2C-B-Fly-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C. elegans: Confirmation with Synthesized Analytical Standards
Abstract
:1. Introduction
2. Results
2.1. Untargeted Analysis and Synthesis of Reference Standards
2.2. LC–MS Analysis of the Synthesized Standards of Proposed Metabolites
2.3. Detection of Metabolites
3. Discussion
3.1. Comparison of 2C-B-Fly-NBOMe Metabolism in Different Species
3.2. Proposed Metabolic Pathways
4. Materials and Methods
4.1. Human Liver Microsomes
4.2. Cunninghamella elegans
4.3. Animals
4.4. Chemistry
4.5. Sample Preparation
4.5.1. Human Liver Microsomes
4.5.2. C. elegans Culture Medium
4.5.3. Rat Urine
4.6. LC-MS Analysis
4.6.1. Untargeted Screening
4.6.2. Fragmentation in MS3
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- United Nations Office on Drugs and Crime. World Drug Report 2021 (Sales No. E.21.XI.8); United Nations Office on Drugs and Crime: Vienna, Austria, 2021. [Google Scholar]
- Poulie, C.B.M.; Jensen, A.A.; Halberstadt, A.L.; Kristensen, J.L. Dark Classics in Chemical Neuroscience: NBOMes. ACS Chem. Neurosci. 2019, 10, 2160–2175. [Google Scholar] [CrossRef]
- Halberstadt, A.L. Pharmacology and toxicology of N-Benzylphenethylamine (“NBOMe”) hallucinogens. In Neuropharmacology of New Psychoactive Substances; Springer: Berlin/Heidelberg, Germany, 2017; pp. 283–311. [Google Scholar] [CrossRef]
- Elz, S.; Kläβ, T.; Heim, R.; Warnke, U.; Pertz, H.H. Development of highly potent partial agonists and chiral antagonists as tools for the study of 5-HT2A-receptor mediated function. In Proceedings of the 43rd Spring Meeting Deutsche Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie, Mainz, Germany, 12–14 March 2002. [Google Scholar]
- Heim, R.; Elz, S. Novel Extremely Potent Partial 5-HT2A-Receptor Agonists: Successful Application of a New Structure-Activity Concept. Arch. Pharm. Pharm. Med. Chem. 2000, 333, 39. [Google Scholar]
- Pertz, H.H.; Heim, R.; Elz, S. N-Benzylated phenylethanamines are highly potent partial agonists at 5-HT2A receptors (abstract). Arch. Pharm. Pharm. Med. Chem. 2000, 333, 30. [Google Scholar]
- Heim, R. Synthese und Pharmakologie Potenter 5-HT2A-Rezeptoragonisten mit N-2-Methoxybenzyl-Partialstruktur. Ph.D. Thesis, Freie Universität Berlin, Berlin, Germany, 2003. [Google Scholar]
- Eshleman, A.J.; Wolfrum, K.M.; Reed, J.F.; Kim, S.O.; Johnson, R.A.; Janowsky, A. Neurochemical pharmacology of psychoactive substituted N-benzylphenethylamines: High potency agonists at 5-HT2A receptors. Biochem. Pharmacol. 2018, 158, 27–34. [Google Scholar] [CrossRef] [PubMed]
- Ettrup, A.; Hansen, M.; Santini, M.A.; Paine, J.; Gillings, N.; Palner, M.; Lehel, S.; Herth, M.M.; Madsen, J.; Kristensen, J.; et al. Radiosynthesis and in vivo evaluation of a series of substituted 11C-phenethylamines as 5-HT2A agonist PET tracers. Eur. J. Nucl. Med. Mol. Imaging 2011, 38, 681–693. [Google Scholar] [CrossRef] [PubMed]
