Comprehensive UHPLC-HR-MSE screening workflow optimized for use in routine laboratory medicine: Four workflows in one analytical method

•LC-HR-MS screening workflow developed for flexible drug screening.•Allows for component-resolved screening of up to thousands of bioactive compounds.•Combined screening library from a commercial and a crowd-sourced NPS libraries. A comprehensive HR-MS screening can be used to identify thousands of drugs from a single analysis, which makes it a valuable tool for broad-scope component-resolved toxicological analysis. However, it is common practice in clinical toxicology to perform restricted data analysis to avoid examining and/or reporting data not requested for examination. In this study, a HR-MS screening workflow was developed to allow a comprehensive toxicological evaluation, but also restricted and levelled data analysis to fit in a clinical setting. Following precipitation and reconstitution, samples were injected on an UHPLC-HR-MS and data were analyzed with the data processing software UNIFI. Analytical validation of 38 selected drugs of abuse (DoA), included determination of matrix effect, recovery, process efficiency, and limit of identification (LOI). The method was tested on 49 authentic samples and matrix-matched ranges of calibrators for 95 drugs. The LOI ranged from 0.3 to 1426.7 ng mL−1 for most analytes which was within expected concentration range for authentic samples with THC-COOH (>1722.0 ng mL−1) and morphine (1426.7 ng mL−1) as notable exceptions. Four individual screening workflows were developed: 1) a targeted workflow to serve as orthogonal identification of the 38 selected DOAs from another in-house method, 2) a general toxicology workflow, 3) an extended toxicology workflow including new psychoactive substances (NPS), and 4) a workflow for NPS based on the online HighResNPS library. Our study presents a comprehensive LC-HR-MS toxicology screening method optimized for laboratory medicine. The workflow allows for levelled data reviewing when requested without compromising the ability to perform full toxicological analyses.