Method of Confirmation (MoC) and Predictive Metabolomics: A Novel Multidimensional Analytical Framework for Characterizing New Psychoactive Substances

of Confirmation (MoC) and Predictive Metabolomics: A Novel Multidimensional Analytical Framework for Characterizing New Psychoactive Substances

Rationale: The rapid emergence and structural diversity of new psychoactive substances (NPS) present significant challenges for forensic drug analysis. Existing targeted methods often fall short in accurately identifying novel compounds, increasing the risk of false positives or missed detections. This study aims to address this gap by developing a robust, non-targeted analytical approach capable of confidently screening, identifying, and confirming a broad range of NPS. Method: A method was developed using Ultra-Performance Liquid Chromatography coupled with Quadrupole Time-of-Flight Mass Spectrometry (UPLC-QTOF-MS) to analyze four selected NPS: Mephedrone (4-methylmethcathinone), Ephylone, 2C-B, and Methamphetamine. Analytical parameters such as retention time, mass accuracy (±5 ppm), diagnostic fragments, ion ratios, and isotope patterns were evaluated. The method was validated against critical criteria including precision, stability, and reproducibility. The limit of confirmation (LOC) for each analyte was established, and predictive metabolomics were performed to investigate metabolic transformations. Results: The UPLC-QTOF-MS method demonstrated excellent precision and reproducibility, with consistent retention times and diagnostic fragment ions within acceptable limits. Mass accuracy remained within ±5 ppm, confirming high analytical performance. The established LOC for all analytes was 50 ng/mL. The study also revealed significant metabolic transformations through predictive metabolomics. Compared to conventional tandem MS techniques, QTOF-MS offered enhanced non-targeted screening capabilities and the advantage of retrospective data analysis. Conclusion: The validated UPLC-QTOF-MS method provides a powerful, reliable tool for the rapid detection and confirmation of emerging NPS in forensic samples. Its high mass accuracy, ability to perform non-targeted analysis, and support for retrospective interrogation make it a superior alternative to traditional mass spectrometric methods. This approach enhances forensic laboratories’ ability to keep pace with evolving drug trends and strengthens the integrity of drug-related investigations. Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License. Collection

Authors Metrics & Citations Metrics Article Usage 360views 210downloads Citations Download citation Anirudha Dixit, Aakanksha Soni, Astha Pandey.

of Confirmation (MoC) and Predictive Metabolomics: A Novel Multidimensional Analytical Framework for Characterizing New Psychoactive Substances. Authorea. 19 May 2025. DOI: https://doi.org/10.22541/au.174769046.65846884/v1 DOI: https://doi.org/10.22541/au.174769046.65846884/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.