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.