- Yoon, K.S.; Yun, J.; Kim, Y.-H.; Shin, J.; Kim, S.J.; Seo, J.-W.; Hyun, S.-A.; Suh, S.K.; Cha, H.J. 2-(2,5-Dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine (25D-NBOMe) and N-(2-methoxybenzyl)-2,5-dimethoxy-4-chlorophenethylamine (25C-NBOMe) induce adverse cardiac effects in vitro and in vivo. Toxicol. Lett. 2019, 304, 50–57. [Google Scholar] [CrossRef] [PubMed]
- Zwartsen, A.; Hondebrink, L.; Westerink, R.H.S. Changes in neuronal activity in rat primary cortical cultures induced by illicit drugs and new psychoactive substances (NPS) following prolonged exposure and washout to mimic human exposure scenarios. Neurotoxicology 2019, 74, 28–39. [Google Scholar] [CrossRef] [PubMed]
- Xu, P.; Qiu, Q.; Li, H.; Yan, S.; Yang, M.; Naman, C.B.; Wang, Y.; Zhou, W.; Shen, H.; Cui, W. 25C-NBOMe, a Novel Designer Psychedelic, Induces Neurotoxicity 50 Times More Potent Than Methamphetamine In Vitro. Neurotox. Res. 2019, 35, 993–998. [Google Scholar] [CrossRef]
- Schetz, D.; Waldman, W.; Kocic, I.; Sein, J. Case Report A Case of Laboratory Confirmed 25I-Nbome Intoxication Associated with Massive Rhabdomyolysis and Multi-Organ Failure. Adv. J. Toxicol. Curr. Res. 2017, 1, 43–48. [Google Scholar]
- Shanks, K.; Sozio, T.; Behonick, G. Fatal intoxications with 25B-NBOMe and 25I-NBOMe in Indiana during 2014. J. Anal. Toxicol. 2015, 39, 602–606. [Google Scholar] [CrossRef] [Green Version]
- Chia, X.W.S.; Ong, M.C.; Yeo, Y.Y.C.; Ho, Y.J.; Binte Ahmad Nasir, E.I.; Tan, L.-L.J.; Chua, P.Y.; Yap, T.W.A.; Lim, J.L.W. Simultaneous analysis of 2Cs, 25-NBOHs, 25-NBOMes and LSD in seized exhibits using liquid chromatography–tandem mass spectrometry: A targeted approach. Forensic Sci. Int. 2019, 301, 394–401. [Google Scholar] [CrossRef]
- Bersani, F.S.; Corazza, O.; Albano, G.; Valeriani, G.; Santacroce, R.; Bolzan Mariotti Posocco, F.; Cinosi, E.; Simonato, P.; Martinotti, G.; Bersani, G.; et al. 25C-NBOMe: Preliminary Data on Pharmacology, Psychoactive Effects, and Toxicity of a New Potent and Dangerous Hallucinogenic Drug. Biomed Res. Int. 2014, 2014, 734749. [Google Scholar] [CrossRef] [PubMed]
- Zawilska, J.B.; Kacela, M.; Adamowicz, P. NBOMes–Highly Potent and Toxic Alternatives of LSD. Front. Neurosci. 2020, 14, 78. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Leth-Petersen, S.; Gabel-Jensen, C.; Gillings, N.; Lehel, S.; Hansen, H.D.; Knudsen, G.M.; Kristensen, J.L. Metabolic Fate of Hallucinogenic NBOMes. Chem. Res. Toxicol. 2016, 29, 96–100. [Google Scholar] [CrossRef]
- Richter, L.H.J.; Menges, J.; Wagmann, L.; Brandt, S.D.; Stratford, A.; Westphal, F.; Flockerzi, V.; Meyer, M.R. In vitro toxicokinetics and analytical toxicology of three novel NBOMe derivatives: Phase I and II metabolism, plasma protein binding, and detectability in standard urine screening approaches studied by means of hyphenated mass spectrometry. Forensic. Toxicol. 2020, 38, 141–159. [Google Scholar] [CrossRef]
- Chambers, J.J.; Kurrasch-Orbaugh, D.M.; Parker, M.A.; Nichols, D.E. Enantiospecific Synthesis and Pharmacological Evaluation of a Series of Super-Potent, Conformationally Restricted 5-HT2A/2C Receptor Agonists. J. Med. Chem. 2001, 44, 1003–1010. [Google Scholar] [CrossRef] [PubMed]
