Influence of Temperature and Freeze-thaw Cycles on the Stability of Flunitrazepam and Clonazepam in Blood

This study investigated the effects of storage temperature and freeze-thaw cycles on the stability of flunitrazepam, clonazepam, and their 7-amino metabolites in whole blood, and clarified the patterns of concentration changes of the four target analytes in the whole-blood matrix over storage time, thereby providing scientific data support for interpreting discrepant results between initial and re-testing in actual cases arising from different storage conditions. In the experiments, blank whole blood was used as the matrix and spiked with reference standards to prepare samples at two concentration levels (10 ng/mL and 100 ng/mL). The samples were stored under refrigeration at 4°C and frozen at –20°C. At various time points, aliquots were taken to determine the relative peak?area ratios of each target analyte to the internal standard diazepam-D 5 . In parallel, the changes in relative content of the four drugs after 1 to 5 freeze?thaw cycles under short-term storage conditions were also evaluated. The results demonstrated that storage temperature significantly influenced drug stability; higher temperatures accelerated the degradation rate and led to more decreases in the concentrations of the target analytes in whole blood. Notably, at the early stage of storage, flunitrazepam and clonazepam showed a slight increase in concentration, which could be attributed to protein?binding effects. However, under short?term storage at –20°C with 1–5 repeated freeze-thaw cycles, the concentrations of the four target analytes did not exhibit any significant decline and remained relatively stable overall. Based on these findings, it is recommended that biological specimens collected after an incident should undergo qualitative and quantitative testing as soon as possible, and that low?temperature conditions be maintained throughout transportation and storage to minimize drug degradation. For samples requiring long-term preservation, the potential impact of storage duration on drug concentrations should be fully considered when interpreting the final results, and conclusions should be drawn cautiously to avoid misinterpretation.