Postmortem analysis of three methoxyacetylfentanyl-related deaths in Denmark and in vitro metabolite profiling in pooled human hepatocytes

• Published in: Forensic science international Vol. 290; pp. 310 - 317
• Main Authors: Mardal, M., Johansen, S.S., Davidsen, A.B., Telving, R., Jornil, J.R., Dalsgaard, P.W., Hasselstrøm, J.B., Øiestad, Å.M., Linnet, K., Andreasen, M.F.
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier B.V 01.09.2018; Elsevier Limited
• Subjects: Autopsy; Bile; Biological properties; Biological samples; Blood; Brain; Calibration; Case reports; Chromatography; Dealkylation; Deamination; Demethylation; Drug abuse; Fatalities; Femur; Fentanyl; Fentanyl analogue; Fluorides; Forensic sciences; Hepatocytes; High performance liquid chromatography; Hydroxylation; Intoxication; LC-HR-MS/MS; Liquid chromatography; Liver; Mass spectrometry; Mass spectroscopy; Metabolic pathways; Metabolism; Metabolites; Methoxyacetylfentanyl; Narcotics; Poisoning; Postmortem; Urine; Denmark; United States > US; Norway; Germany; Methoxyacetylfentanyl; LC-HR-MS/MS; Hepatocytes; Metabolism; Fentanyl analogue; Postmortem
• ISSN: 0379-0738; 1872-6283
• DOI: 10.1016/j.forsciint.2018.07.020

•Three postmortem cases tested positive for consumption of methoxyacetylfentanyl.•Quantification in whole blood, homogenized brain, and urine by UHPLC-MS/MS.•In vitro metabolite profiling by UHPLC-HR-MS/MS after hepatocyte incubations.•In vivo metabolite profiling by UHPLC-HR-MS/MS in postmortem biological samples. Methoxyacetylfentanyl belongs to the group of fentanyl analogues and has been associated with several deaths in recent years. We present three case reports of deceased individuals that tested positive for methoxyacetylfentanyl consumption, as well as in vitro and in vivo metabolite profiles. Methoxyacetylfentanyl was quantified by ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) in femoral blood, as well as in urine and brain tissue when these were available. Metabolite profiling was performed by incubating methoxyacetylfentanyl with pooled human hepatocytes (pHH) in Leibovitz's L-15 medium supplemented with fetal bovine serum. Metabolites were identified in vivo and in vitro using UHPLC–high resolution (HR)–MS/MS. The measured methoxyacetylfentanyl concentration was 0.022–0.056mg/kg (N=3) in femoral blood, 0.12mg/kg (N=1) in urine, and 0.074mg/kg (N=1) in brain tissue homogenate. A total of 10 metabolites were identified. The observed metabolic pathways were: hydroxylation(s), N-dealkylation, O-demethylation, deamination, glucuronidation, and combinations thereof. Major analytical targets in vitro and across measured biological samples in vivo were methoxyacetylfentanyl, the O-demethyl- metabolite, and the deamide-metabolite. Intoxication with methoxyacetylfentanyl was judged as the cause of death or a major contributing factor in all three presented cases.