Characterization and in vitro phase I microsomal metabolism of designer benzodiazepines: An update comprising flunitrazolam, norflurazepam, and 4'‐chlorodiazepam (Ro5–4864)

Abstract

The number of newly appearing benzodiazepine derivatives on the new psychoactive substances (NPS) drug market has increased over the last couple of years totaling 23 'designer benzodiazepines' monitored at the end of 2017 by the European Monitoring Centre for Drugs and Drug Addiction. In the present study, three benzodiazepines [flunitrazolam, norflurazepam, and 4′-chlorodiazepam (Ro5-4864)] offered as 'research chemicals' on the Internet were characterized and their main in vitro phase I metabolites tentatively identified after incubation with pooled human liver microsomes. For all compounds, the structural formula declared by the vendor was confirmed by gas chromatography−mass spectrometry (GC-MS), liquid chromatography−tandem mass spectrometry (LC MS/MS), liquid chromatography−quadrupole time of flight−mass spectrometry (LC−QTOF−MS) analysis and nuclear magnetic resonance (NMR) spectroscopy. The metabolic steps of flunitrazolam were monohydroxylation, dihydroxylation, and reduction of the nitro function. The detected in vitro phase I metabolites of norflurazepam were hydroxynorflurazepam and dihydroxynorflurazepam. 4'-Chlorodiazepam biotransformation consisted of N-dealkylation and hydroxylation. It has to be noted that 4′-chlorodiazepam and its metabolites show almost identical LC-MS/MS fragmentation patterns to diclazepam and its metabolites (delorazepam, lormetazepam, and lorazepam), making a sufficient chromatographic separation inevitable. Sale of norflurazepam, the metabolite of the prescribed benzodiazepines flurazepam and fludiazepam, presents the risk of incorrect interpretation of analytical findings.

Designer Benzodiazepines: A Review of Toxicology and Public Health Risks

“…Furthermore, the newest DBDZ may have high cross-reactivity with common BZD immunoassays, which often do not distinguish between designer and prescribed BZD. Metabolism to licensed BZD, the sale of metabolites of prescribed BZD and the unavailability of confirmatory testing in health care centers pose the risk of an incorrect interpretation of analytical findings [5,127,[155][156][157]. The roles DBZD play in deaths remains poorly understood, and how different pathologists and toxicologists attribute and interpret cause of death is largely unknown.…”

The Evolution Toward Designer Benzodiazepines in Drug-Facilitated Sexual Assault Cases

“…Metabolites can then be identified using high-resolution mass spectrometry (MS) based techniques and nuclear magnetic resonance. HLMs have been used to study the metabolism of clonazolam ( 63 ), diclazepam ( 63 ), deschloroetizolam ( 63 ), etizolam ( 63 ), flualprazolam ( 64 ), flubromazepam ( 63 ), flubromazolam ( 63 , 65 ), flunitrazolam ( 66 ), meclonazepam ( 63 , 67 ), metizolam ( 68 ), nifoxipam ( 63 ) and norflurazepam ( 66 ). Another strategy relies on the analysis of urine samples obtained from a large cohort of users.…”

“…A striking example is flunitrazolam ( Table VI ), where the in vitro experiments conducted by Moosman et. al ( 66 ) identified hydroxylation reactions that could not be confirmed in vivo in the study of Ameline and colleagues ( 74 ). Conversely, the metabolite 7-acetamidoflunitrazolam could be detected in vivo but not in vitro .…”

Peripheral benzodiazepine receptor TSPO needs to be reconsidered before using as a drug target for a pigmentary disorder

“…15 At present, the exogenous ligand studied mainly includes PK 11195, Ro5-4864, Etifoxine, SSR-180575, ZBD-2, YL-IPA08, AC-5216, DAA-1097, etc. [16][17][18][19] Previous studies have shown that these ligands have regulatory activities in different biological functions. Therefore, TSPO is also considered a potential drug target.…”

“…Cholesterol and porphyrins are endogenous ligands of TSPO and have a high affinity for TSPO 15 . At present, the exogenous ligand studied mainly includes PK 11195, Ro5‐4864, Etifoxine, SSR‐180575, ZBD‐2, YL‐IPA08, AC‐5216, DAA‐1097, etc 16–19 . Previous studies have shown that these ligands have regulatory activities in different biological functions.…”