Postmortem redistribution and quantitative analysis of bromazolam, etizolam, flualprazolam, and flubromazolam

Novel Psychoactive Substances (NPS) have been consistently prevalent in postmortem casework at the Miami-Dade County Medical Examiner (MDME) for over a decade. Between May 2019 and September 2025, 88 positive identifications of NPS benzodiazepines were reported, including bromazolam (n=47), etizolam (n=25), flualprazolam (n=15), and flubromazolam (n=1). To improve interpretation of these findings, a quantitative method for these four analytes was developed and fully validated for postmortem blood and tissues utilizing solid phase extraction followed by ultra-high performance liquid chromatography-tandem mass spectrometry. Postmortem redistribution (PMR) was also evaluated for each analyte. In total, 180 specimens from 81 cases were analyzed, including 70 peripheral bloods, 72 central bloods, 18 liver homogenates, 16 brain homogenates, and 4 antemortem bloods. Peripheral blood concentrations for bromazolam (n=26) and etizolam (n=14) ranged from 2.5-168 ng/mL (mean: 33.4 ng/mL; median: 16.9 ng/mL), and 2.3-350 ng/mL (mean: 59.4 ng/mL; median: 12.1 ng/mL), respectively. Central blood concentrations for bromazolam (n=27) and etizolam (n=15) ranged from 3.0-153 ng/mL (mean: 38.3 ng/mL; median: 17.6 ng/mL), and 2.0-295 ng/mL (mean: 67.5 ng/mL; median: 15.3 ng/mL), respectively. Flualprazolam concentrations were notably lower with peripheral blood (n=3) concentrations ranging from 2.2-9.0 ng/mL (mean: 5.6 ng/mL; median: 5.7 ng/mL), and central blood (n=5) concentrations ranging from 2.4-10.6 ng/mL (mean: 6.0 ng/mL; median: 6.5 ng/mL). PMR ratios were calculated for central to peripheral blood (C/P), liver to peripheral blood (L/P), liver to central blood (L/C) and postmortem to antemortem blood (PM/AM). Median C/P and PM/AM ratios were ≤1.3, while median L/P ratios were ≤5.2. The observed PMR ratios are comparable to those reported for traditional benzodiazepines, suggesting limited PMR. The observed C/P ratios further suggest that central blood may be suitable for quantitative interpretation when peripheral blood is unavailable. This study represents the first systematic evaluation of C/P, L/P, L/C, and PM/AM ratios for NPS benzodiazepines across multiple cases.