Stability of 22 sedative-type drugs and metabolites in human urine under variable pH, temperature, and freeze–thaw conditions

Ensuring analyte stability is essential for accurate forensic and clinical detection of sedative-type drugs. This study systematically evaluated the stability of 22 sedative-type drugs and metabolites in human urine under controlled conditions varying by pH (4.0, 7.0), temperature (25 °C, 4 °C, -20 °C), and freeze-thaw cycles (5 cycles), using a fully validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. While compounds such as midazolam, clobazam, and zolpidem remained highly stable, others-including alprazolam, triazolam, and lorazepam-exhibited notable degradation, particularly under acidic pH and elevated temperature. Flunitrazepam and clonazepam showed distinct degradation with the formation of 7-amino metabolites at neutral pH. Notably, this transformation occurred only in urine and not in phosphate-buffered saline, suggesting a urine-specific mechanism. These findings highlight the importance of compound-specific preservation strategies. In scenarios where analyte identity or sample pH cannot be verified promptly, immediate refrigeration or freezing (ideally at -20 °C), along with minimizing freeze-thaw cycles, is strongly recommended to preserve sample integrity and ensure reliable toxicological interpretation.