In vivo detection of the new psychoactive substance AM-694 and its metabolites

• Blood and urine from a real case were screened by LC−MS/MS for NPS.
• AM-694 was present in urine at 0.084 μg/L; it was not detected in blood.
• AM-694 main metabolites were tentatively identified in urine.
• In Italy, AM-694 had never been reported before in real cases.
• Few documented cases can be due to a lack of routine analytical methods for NPS.

AM-694 or 1-(5-fluoropentyl)-3-(2-iodobenzoyl)indole is a synthetic cannabinoid that acts as a selective and a powerful agonist for CB1 receptor, inducing cannabinoid-like effects (euphoria, sedation, hallucinations and anxiety). Its spread, like for other synthetic cannabinoids, has increased in recent years and many web sources freely supply these kinds of new drugs. It can be taken by smoking or through oral consumption. A 25-years-old man was hospitalized at the local hospital following a major trauma after ingestion of alcohol and an unknown pill. Urine and blood samples were sent to our Forensic Toxicology Division to investigate on possible substance abuse. A general unknown screening of biological samples, extracted by liquid–liquid extraction (ethylacetate and dichloromethane) in basic, acidic and neutral conditions, was achieved to verify the presence of drugs of abuse and/or their metabolites, both in gas chromatography-mass spectrometry (GC–MS) and liquid chromatography-tandem mass spectrometry (LC–MS/MS). For the quantification of AM-694, urine was extracted by solid phase extraction (SPE) on a Bond Elut Certify cartridge; an acidic hydrolysis (HCl 30%, 95 °C, 60 min) was necessary before liquid–liquid extraction of metabolites. For the detection of benzodiazepines and their metabolites, an enzymatic hydrolysis was applied (β-glucuronidase, pH 4.5, 50 °C, 18 h). Quantification of AM-694 (internal standard AM-2201), midazolam and α-hydroxymidazolam (internal standard halazepam) were performed by LC–MS/MS analysis in multiple reaction monitoring ([M + H]+: m/z 436→190, 272, AM-694; m/z 360→155, 127, AM-2201; m/z 326→291, 223, midazolam; m/z 342→168, 203, α-hydroxymidazolam; m/z 353→241, 222, halazepam). The general unknown screening revealed the presence of AM-694 (urine sample) and benzodiazepines (urine and blood). The concentration of AM-694, obtained by LC–MS/MS, was 0.084 μg/L. Midazolam and α-hydroxymidazolam were detected in urine (0.97 and 74.58 μg/L, respectively) and in blood (34.84 and 23.15 μg/L, respectively). Qualitative information about the AM-694 metabolites was obtained by LC–MS/MS in selected-ion monitoring for the putative [M+H]+ ions: m/z 448, carboxylated metabolite; m/z 434, defluorinated metabolite; quantification was not possible since reference standards are not available. Our report is the first case of detection of AM-694 and its metabolites in human biological fluids in Italy. For this reason, this case constitutes a first worrisome alarm about the spread of this substance.

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