolome, even if they do not match those of a controlled study.
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Data availability
The datasets generated and/or analyzed during the current study are not publicly available due to ethical constriction regarding the private information present in routine data. Data can only be made available via the corresponding author upon reasonable request.
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Acknowledgements
The authors would like to thank Maja Keller for her support and express their gratitude to Emma Louise Kessler, MD for her generous legacy she donated to the Institute of Forensic Medicine at the University of Zurich, Switzerland for research purposes.
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Annina Bovens conducted the experiments, the statistical analysis and wrote the manuscript. Claudio Leu conducted the experiments. Lana Brockbals wrote the manuscript. Friederike Holze and Matthias E. Liechti provided the samples from the controlled administration study. Thomas Kraemer wrote the manuscript. Andrea E. Steuer had the organizational lead and wrote the manuscript.
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Informed consent was obtained for all data used from the placebo-controlled study (study A) performed by Holze et al. The study was registered at ClinicalTrials.gov (NCT03019822) and was in full accordance with the Declaration of Helsinki, as well as approved by the Ethics Committee Northwest Switzerland (EKNZ). Due to the retrospective nature of the routine data used for studies B and C a waiver and a declaration of no objection for ethical approval of the Cantonal Ethics Board of the Canton of Zurich were obtained (KEK waiver no. 42.2005 and BASEC-Nr. Req2017-00946).
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Bovens, A., Leu, C., Brockbals, L. et al. Comparison of human metabolome changes identified in a placebo-controlled amphetamine administration study versus those using forensic toxicology routine data. Sci Rep (2026). https://doi.org/10.1038/s41598-026-34985-w
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DOI: https://doi.org/10.1038/s41598-026-34985-w
