Stability Study of Six 1,4-Benzodiazepines in Bio-fluids Stored at -20oC

Abstract

An approach has been undertaken to study the stability of six 1,4-benzodiazepines; alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam and lorazepam in spiked plasma, urine and saliva samples at defined time intervals. Quantification was carried out using high-performance liquid chromatography equipped with a photodiode-array ultraviolet detector while extraction was performed using SPE. Spiked samples (0.5, 2.0 ng μL-1) were stored at-20oC and analysed at selected times during 180 days for both plasma and urine, and 120 days for saliva. At -20oC, at the end of the observation period decrease was measured around 40% for flunitraepam and clonazepam and around 20% for remaining four benzodiazepines of the original levels. At end of study period for low concentrations (0.5 ng μL-1) all benzodiazepines were though detectable but a clear pattern of breakdown could not be established. Results of stability study through freeze-thaw cycle during seven successive days for spiked samples stored at -20oC were also reported. All analytes were seemed to be completely stable up to five cycles in plasma, urine, and up to three cycles in saliva. Moreover, stability study of all analytes over a period of one year revealed that standard solution in methanol stored at 4oC can be used without any remarkable degradation. The data collected suggest that quantitative results concerning long-term stored samples should be interpreted with caution in forensic cases.