Validation of a Dried Blood Spot (DBS) Liquid Chromatography – High-Resolution Mass Spectrometry (LC-HRMS) workflow for simultaneous identification of emerging opioids

: The rapid emergence of novel synthetic opioids, including fentanyl analogues and nitazene-class substances, represents a critical public health concern. Their high potency and continuous structural modification demand highly sensitive and adaptable analytical approaches for reliable forensic detection. This study evaluates dried blood spot (DBS) sampling as a robust alternative to conventional liquid matrices for the identification of synthetic opioids. A method based on liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) was developed and validated. Data acquisition was performed in full-scan data-dependent acquisition (DDA) mode. The combined DBS-LC-HRMS methodology is suitable for the detection of known fentanyl analogues and nitazenes, while also allowing the identification of potentially unknown compounds through data analysis. The limits of detection (LOD) and quantification (LOQ) ranged from 0.05 to 0.2 ng/mL and 0.1-0.75 ng/mL, respectively. The method established a linear range of 0.5-3 ng/mL for all analytes, with validation results supporting the suitability of DBS as a sample storage and pre-treatment approach, with simplified collection, storage and transport compared to traditional matrices. Stability was also assessed, demonstrating that DBS is a more robust and cost-effective approach for analyte preservation than frozen whole blood over a 7 days period. Furthermore, the method was successfully applied to real samples, confirming its suitability as an effective tool for forensic toxicology screening and analysis. The proposed workflow demonstrated satisfactory analytical performance, enabling the identification and quantification of 37 synthetic opioids; Overall, this strategy provides a flexible and efficient tool for routine forensic toxicology, with the capacity to adapt to the dynamic and evolving landscape of illicit synthetic opioids.