Dual-ionization GC-Orbitrap HRMS workflow for screening, confirmation, and quantitation of selected psychoactive substances including novel psychoactive substances.

The rapid emergence of novel psychoactive substances (NPS) presents significant analytical challenges due to their structural diversity, continuous evolution, and occurrence at trace levels in complex matrices. In this study, an analytical workflow integrating orthogonal ionization strategies based on gas chromatography coupled with high-resolution Orbitrap mass spectrometry (GC-HRMS) was developed for reliable screening, confirmation, and quantitation of selected psychoactive substances. This study demonstrates the application of combining electron ionization-based screening with positive chemical ionization-based confirmation and quantitation, enabling complementary acquisition of fragmentation and molecular ion information for improved confirmatory confidence. The GC-EI-Orbitrap approach provided high-resolution full-scan data with excellent mass accuracy (±3 ppm), enabling confident identification through accurate mass measurement, isotopic pattern fidelity, and ion ratio confirmation (±20%). The method demonstrated consistent performance for selected classes representing amphetamine-type stimulants, synthetic cathinones, and phenethylamine derivatives, with reproducible fragmentation patterns and acceptable matrix effects. Quantitative analysis using GC-CI-PRM exhibited good linearity (R2 > 0.995) over the range of 1-100 ng mL-1, with limits of detection as low as 2.5 ng mL-1. Application to spiked forensic powder matrices confirmed the robustness and reliability of the workflow. This dual-ionization GC-Orbitrap HRMS strategy enhances confirmatory capability by combining fragmentation-based identification with molecular ion preservation, providing a robust and complementary approach for the analysis of emerging NPS and demonstrating the utility of dual-ionization strategies for forensic drug analysis.