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.

Characterization and tentative identification of new flunitrazepam metabolites in authentic human urine specimens using liquid chromatography‐Q exactive‐HF hybrid quadrupole‐Orbitrap‐mass spectrometry (LC‐QE‐HF‐MS)

“…The fragmentation of M6 included the elimination of the glucuronic acid moiety ( m/z of 316.11041, C 16 H 14 FN 3 O 3 ). We thus determined that the positions of the two hydroxyl groups in the benzene fluoride ring were dependent on the m/z 168.04747 (C 8 H 7 FNO 2 ) ion, which was generated by the fracture of the diazepine core and the elimination of an anilino group . Therefore, the addition of two hydroxyl groups in the benzene fluoride ring and the subsequent glucuronidation, conjugated with 7‐amino‐FNZ, was the most feasible explanation for M6 .…”

Testing for midazolam and oxycodone in blood after formalin‐embalmment: About a complex medico‐legal case

“…Chromatography was achieved using a Waters Acquity HSS C18 column (150 x 2.1 mm x 1.8 μm) maintained at 50°C in a thermostatically controlled oven, according to previous applications . A gradient elution was performed using formate buffer adjusted to pH 3 (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B).…”

Identification of adrafinil and its main metabolite modafinil in human hair. Self-administration study and interpretation of an authentic case

“…In order to confirm the nature, the purity and the presence of possible other organic substances of the adrafinil powder bought on the Internet, a nuclear magnetic resonance (NMR) analysis was achieved, following method previously published [4,5]. Briefly, the NMR spectra were recorded on AVANCE 300 (Bruker Biospin, France) operating at 300 MHz equipped with a 5 mm quadruple nucleus probe (QNP) at 295 K. The 1 H spectra were recorded with 64 scans, 32-K time-domain data points with 4 800 Hz spectral width, an acquisition time of 3.42 s, a relaxation delay of 2 s and a flip angle of 30 .…”