Fast, accurate and sensitive detection of drugs in human tissue is of crucial importance in an investigation of a suspicious death. Here, we aimed to screen cocaine, diazepam, methadone and morphine in post-mortem muscle samples without sample preparation and in quasi-real time using rapid evaporative ionisation mass spectrometry (REIMS). REIMS enables the online MS analysis of vapours generated from tissue dissection by a diathermic knife. Human muscle samples were soaked in solutions of 4

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... s at different concentrations and multiple incubation times to check the feasibility of REIMS for this innovative application. Muscle samples soaked in blank saline were used as a control. The classification model was able to distinguish between 30 μg g-1 cocaine (m/z 304.2), 200 μg g-1 morphine (m/z 286.2), 10 μg g-1 methadone (m/z 310.2) and 10 μg g-1 muscle of diazepam (m/z 285.1). REIMS tandem MS confirmed that the mass peaks that contributed to the class separation, originated from the drugs of interest. As a proof-of-concept, a forensic case muscle sample from a methadone overdose was investigated using REIMS. Here, using our classification model, the recognition software was able to detect methadone, demonstrating that the REIMS method opens new possibilities in forensic toxicology and during autopsy, leading to faster crime solving and decreased costs.