Short-term stability evaluation of synthetic opioids, cathinones and ketamine in dried urine spots. Method validation and application to real cases

Dried urine spots (DUS) are emerging as a practical alternative to traditional urine collection in forensic and clinical toxicology, offering advantages in storage, handling, and biosafety. However, limited data exist on the stability of psychoactive substances in this matrix, particularly for opioids, synthetic cathinones, and dissociative anaesthetics. This study validates an LC-MS/MS method for the simultaneous quantification of twelve psychoactive drugs in DUS: methadone, EDDP, oxycodone, tapentadol, tramadol, ketamine, norketamine, fentanyl, carfentanyl, furanyl fentanyl, 3,4-MD-α-PHP, and MDPV. We also assessed their short-term stability under ambient conditions, using both fortified and authentic samples from forensic and clinical cases. Validation followed SWGTOX guidelines, ensuring sensitivity, linearity, accuracy, precision, recovery, matrix effects, and carryover. Stability was assessed over a 3-week period at room temperature (DUS) and −20°C (urine) at four time points (T0–T3). Our results showed strong linearity (R² > 0.99), with LODs below expected thresholds (0.10 ng/mL in urine and 0.27 ng/mL in DUS), and acceptable accuracy and precision across all QC levels. Matrix effects were negligible; however, recovery was lower at minimal concentrations in DUS. Stability tests revealed analyte-dependent degradation, with synthetic cathinones showing higher resilience, and opioids such as EDDP, tramadol, and oxycodone demonstrating more rapid degradation in real-case samples. Our findings highlight the utility of DUS as reliable matrix for forensic toxicology, especially when a small amount of specimen occurs, although some limitations remain. Nevertheless, molecule-specific stability profiles necessitate tailored validation strategies. Future studies should expand the analyte range and assess long-term stability under variable environmental conditions.