Purpose <p>Flunitrazepam is a widely used hypnotic agent detected in many fatal intoxication and suicide cases. However, its concentration in blood during autopsy differs from that at the time of death; it is either undetected or detected at lower concentrations than expected in post-mortem blood. In this study, we evaluated interactions between flunitrazepam and human blood using liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry (LC-Q-Orbitrap-MS).</p> Methods <p>We measured flunitrazepam, 7-aminoflunitrazepam via reduction by aldehyde oxidase 1 (AOX1), and the degradation product of 7-aminoflunitrazepam via the Fenton reaction by hemoglobin (Hb) in human blood. In addition, we also detected flunitrazepam, 7-aminoflunitrazepam, and their degradation products in the blood of individuals who died from intentional ingestion of flunitrazepam.</p> Results <p>We found that flunitrazepam was reduced to 7-aminoflunitrazepam by AOX1 in blood, and 7-aminoflunitrazepam was oxidized by Hb via the Fenton reaction. The mass spectra obtained by LC-Q-Orbitrap-MS revealed the formation of 7-amino-5-(2-fluorocyclohexa-1,3-dien-1-yl)-3-hydroxy-1-methyl-1,3-dihydro-2<i>H</i>-benzo[e][1,4]diazepin-2-one (7-amino-3-hydroxyflunitrazepam) in the Hb/H₂O₂ reaction mixture containing 7-aminoflunitrazepam, as well as in the blood of some individuals who died from intentional ingestion of flunitrazepam.</p> Conclusion <p>7-Amino-3-hydroxyflunitrazepam is the degradation product of flunitrazepam at various temperatures. The degradation is brought about by post-mortem changes via AOX1-mediated reduction and Hb-mediated Fenton reaction. Thus, 7-amino-3-hydroxyflunitrazepam may be a valuable biomarker for correcting blood flunitrazepam concentration at the time of death in clinical cases.</p>