Method of Confirmation 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 using bulk powder spiking and spiked urine matrices.

Results

The UPLC-QTOF-MS method demonstrated excellent precision and reproducibility, with consistent retention times and diagnostic fragment ions within acceptable limits as per SANTE and WADA TD2023IDCR guidelines. 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.