Recovery and Analysis of Diazepam from Dried Blood Stains

This study aims to determine whether it is possible to detect diazepam in bloodstains dried on a glass
surface, to assess stability of diazepam in bloodstains over several days and lastly to determine whether dried bloodstains can give reliable quantitative information. One millilitre of blood fortified with different concentrations of diazepam (1, 5, 10, 50 μg/ml) was spotted on a glass surface and allowed to dry at room temperature. Liquid-liquid extraction (LLE) was completed and 100μL of internal standard (flurazepam) was added to the sample, vortexed and allowed to equilibrate for 15 min followed by the addition of 1ml of carbonate-bicarbonate buffer. Diazepam was extracted with 1ml of toluene: heptane (9:1, v/v) and centrifuged for 10min at 4500 rpm. The organic layer collected was analysed by gas chromatography mass spectrometry (GC/MS) using selective ion monitoring (SIM) mode.
The method was checked for linearity, limit of detection (LOD), limit of quantitation (LOQ), precision,
accuracy, extraction recovery and stability. Good linearity was obtained between 1-100 μg/ml (r2=0.9983) for diazepam in dried bloodstains. LOD and LOQ in bloodstains were 0.5-1 μg/ml and 1 μg/ml
respectively. Interday precision was 2-10% and accuracy ranged from -66.3% to -39.3%. A scatter plot showed results obtained from dried bloodstains and whole blood was comparable. However, stability study conducted on five days showed that diazepam was not stable in dried bloodstains since an apparent decrease in the measured concentration of diazepam was observed on each day. It was shown that
detection of diazepam in dried bloodstains was possible. Stability study showed that diazepam was not stable in dried bloodstains. Therefore, toxicological analysis of diazepam in bloodstains can assist the court based on qualitative information but not on quantitative information since measured concentration varied on different days. Toxicological analysis of dried bloodstains can be important for the police and lawyers since it can help to determine the chronology of events in a crime and helped the court to
reconstruct that crime. The new ideas raised in this study compared to other studies are: glass used as a substrate, diazepam drug, stability and recovery study were assessed on 5 days.