In vitro Phase I and Phase II metabolism of the new designer benzodiazepine cloniprazepam using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

Abstract

Designer benzodiazepines have recently emerged as a class of new psychoactive substances. These substances are used in recreational settings and as alternatives to prescription benzodiazepines as self-medication for patients suffering from anxiety or other mental disorders. Due to the limited information available on the metabolic fate of these new substances, it is challenging to reliably detect their usage in bioanalytical (e.g. clinical and forensic) settings. The objective of this study was to investigate the in vitro Phase I and Phase II metabolism of the new designer benzodiazepine cloniprazepam and identify potential biomarkers for its detection in human biological fluids. Cloniprazepam was incubated with human liver microsomes and cytosolic fractions to generate both Phase I and II metabolites. The extracts were analysed using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Identification of the metabolites was performed using two complementary workflows, including a suspect screening based on in silico predictions and a non-targeted screening. A total of nine metabolites were identified, eight Phase I metabolites and one Phase II metabolite, of which five were specific for cloniprazepam. Clonazepam was the major metabolite of cloniprazepam. Hydroxy-cloniprazepam, dihydroxy-cloniprazepam, 3-keto-cloniprazepam, 7-amino-cloniprazepam, hydroxy-clonazepam, 7-amino-clonazepam and 3-hydroxy-7-amino-clonazepam were formed through oxidation, hydroxylation, and/or reduction of the nitro-group. Glucuronidated hydroxy-cloniprazepam was the only Phase II metabolite detected. Five metabolites were specific for cloniprazepam. This study provided a set of human in vitro biotransformation products which can assist specific detection of cloniprazepam consumption in future studies.

Simulated Gastrointestinal Biotransformation of Chlorogenic Acid, Flavonoids, Flavonolignans and Triterpenoid Saponins in Cecropia obtusifolia Leaf Extract

“…Identification was carried out based on accurate mass obtained in the full MS mode, fragmentation pattern (MS/MS) of the product ions, and comparison with analytical standards, if available, and/ or fully identified compounds isolated from C. obtusifolia. Acceptance requirements for identification of biotransformation products were set as in similar studies: mass tolerance of ± 5 ppm between theoretical and measured monoisotopic mass; product ions may not exceed the maximal mass variation of ± 10 ppm; compounds detected in the samples that were also present in the MB and NC were ignored (allowing the interpretation of only those specific metabolites produced during the GIM and by action of microbiota), and the detected metabolites were present in at least two out of the three replicate samples [19]. Biotransformation products are shown in ▶ Table 2.…”

“…▶ Fig. 7 In vitro biotransformation of saponin triterpenoids (19)(20)(21)(22). 19 = Niga-ichigoside F2; 20 = buergericic acid 28-O-glucoside; 21 = kaji-ichigoside F1; 22 = tormentoside; T1 = 2α,3α,19α,23-tetrahydroxy-12-ursen-28-oic acid; T2 = buergericic acid; T3 = euscaphic acid and tormentic acid.…”

Suspect and non‐target screening workflows to investigate the in vitro and in vivo metabolism of the synthetic cannabinoid 5Cl‐THJ‐018

“…[23][24][25][26][27] An overview of the experimental setup can be found in the Supporting Information (Figure SI-1). [23][24][25][26][27] An overview of the experimental setup can be found in the Supporting Information (Figure SI-1).…”

“…This study employed the in vitro assay optimized and used in previous studies. [23][24][25][26][27] An overview of the experimental setup can be found in the Supporting Information (Figure SI-1). All tested sample sets contained three replicates.…”

Toxicometabolomics of the new psychoactive substances α-PBP and α-PEP studied in HepaRG cell incubates by means of untargeted metabolomics revealed unexpected amino acid adducts

“…Metabolomics techniques are applied to investigate the response to pathophysiological stimuli or genetic modifications (Lindon et al 2000) such as cancer (Dang et al 2009), diseases (Wild et al 2019), or intoxications (D'Elia et al 2019). Metabolomics has lately also been used to study DOA or NPS, either with a focus on the drug metabolism (Manier et al 2019a;Mortele et al 2018;Vervliet et al 2019a) or the wider changes of the metabolome in abusers (Boxler et al 2018). The huge potential of these techniques for the identification of biomarkers of DOA or NPS abuse can be found in the enhanced number of metabolites that might be detected and the identification of compounds that at first do not seem to be related to the drug (Manier et al 2019a;Vervliet et al 2019b).…”