- Monte, A.P.; Marona-Lewicka, D.; Parker, M.A.; Wainscott, D.B.; Nelson, D.L.; Nichols, D.E. Dihydrobenzofuran analogues of hallucinogens. 3. Models of 4-substituted (2,5-dimethoxyphenyl)alkylamine derivatives with rigidified methoxy groups. J. Med. Chem. 1996, 39, 2953–2961. [Google Scholar] [CrossRef]
- Caspar, A.T.; Helfer, A.G.; Michely, J.A.; Auwärter, V.; Brandt, S.D.; Meyer, M.R.; Maurer, H.H. Studies on the metabolism and toxicological detection of the new in psychoactive designer drug 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) human and rat urine using GC-MS, LC-MS n, and LC-HR-MS/MS. Anal. Bioanal. Chem. 2015, 407, 6697–6719. [Google Scholar] [CrossRef]
- Caspar, A.T.; Brandt, S.D.; Stoever, A.E.; Meyer, M.R.; Maurer, H.H. 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–MSn, and LC–HR–MS/MS approaches. J. Pharm. Biomed. Anal. 2017, 134, 158–169. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wagmann, L.; Hempel, N.; Richter, L.H.J.; Brandt, S.D.; Stratford, A.; Meyer, M.R. Phenethylamine-derived new psychoactive substances 2C-E-FLY, 2C-EF-FLY, and 2C-T-7-FLY. Investigations on their metabolic fate including isoenzyme activities and their toxicological detectability in urine screenings. Drug Test. Anal. 2019, 11, 1507–1521. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Noble, C.; Holm, N.B.; Mardal, M.; Linnet, K. Bromo-dragonfly, a psychoactive benzodifuran, is resistant to hepatic metabolism and potently inhibits monoamine oxidase A. Toxicol. Lett. 2018, 295, 397–407. [Google Scholar] [CrossRef]
- Nielsen, L.M.; Holm, B.; Leth-Petersen, S.; Kristensen, J.L.; Linnet, K. Characterization of the hepatic cytochrome P450 enzymes involved in the metabolism of 25I-NBOMe and 25I-NBOH. Drug Test. Anal. 2017, 9, 671–679. [Google Scholar] [CrossRef] [PubMed]
- Grafinger, K.E.; Stahl, K.; Wilke, A.; König, S.; Weinmann, W. In vitro phase I metabolism of three phenethylamines 25D-NBOMe, 25E-NBOMe and 25N-NBOMe using microsomal and microbial models. Drug Test. Anal. 2018, 10, 1607–1626. [Google Scholar] [CrossRef] [PubMed]
- Parker, G.C.; McKee, M.E.; Bishop, C.; Coscina, D.V. Whole-body metabolism varies across the estrous cycle in Sprague-Dawley rats. Physiol. Behav. 2001, 74, 399–403. [Google Scholar] [CrossRef]
- Šuláková, A.; Nykodemová, J.; Palivec, P.; Jurok, R.; Rimpelová, S.; Leonhardt, T.; Šíchová, K.; Páleníček, T.; Kuchař, M. 25CN-NBOMe metabolites in rat urine, human liver microsomes and C. Elegans—Structure determination and synthesis of the most abundant metabolites. Metabolites 2021, 11, 212. [Google Scholar] [CrossRef] [PubMed]
- Linhart, I.; Himl, M.; Židková, M.; Balíková, M.; Lhotková, E.; Páleníček, T. Metabolic profile of mephedrone: Identification of nor-mephedrone conjugates with dicarboxylic acids as a new type of xenobiotic phase II metabolites. Toxicol. Lett. 2016, 240, 114–121. [Google Scholar] [CrossRef]
- Zeng, X.; Yao, H.; Zheng, Y.; Chen, T.; Peng, W.; Wu, H.; Su, W. Metabolite Profiling of Naringin in Rat Urine and Feces Using Stable Isotope-Labeling-Based Liquid Chromatography-Mass Spectrometry. J. Agric. Food Chem. 2020, 68, 409–417. [Google Scholar] [CrossRef] [PubMed]
