Metabolic fate and detectability of the new psychoactive substances 2-(4-bromo-2,5-dimethoxyphenyl)- N- [(2-methoxyphenyl)methyl]ethanamine (25B-NBOMe) and 2-(4-chloro-2,5-dimethoxyphenyl)- N- [(2-methoxyphenyl)methyl]ethanamine (25C-NBOMe) in human and rat urine by GC–MS, LC–MS n , and LC–HR–MS/MS approaches

Abstract

25B-NBOMe and 25C-NBOMe are potent 5-HT receptor agonists that have been associated with inducing hallucinogenic effects in drug users and severe intoxications. This paper describes the identification of their metabolites in rat and human urine by liquid chromatography (LC)-high resolution (HR)-MS/MS, the comparison of metabolite formation in vitro and in vivo and in different species, the general involvement of human cytochrome-P450 (CYP) isoenzymes on their metabolism steps, and their detectability by standard urine screening approaches (SUSAs) using GC-MS, LC-MS, or LC-HR-MS/MS. Both NBOMe derivatives were mainly metabolized by O-demethylation, O,O-bis-demethylation, hydroxylation, and combinations as well as by glucuronidation and sulfation of the main phase I metabolites. For 25B-NBOMe, 66 metabolites could be identified and 69 for 25C-NBOMe. After application of low doses of both substances to rats, they were detectable mainly via their metabolites by both LC-based SUSAs. In case of acute intoxication, it was possible to detect 25B-NBOMe and its metabolites in an authentic human urine sample when using the GC-MS SUSA in addition to the LC-based SUSAs. Initial CYP activity screening revealed the involvement of CYP1A2 and CYP3A4 in hydroxylation and CYP2C9 and CYP2C19 in O-demethylation. The presented study demonstrated that 25B-NBOMe and 25C-NBOMe were extensively metabolized and detectable by both LC-based SUSAs.

In vitro phase I metabolism of three phenethylamines 25D‐NBOMe, 25E‐NBOMe and 25N‐NBOMe using microsomal and microbial models

“…The product ions resulted due to amine cleavage of the NBOMe moiety forming [C 8 H 9 O] + ( m/z 121.0653) and further demethoxylation via neutral loss of formaldehyde forming the tropylium ion [C 7 H 7 ] + ( m/z 91.0548) . These product ions were consistent with previously published data on 25B‐NBOMe, 25C‐NBOMe, and 25I‐NBOMe, because the alterations between the 25X‐NBOMe are on the 2C part at the 4‐position . The following observations can be made for all metabolites of 25X‐NBOMe substances: If product ions with m/z 121.0653 and m/z 91.0534 are present, no biotransformation occurs at the NBOMe part.…”

“…Chatwin et al define NPS as “chemical compounds that have been modified and developed to mimic the effects of drugs which are already prohibited.” Currently, the European Union (EU) Early Warning System, coordinated by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), is monitoring over 620 NPS, with one‐sixth of them belonging to the chemical class of phenethylamines . Prominent substances belonging to this class are 2C‐type phenethylamines, which are 2,5‐dimethoxyphenethylamines, substituted at the 4‐position, typically with halogens or alkyl groups, and also 2,5‐dimethoxy‐ N‐ benzylphenethylamines, so‐called NBOMes which have emerged in recent years . Substitutions on the 4‐position include bromine (25B‐NBOMe), chlorine (25C‐NBOMe), methyl (25D‐NBOMe), ethyl (25E‐NBOMe), iodine (25I‐NBOMe), and nitro (25N‐NBOMe) groups but the unsubstituted form (25H‐NBOMe) is also available, which has been detected as an impurity in blotter papers .…”

“…It was recently shown by Nielsen et al that the main enzymes involved in the 25I‐NBOMe metabolism are CYP3A4 and CYP2D6. Caspar et al reported that for 25B‐NBOMe and 25C‐NBOMe the enzymes CYP1A2, CYP3A4, CYP2C9, and CYP2C19 play a crucial role.…”

Metabolism of the tryptamine‐derived new psychoactive substances 5‐MeO‐2‐Me‐DALT, 5‐MeO‐2‐Me‐ALCHT, and 5‐MeO‐2‐Me‐DIPT and their detectability in urine studied by GC–MS, LC–MSn, and LC‐HR‐MS/MS

“…Due to the lack of authentic human urine samples, in vitro testing was performed to evaluate the transferability of rat metabolism results to humans. The combination of rat urine studies and the incubation of pHLM and/or pHLC with the corresponding cofactors is a common technique that produced reliable results and data for detection methods in human urine as shown in previous studies . However, it should be kept in mind, that the comparison of in vitro and in vivo models could be challenging due to sampling time of urine (24 h), time, amount, and route of application, the used incubation conditions, and/or genetic variations.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing

“…In 2017, WADA's Prohibited List sustained the existence of 2 groups of stimulants namely non‐specified and specified compounds. Of note, despite the ever‐growing plethora of “designer” psychoactive substances (including psychostimulants), which have urged forensics and toxicology laboratories to continuously refine testing capabilities, the frequency at which such designer compounds are determined in doping controls is still comparably low.…”