Postmortem distribution of flunitrazepam and its metabolite 7-aminoflunitrazepam in body fluids and solid tissues in an autopsy case: Usefulness of bile for their detection

• In an autopsy of asphyxia case, we identified fatal level flunitrazepam in blood.
• Various body fluids and solid tissue containing bile were collected in the autopsy.
• Detailed distribution of flunitrazepam and its metabolite was determined.
• We used modified QuEChERS method for extracting analytes and LC–MS–MS for analysis.
• We showed the bile useful for detection of flunitrazepam and its metabolite.

We experienced an autopsy case of a woman in her 70s, in which the direct cause of her death was judged as asphyxia due to the occlusion of food in the trachea. The postmortem interval was estimated at about 2 days. The specimens dealt with were femoral vein blood, right heart blood, left heart blood, bile, brain, lung, heart muscle, liver, spleen, kidney, pancreas, skeletal muscle, and adipose tissue. By tentative drug screening, we found a high concentration of 7-aminoflunitrazepam in the femoral vein blood, which lead us to examine the postmortem distribution of flunitrazepam and its metabolite 7-aminoflunitrazepam in her body fluids and solid tissues. The extraction of flunitrazepam, 7-aminoflunitrazepam and internal standard nimetazepam was performed by a modified QuEChERS method, followed by the analysis by liquid chromatography–tandem mass spectrometry. Because this study included various kinds of human matrices with quite different properties, we used the standard additional method to overcome the matrix effects. The concentration of 7-aminoflunitrazepam were generally much higher than those of the parent drug flunitrazepam for most specimens except for the adipose tissue, showing that flunitrazepam is readily metabolized to its 7-amino metabolite after absorption into the body both antemortem and postmortem. The outstandingly highest concentration of 7-animoflunitrazepam was found in the bile, followed by the kidney, pancreas, left heart blood, spleen and liver. Although a majority of flunitrazepam was converted to 7-aminoflunitrazepam, the flunitrazepam concentration was highest in the pancreas, followed by the spleen, bile, left heart blood, and brain. In contrast to the results on synthetic cannabinoids, the levels of flunitrazepam and 7-animoflunitrazepam in the adipose tissue were relatively low. The present study showed that the bile may be a useful specimen for detection of unchanged benzodiazepines/their metabolites to be collected at autopsy.