Testing for drugs in formalin-fixed tissues: About a complex medicolegal case involving midazolam and oxycodone

Abstract

Establish the analytical and interpretation criteria when the body has been subjected to formalin preservation techniques. Death is accompanied by various body modifications: lividities, rigidity, cyanosis, swelling, etc. Thanatopraxy refers to all the techniques that can delay the degradation of a body and keep it in a state close to living. This operation aims to stop the invasive bacterial evolution and to slow down the decomposition. Formalin and methanol (used to stabilize formalin) may change the characteristics of xenobiotics present in the body, modify the pH and dilute the compounds. The toxicological results are therefore not characteristic of the moment of death. The difficulty lies in the interpretation of the results since neither the exact volume of conservation liquid injected nor the volume of blood removed are known. The authors report the case of a 35-year-old man who died as a result of cancer. The body was embalmed the day after his death, at the request of his family, and buried. After a deeper investigation of the facts, it was envisaged that Mr. X. died as a result of an overdose of oxycodone, due to a dilution error of the pump, not correctly fixed by his nurse. A complete toxicological analysis was therefore requested to measure the concentrations, as the subject was simultaneously under midazolam and oxycodone. A standard analysis of blood was performed, using validated laboratory procedures. In addition, stability studies of oxycodone and midazolam were conducted in the presence of formalin to determine possible influence. A 30 mL pool of blank blood containing 1 mg/L of oxycodone and midazolam 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. The tubes were stored at + 4 °C until analysis. On day 1, the first set of tubes (without formalin) was extracted and analyzed on ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS ® , Waters). The stability studies were spread over 21 days, and each analysis was done three times ( n = 3). The femoral blood analyzes showed the presence of methanol (1.31 g/L) but no ethanol, midazolam at therapeutic concentration (74 ng/mL) and oxycodone at a toxic concentration (152 ng/mL). No other compound was detected. It can be concluded that the concentrations measured in blood were certainly lower than those at the time of death, without being able to evaluate the percentage of degradation and/or dilution. The stability study of oxycodone showed a rapid and complete degradation of the molecule, in contact with formalin (−99% in 9 days). Midazolam was less affected, although instability was evident (−48% and −73% in 9 and 21 days, respectively). These results should be taken into account when interpreting the concentrations. The results of the stability studies prove the degradation of both midazolam and oxycodone in the presence of formalin. It is therefore assumed that the victim was exposed to higher concentration of oxycodone, confirming the mistake of the nurse. This was also documented when evaluating the oxycodone pump records. The concentrations obtained during the toxicological analyzes are therefore underestimated in relation to the concentrations at the time of death, but the level of variation cannot be quantified. Formalin preservation techniques have negative consequences for toxicological analyses and their interpretations. Stability studies of oxycodone and midazolam were instrumental in understanding the probable causes of death of Mr. X. These observations constitute the first stability studies of these two compounds in the presence of formalin.