Effect of hemoglobin on the concentration of etizolam in postmortem blood determined by liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry

Etizolam (EZM), a benzodiazepine drug, is a derivative of thienodiazepine. EZM displays an array of biological activities, including as an amnesic, anxiolytic, hypnotic, and muscle relaxant. Given that EZM is associated with instances of lethal intoxication and suicide, it is crucial to establish its exact levels in postmortem (PM) blood. However, EZM concentration at autopsy often diverges from that at the point of death. Here, we demonstrate EZM undergoes hydroxylation and/or oxidation in a mixture of hemoglobin (Hb) and hydrogen peroxide (H2O2) at temperatures between 4 to 45 °C. Mass spectrometry combined with liquid chromatography analysis showed the formation of 1-(4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1, 2, 4]triazolo[4,3-a][1, 4]diazepin-2-yl)ethan-1-ol (α-hydroxyetizolam, M1), 4-(2-chlorophenyl)-2-ethyl-9-methyl-6H-thieno[3,2-f][1, 2, 4]triazolo[4,3-a][1, 4]diazepin-6-ol (M2) and 1-(4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1, 2, 4]triazolo[4,3-a][1, 4]diazepin-2-yl)ethan-1-one when EZM was incubated with Hb/H2O2. M1 and M2 were detected in the PM blood of individuals who had died after ingestion of drug, carbon monoxide poisoning, heart attack or choking, following deliberate ingestion of EZM. Our results show that M1 and M2, formed by Hb/H2O2-mediated PM EZM decomposition, are potential biomarkers that can be used to correct the EZM concentration in PM blood.