Blanco García, LauraCabarcos Fernández, PamelaÁlvarez Freire, IvánTabernero Duque, María JesúsBermejo Barrera, PilarMoreda Piñeiro, AntonioBermejo Barrera, Ana María2025-10-152025-10-152025-05-28Microchemical Journal Volume 214, July 2025, 1141381095-9149https://hdl.handle.net/10347/43088Tramadol is an opioid used to treat mild to moderate pain. It has a dual mechanism of action, being not only a pure, non-selective agonist of µ-opioid receptors, but also an inhibitor of neurotransmitter reuptake. In addition, tramadol’s main metabolite, O-desmethyltramadol (O-DMT), is also active and contributes to tramadol’s effects through the same mechanisms. The use of tramadol became relevant because of its lower rate of respiratory depression as a side effect compared to other opioids and its low potential for abuse. The belief that tramadol is a safer opioid has led to its widespread use, but also to its misuse and abuse. Therefore, the number of deaths reported to forensic toxicology laboratories due to tramadol abuse/poisoning, in combination or not with other substances of abuse, has increased. For this reason, methods for the determination of tramadol in alternative forensic specimens, such as pericardial fluid, which are useful post-mortem clinical specimens, are needed. A correlation between the concentration of several compounds in this fluid and in blood has already been established. Thus, pericardial fluid has been proposed as an alternative forensic sample and is of considerable use when blood cannot be obtained or is affected by post-mortem redistribution. A novel sample pretreatment method based on dispersive liquid–liquid microextraction (DLLME) was developed for the first time for the isolation of tramadol and its metabolite O-DMT from pericardial fluid. Acetone was used as a dispersant and chloroform as an extractant. Determinations were performed by gas chromatography-mass spectrometry (GC–MS) and method validation was performed according to FDA validation guidance. Results showed target linearity in the range 0.05–5.0 µg mL−1 for tramadol and 0.3–5.0 µg mL−1 for O-DMT with limits of detection (LOD) of 0.02 µg mL−1 and 0.1 µg mL−1 for tramadol and O-DMT, respectively.eng© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. Attribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/TramadolO-desmethyltramadolPericardial fluidDispersive liquid–liquid microextractionGas chromatography-mass spectrometry2301 química analíticajournal article10.1016/j.microc.2025.114138open access