Automated Workflow for High-Throughput LC–MS/MS Therapeutic Monitoring of Cannabidiol and 7-Hydroxy-cannabidiol in Patients with Epilepsy

his study describes the development and validation of a fully automated workflow serum sample preparation for the quantitative determination of cannabidiol (CBD) and its active metabolite 7-hydroxy-CBD, using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Implemented on an automated platform, the workflow performs key steps such as solvent dispensing, mixing, centrifugation, filtration, and supernatant transfer, producing 96-well plates ready for analysis. Human serum samples were obtained from patients with epilepsy treated with CBD. All samples were processed using both manual and automated methods to evaluate method agreement. Quantification was performed by LC–MS/MS with CBD-d3 as the internal standard (IS). Method validation was conducted in accordance with European Medicines Agency (EMA) guidelines, confirming that the automated protocol meets the recommended acceptance criteria for both intraday and interday precision and accuracy. Calibration curves demonstrated excellent linearity across the concentration ranges. Comparative analysis using Passing–Bablok regression and Bland–Altman plots demonstrated strong agreement between the methods. These findings support the clinical applicability of the automated method for the therapeutic drug monitoring (TDM) of CBD and 7-hydroxy-CBD, and its robust performance and scalability provide a solid foundation for the development of an expanded analytical panel covering a broader range of antiseizure medications (ASMs), enabling more standardized TDM protocols in clinical practice.