Synthesized Standard | Precursor Ion [m/z] | MS2 Fragment Ions [m/z] 1 | MS3 Fragment Ions [m/z] 1 | HPLC Method | RT [min] |
---|---|---|---|---|---|
2C-B-Fly-NBOMe (1) | 404 | 91 (42), 121 (100), 173 (1), 188 (2), 267 (8), 296 (8), 325 (5) | 121: 77 (9), 91 (100), 93 (94), 121 (2) 267: 145 (3), 159 (7), 173 (24), 188 (100) | method I | 6.0 |
C4’-hydroxy-2C-B-Fly-NBOMe (15) | 420 | 107 (15), 137 (100), 188 (5), 267 (92), 284 (34) | 137: 77 (14), 79 (21), 107 (100), 109 (16), 137 (33) 284: 173 (2), 188 (12), 267 (100), 284 (40) | method I method II | 5.1 8.8 |
C5’-hydroxy-2C-B-Fly-NBOMe (14) | 420 | 77 (10), 107 (70), 137 (100), 188 (4), 203 (2), 267 (15), 312 (12), 341 (5), 420 (11) | 312: 147 (16), 175 (100), 188 (11), 279 (8), 312 (47) 341: 175 (43), 203 (6), 310 (27), 312 (78), 341 (100) | method I method II | 5.2 8.8 |
C6’-hydroxy-2C-B-Fly-NBOMe (13) | 420 | 77 (5), 107 (22), 109 (14), 137 (100), 188 (8), 267 (98), 284 (78) | 267: 145 (3), 159 (5), 173 (22), 188 (100), 267 (74) 284: 173 (1), 188 (7), 267 (100), 284 (4) | method II | 9.3 |
2C-H-Fly-NBOMe-d3 (16) | 329 | 91 (86), 124 (100), 176 (5), 187 (13), 189 (49), 204 (8), 312 (32), 329 (27) | 189: 133 (9), 161 (26), 174 (17), 187 (100), 189 (23) 312: 124 (93), 150 (53), 175 (40), 201 (73), 270 (93), 312 (100) | method I | 4.2 |
2C-B-Fly-NBOH (12) | 390 | 77 (3), 107 (64), 173 (1), 188 (13), 254 (1), 267 (100), 284 (40), 390 (12) | 267: 145 (4), 159 (6), 173 (26), 188 (100), 267 (94) 284: 173 (1), 188 (10), 267 (100), 284 (11) | method I | 5.1 |
2C-B-Fly (11) | 284 | 145 (1), 159 (2), 173 (9), 188 (48), 267 (100), 284 (2) | 188: 131 (5), 145 (9), 159 (20), 173 (100), 188 (23) 267: 145 (3), 159 (5), 173 (19), 188 (100), 267 (29) | method II | 7.7 |
hydroxy-2C-B-Fly (8) | 300 | 174 (7), 186 (11), 203 (100), 265 (6), 282 (7) | 203: 174 (17), 186 (19), 203 (100) 282: 174 (3), 186 (3), 203 (100) | method II | 6.8 |
2C-H-Fly (10) | 206 | 91 (7), 105 (23), 133 (53), 161 (100), 189 (93) | 161: 91 (2), 105 (9), 133 (27), 161 (100) 189: 105 (15), 133 (44), 161 (100), 171 (16), 189 (27) | method II | 6.4 |
No. | Metabolite | PM [m/z] | RT [min] | RU | HLM | CE |
---|---|---|---|---|---|---|
M1 | 2C-B-Fly-NBOMe | 404.0861 | 8.4 | C | C | C |
M2 | C5′-hydroxy-2C-B-Fly-NBOMe | 420.0810 | 7.8 | C | C | |
M3 | hydroxy-2C-B-Fly-NBOMe isomer 2 | 420.0810 | 6.8 | I | I | I |
M4 | hydroxy-2C-B-Fly-NBOMe isomer 3 | 420.0810 | 7.7 | I | ||
M5 | hydroxy-2C-B-Fly-NBOMe isomer 4 | 420.0810 | 8.1 | I | I | |
M6 | 2C-B-Fly-NBOH | 390.0705 | 8.0 | C | C | |
M7 | 2C-H-Fly-NBOMe | 326.1756 | 7.6 | D | C | |
M8 | 2C-B-Fly | 284.0286 | 6.5 | D | C | C |
M9 | hydroxy-2C-B-Fly | 300.0235 | 4.7 | I | I | D |
M10 | dihydroxy-2C-B-Fly-NBOMe isomer 1 | 436.0760 | 6.1 | I | I | |
M11 | dihydroxy-2C-B-Fly-NBOMe isomer 2 | 436.0760 | 6.2 | I | ||
M12 | dihydroxy-2C-B-Fly-NBOMe isomer 3 | 436.0760 | 6.3 | I | ||
M13 | dihydroxy-2C-B-Fly-NBOMe isomer 4 | 436.0760 | 6.5 | D | I | |
M14 | dihydroxy-2C-B-Fly-NBOMe isomer 5 | 436.0760 | 7.1 | D | D | |
M15 | dihydroxy-2C-B-Fly-NBOMe isomer 6 | 436.0760 | 7.3 | I | ||
