This study aimed to optimize, validate, and apply a toxicological screening method using Dried Blood Spot (DBS) coupled with high-resolution mass spectrometry (HRMS) in forensic toxicology. The primary goals included method refinement for extraction and analysis, validation based on critical performance parameters, and testing the method with post-mortem blood samples.

Samples containing 20 xenobiotics from diverse pharmacological classes were prepared for DBS analysis. Targeted and non-targeted toxicological screening was performed by LC-HRMS (Orbitrap Exploris 120, ThermoFisher Scientific). Optimization included the extraction process (sonication time, recovery volume) and analytical parameters (injection volume, ion intensity thresholds for data dependent analysis). Validation followed European Medicines Agency (EMA) guidelines, assessing repeatability, reproducibility, stability, linearity, matrix effect, extraction yield, and lower limits of quantification (LLOQ). The limit of identification (LOI) was also evaluated with 135 compounds. Post-mortem blood samples (n=20) were analyzed to compare the method's accuracy and sensitivity with the laboratory's standard LC-HRMS procedure.

Optimization yielded a final DBS method with a recovery volume of 70μL, 30-minute sonication, and a 25μL injection volume. Validation demonstrated reproducibility, linearity, and acceptable LLOQ for most compounds, while some issues arose with stability at room temperature (mephedrone and 6-acetylmorphine) and matrix effect (ketamine). Extraction yields were low overall (15–85%), but sensitivity was sufficient for the detection of therapeutic concentrations (mean LOI of 48ng/mL for 135 compounds). Comparative analysis with LC–HRMS showed similar identification results (65 compounds), except for three discrepancies (bisoprolol (6ng/mL), codeine (<5ng/mL) and oxazepam (5ng/mL) were not detected using DBS) confirming the DBS method's detection capability.

The DBS method effectively balances small sample volumes and analytical robustness, presenting a reproducible, linear, and sensitive alternative for toxicological screenings. While unsuitable for precise quantification in forensic settings, DBS offers potential for qualitative analyses and broader toxicological applications. Further validations, including inter-sample contamination and extended compound library integration, should be done to make this method suitable for routine analysis of post-mortem blood samples.