Fate of fentanyl after formalin-fixed tissues: About an authentic case and a stability study

Abstract

Death leads to various body modifications: lividities, rigidities, cyanosis, swelling, etc. In order to delay the degradation of a body and to keep it in a state close to living, formalin-fixed can be done, in order to stop invasive bacterial growth and slow down decomposition. Formalin is the main preservative liquid used. Formalin will modify the characteristics of the xenobiotics present in the organism: modification of the pH, dilution of the compounds and/or chemical transformations. Thus, the toxicological results after formalin-fixed are therefore not characteristic of the time of death. The difficulty lies in the interpretation of the results. The authors report the case of a 41-year-old man, found unconscious at his home. Resuscitation manoeuvers were unsuccessful and death was pronounced shortly after. An external examination of the body was carried out, revealing no lesion suggestive of violence or cause of death. Femoral blood was collected in a dry tube (blood 1). An autopsy, performed a few days later, also failed to identify a cause of death. In the interval between the external examination and the autopsy, body preservation treatments were carried out. After a deeper investigation of the circumstances, fentanyl transdermal patches (Durogesic®) were discovered at the home of the victim. It was envisaged that the victim died from an overdose of fentanyl, linked of transdermal patches misuse. A complete toxicological analysis was requested on the samples collected during the autopsy, including femoral blood (blood 2). A standard analysis of blood 2 was performed, using validated laboratory procedures. In addition, stability study of fentanyl was conducted in the presence of formalin to determine possible influence. A 30 mL pool of blank blood containing 1 mg/L of fentanyl was prepared and then aliquoted into 30 tubes of 1 mL. Except for the first tube, 20 μL of formalin (20%) was added to each tube. On day 1, the first set of tubes (without formalin) was extracted and analyzed on UPLC-MS/MS. The stability study was spread over 4 months, and each analysis was done in duplicate ( n = 2). The femoral blood 2 analyzes showed the presence of methanol (present in the preservative liquid), fentanyl at toxic concentration (30 ng/mL) and norfentanyl (4 ng/mL). No other compound was detected. It is possible that the concentrations measured in blood 2 were lower than those at the time of death, due to preservative treatment, without being able to evaluate the percentage of degradation or dilution. However, the stability study of fentanyl over 4 months did not show any fentanyl degradation in the presence of formalin, as the concentration remained stable throughout the study. The blood 1 collected during the external examination was also analyzed. Fentanyl and norfentanyl were measured at 16 ng/mL and 1 ng/mL, respectively. The differences in concentrations between the two femoral blood samples confirm that there is no degradation of fentanyl in presence of formalin. Moreover, the higher concentrations measured after preservative treatments are in favor of a redistribution of fentanyl from the skin where the patches were worn (relocation of fentanyl linked to preservation treatments operations). The results can also be explain by the breaking of the plasma protein binding of fentanyl to albumin (estimated at 80–85%) in presence of formalin, and thus a significant increase in the concentration of free-fentanyl. A stability study showed that fentanyl does not degrade in the presence of formalin. Nevertheless, other formalin-related phenomena can have an impact on the concentrations (transfer of molecules and breaking of protein bonds), making interpretation very difficult, if not impossible.