Detection of the designer benzodiazepine flunitrazolam in urine and preliminary data on its metabolism

Abstract Designer benzodiazepines have emerged as recreational drugs. They are available via the Internet without control and are found in the form of falsified (fake) medicines. For some of them, limited information concerning their effects, their toxicity, and their detection in bio fluids is available in the literature. For others, nothing has been published, as in the case of flunitrazolam (FNTZ). To gain preliminary data on its elimination parameters in urine and to investigate its metabolism, one of the authors ingested one pink tablet bought on the Internet, after confirming the absence of other compounds and agreement with the labeled dosage (0.25 mg) by nuclear magnetic resonance (NMR). A software algorithm (MetaboLynx, Waters, Milford, MA, USA) was used to predict FNTZ biotransformation and four potential metabolites were proposed: 4‐hydroxy‐FNTZ, desnitro‐FNTZ, 7‐amino‐FNTZ, and 7‐acetamido‐FNTZ. Urine samples were collected over 72 hours following oral administration of one tablet. After liquid/liquid extraction at pH 9.5, FNTZ concentrations were determined using ultra performance liquid chromatography–triple quadrupole–mass spectrometry (UPLC–QqQ–MS/MS). FNTZ remained detectable in hydrolyzed urine for 21 hours after ingestion, with concentrations ranging between 1 and 18 ng/mL. About 3% of the initial dose was excreted in urine as total unchanged FNTZ during this period. In vitro experiments (HLM incubations) were performed using ultra performance liquid chromatography–quadrupole time of flight‐mass spectrometry (UPLC–QTOF–MS) in order to investigate the potential CYP‐ and UGT‐dependent metabolites where only 7‐amino‐FNTZ was detected as the only metabolite. However, in the urine specimens, desnitro‐FNTZ, 7‐acetamido‐FNTZ and 7‐amino‐FNTZ were the main detected compounds. The identification of FNTZ metabolites dramatically improves the detection windows of the drug up to 37 hours.