M16 | hydroxy-2C-B-Fly-NBOMe-N-oxide | 436.0760 | 9.0 | D | D | |
M17 | trihydroxy-2C-B-Fly-NBOMe isomer 1 | 452.0709 | 6.3 | I | D | |
M18 | trihydroxy-2C-B-Fly-NBOMe isomer 2 | 452.0709 | 6.9 | D | ||
M19 | hydroxy-2C-H-Fly-NBOMe | 342.1705 | 6.3 | I | D | |
M20 | dihydroxy-2C-H-Fly-NBOMe | 358.1654 | 9.8 | I | ||
M21 | trihydroxy-2C-H-Fly-NBOMe | 374.1604 | 9.1 | I | ||
M22 | hydroxy-2C-B-Fly-NBOH isomer 1 | 406.0654 | 6.4 | I | D | |
M23 | hydroxy-2C-B-Fly-NBOH isomer 2 | 406.0654 | 7.5 | I | ||
M24 | dihydroxy-2C-B-Fly-NBOH | 422.0603 | 5.8 | I | ||
M25 | trihydroxy-2C-B-Fly-NBOH | 438.0552 | 4.9 | D | ||
M26 | dehydro-2C-B-Fly-NBOMe isomer 1 | 402.0705 | 8.1 | I | I | |
M27 | dehydro-2C-B-Fly-NBOMe isomer 2 | 402.0705 | 8.6 | I | I | I |
M28 | dehydro-2C-B-Fly-NBOMe isomer 3 | 402.0705 | 9.0 | I | I | |
M29 | dehydro-hydroxy-2C-B-Fly-NBOMe | 418.0654 | 6.9 | D | I | D |
M30 | dehydro-2C-B-Fly-NBOMe-N-oxide | 418.0654 | 10.0 | I | ||
M31 | dehydro-dihydroxy-2C-H-Fly-NBOMe | 356.1498 | 10.0 | I | ||
M32 | dehydro-2C-B-Fly-NBOH | 388.0548 | 8.3 | I | D | |
M33 | dehydro-dihydroxy-2C-H-Fly-NBOH isomer 1 | 342.1341 | 9.4 | I | ||
M34 | dehydro-dihydroxy-2C-H-Fly-NBOH isomer 2 | 342.1341 | 9.8 | I | ||
M35 | dihydroxy-2C-B-Fly | 316.0184 | 4.7 | D | ||
M3-G | hydroxy-2C-B-Fly-NBOMe glucuronide isomer 1 | 596.1131 | 6.1 | I | ||
M4-G | hydroxy-2C-B-Fly-NBOMe glucuronide isomer 2 | 596.1131 | 6.4 | I | ||
M9-G | hydroxy-2C-B-Fly glucuronide | 476.0556 | 4.1 | I | ||
M11-G | dihydroxy-2C-B-Fly-NBOMe glucuronide | 612.1080 | 5.9 | D | ||
M19-G | hydroxy-2C-H-Fly-NBOMe glucuronide | 518.2026 | 5.6 | D | ||
M22-G | hydroxy-2C-B-Fly-NBOH glucuronide isomer 1 | 582.0975 | 5.8 | I | ||
M22-G | hydroxy-2C-B-Fly-NBOH glucuronide isomer 2 | 582.0975 | 5.9 | I | ||
M-Ac1 | dehydro-2C-B-Fly acetate | 324.0235 | 6.4 | I | ||
M-Ac2 | dehydro-dihydroxy-2C-B-Fly acetate | 356.0134 | 4.7 | I |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Nykodemová, J.; Šuláková, A.; Palivec, P.; Češková, H.; Rimpelová, S.; Šíchová, K.; Leonhardt, T.; Jurásek, B.; Hájková, K.; Páleníček, T.; et al. 2C-B-Fly-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C. elegans: Confirmation with Synthesized Analytical Standards. Metabolites 2021, 11, 775. https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11110775
Nykodemová J, Šuláková A, Palivec P, Češková H, Rimpelová S, Šíchová K, Leonhardt T, Jurásek B, Hájková K, Páleníček T, et al. 2C-B-Fly-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C. elegans: Confirmation with Synthesized Analytical Standards. Metabolites. 2021; 11(11):775. https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11110775
Chicago/Turabian StyleNykodemová, Jitka, Anna Šuláková, Petr Palivec, Hedvika Češková, Silvie Rimpelová, Klára Šíchová, Tereza Leonhardt, Bronislav Jurásek, Kateřina Hájková, Tomáš Páleníček, and et al. 2021. "2C-B-Fly-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C. elegans: Confirmation with Synthesized Analytical Standards" Metabolites 11, no. 11: 775. https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11110775