Stability of benzodiazepines in whole blood samples stored at varying temperatures

Study has been undertaken to determine the stability of four benzodiazepines: clonazepam, midazolam, flunitrazepam and oxazepam in whole blood samples. Spiked blood was stored at four different temperatures (room temperature, 4 degrees C, -20 degrees C and -80 degrees C) and analysed at selected times during one year. Determination was performed on the first, third and seventh day during the first week, then once a week for three weeks, once every two weeks for four weeks, then once a month for 4 months and finally, once every 2 months. Extraction was performed using liquid-liquid extraction with 1-chlorobutane, while quantification was carried out using high performance liquid chromatography equipped with a photodiode-array ultraviolet detector. At room temperature, the concentration of all benzodiazepines decreased over one year to 100 and 70% for low and high concentrations, respectively. At 4 degrees C, the decrease was between 90 and 100% for low concentrations and between 50 and 80% for high concentrations. At -20 degrees C, the measured decrease was between 10 and 20% for high and low concentrations, respectively. At -80 degrees C, the measured loss was not significant at high concentration except for midazolam. However, at low concentration the determined decrease was between 5 and 12%. The data collected suggests that quantitative results concerning long-term stored samples should be interpreted with caution in forensic cases. Further investigations concerning the stability of drugs in whole blood or other biological samples, additional methods of identification and determination as well as the establishment of optimal storage conditions should be undertaken in forensic cases.