Stability Study Of Diazepam In Formalin-Fixed Rabbit Liver Tissue And In Rabbit Liver Stored At Different Temperatures

Objective To establish a method for simultaneous determination of diazepam in formalin-fixed rabbit liver tissue. Study on the stability of diazepam in formalin-fixed rabbit liver tissue and in rabbit liver stored at different temperatures. To provide a scientific basis for forensic identification of diazepam abuse poisoning.Methods 1. Study on the stability of diazepam in formalin-fixed rabbit liver tissue: The nine rabbits were equally divided into three groups (5%, 10%, 20% formalin) at random and then given intragastric administration of diazepam with a doze of 704.3mg/kg and executed two hours later. The liver tissues were collected and divided into 85 pieces (0.5g /piece), five of which were extracted at once and the other eighty pieces were equally fixed in water as well as acid, neutral and alkaline formalin solution of different concentration (5%, 10%, 20%). The content of diazepam was determined by GC/MS and GC at different times(1d,7d,14d,28d). 2. Stability study of diazepam in rabbit liver stored at different temperatures: The rabbits were separately given intragastric administration of diazepam with a doze of 704.3mg/kg and executed two hours later. The liver tissues were collected and divided into 42 pieces (0.5g /piece), six of which were extracted at once and the other thirty-six pieces were stored at three different temperatures (room temperature, 4 degrees C, -20 degrees C) and analyzed at selected times(1d,7d,14d,28d).Results 1.The linear range for diazepam in liver was 0.1~64μg/ml. The limit of detection was 0.05μg/ml. 2.The recovery of diazepam had a tendency to decline in 5%, 20% formalin solution and the fixed liver tissue. The same went for the 10% acid condition(P<0.05). It tended to decline then rise in 10% neutral formalin condition(P<0.05), while there was no significant statistics difference in 10% alkaline formalin condition(P>0.05). 3.In formalin solution of different degree of acidity, the recovery of diazepam in the 10% group was higher than that in the 5%and 20% group. 4.The changes of recovery rate of diazepam in the three groups of different temperature all had a tendency to decline but the degrees were of significant difference(P<0.05).Conclusion Diazepam in the rabbit liver tissue was relatively stable in 10% alkaline formalin solution, but instable in 5% formalin solution, 20% formalin solution ,10% acid and neutral formalin solution. Time and temperature both have an influence on the content of diazepam in the sample, and diazepam is relatively stable in -20 degrees C condition.