Comparative stability assessment of designer benzodiazepines and methaqualones in dry blood spots and blood using...

New psychoactive substances (NPS) pose a significant and ongoing threat to public health. Among the most prevalent on the current drug market are designer benzodiazepines, while new groups, such as methaqualone analogs, continue to emerge to bypass legal restrictions. In this study, 26 NPS compounds from these two groups were analyzed to assess their stability in different storage conditions. Samples were secured either as whole blood (stored at 4 °C and -20 °C) or as dried blood spots (DBS, stored at room temperature and 4 °C). Analyses were performed using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry. The stability study lasted 365 days, monitoring both the degradation rate of analytes and the possible formation of degradation products. In addition, the influence of chemical structure on analyte stability was evaluated. Results demonstrated that the stability of analytes depended on both storage method and temperature. Several compounds showed superior long-term stability in DBS samples. For example, 3-hydroxyphenazepam was undetectable in whole blood by day four, while still present in DBS. In many cases, short-term stability was also better in DBS than in blood. Furthermore, specific degradation products were identified, such as diazepam from ketazolam and 7-aminoclonazolam from clonazolam. Overall, the findings highlight that the stability of methaqualone analogs and designer benzodiazepines is strongly related to chemical structure. DBS cards frequently provide equal or improved stability compared to whole blood, particularly in short-term analyses. Consequently, securing biological material in both forms is recommended to maximize analytical options in forensic and toxicological investigations.