Michael Dunn (Presenter)
Newcastle University
Bio: I am a physical chemist applying mass spectrometry based multi-omic methodologies to clinical and toxicological research.
Authorship: Michael Dunn, Margaret Knight, Clair Roper, Simon Thomas, Peter Blain
Medical Toxicology Centre, Newcastle University, United Kingdom
| Short Abstract Untargeted data acquisition is used to identify the metabolites of novel psychoactive substance 25I-NBOMe in clinically relevant samples. Lack of phase I and II metabolite identification studies of emerging psychoactive substances limits effective monitoring in clinical and forensic testing. Data independent SWATH MS/MS acquisition has been used to identify phase I and II metabolites of 25I-N-BOMe. The present study demonstrates that 25I-NBOMe is extensively metabolised, mainly by O-demethylation, O,O-bis-demethylation, debenzylation, acetylation, glutathione adduction, hydroxylation with or without demethylation, as well as by glucuronidation and sulphation of the main phase I metabolites. |
Long Abstract
Introduction: 4-Iodo-2,5-dimethoxy-N-(2-methoxybenzyl)phenethylamine (25I-NBOMe) is a N-methoxybenzyl-substituted phenethylamine with potent serotoninergic effects, and has been involved in several fatal and nonfatal intoxication cases. There are little published data on the absorption, metabolism and elimination of 25I-NBOMe, or any of the other NBOMe derivatives, so there are no definitive metabolite biomarkers of 25I-NBOMe intoxication. The detection and identification of low levels of Novel Psychoactive Substances (NPS), or NPS derived metabolites in complex biological matrices requires analytical methodologies with high selectivity and sensitivity. Forensic and clinical toxicological screening procedures are increasingly employing non-targeted liquid chromatography−tandem mass spectrometry (LC-MS/MS) techniques. With information/data dependent acquisition (IDA/DDA), the complexity of a sample and the IDA/DDA instrument settings can result in irreproducible precursor ion selection, preventing significant compounds from being triggered for analysis. In these circumstances, data-independent acquisition (DIA) methods should be more suitable for systematic toxicological analysis. One such DIA method is sequential window acquisition of all theoretical fragment-ion spectra (SWATH), which utilises the very fast scanning speeds of quadrupole time-of-flight (QTOF) mass spectrometers. SWATH MS is a form of DIA method that repeatedly cycles through consecutive pre-set precursor ion isolation windows, detecting all fragment ion spectra from all the precursor ions contained in a specific window at a given time, at speeds fast enough to generate at least 10 data points over the chromatographic peak. We will demonstrate the utility of this analytical method by detecting 25I-NBOMe metabolites in anonymised clinically relevant urine and plasma specimens. This strategy is suitable for identification of NPS and metabolites present at low concentrations in complex matrices, with the potential to provide a sensitive and selective tool for toxicological screening.
Methods: Blood and urine samples were obtained with consent, from patients presented to hospitals in the northeast of England with clinical toxicity after analytically confirmed use of the recreational drug 25I-NBOMe in January 2013.25I-NBOMe and metabolites were extracted from 250ìL of plasma and 1mL of urine using solid supported liquid/liquid extraction. Samples were analysed by HPLC coupled to a TripleTOF® 5600+ mass spectrometer. Data independent LC/MS/MS analysis was performed using MS/MSALL with SWATH™ Acquisition. Protonated molecular ions were detected via a TOF MS scan covering 100 to 700 Da mass range. SWATH MS/MS data was acquired in high sensitivity mode, with a collision energy spread of 30±15V over a mass range of 30 to 725 Da, with a 20 Da SWATH isolation window. Data were acquired with AnalystTF 1.6. Data processing and evaluation of metabolites was performed with MetabolitePilot™ software, version 2 Alpha. Sets of theoretically possible bio-transformations for 25I-NBOMe were created based on their structures. Processing parameters were: retention time window 2.0-20.0 min; peak finding algorithms, predicted mass, generic, mass defect, based on characteristic product ions/neutral losses (at least 2); mass defect window range ±50 mDa; intensity thresholds, extracted ion chromatogram peak 1,500 cps, MS molecular ion peak 400 cps, MS/MS peak 100 cps; minimum peak width 5 s; maximum number of unexpected metabolites 10. The identified peaks were confirmed as metabolites based on mass accuracy as well as plausible retention time and fragmentation.
Results: Lack of phase I and II metabolite identification studies of emerging psychoactive substances limits effective monitoring in clinical and forensic testing. The aim of this study was to prove the utility of the non-targeted SWATH LC-QqTOF methodology for the identification in clinical samples of the metabolites of the novel N-methoxybenzyl-substituted phenethylamine 25I-N-BOMe. This data independent MS/MS acquisition has successfully been used to identify twenty phase I and II metabolites of 25I-N-BOMe. The present study demonstrates that 25I-NBOMe is extensively metabolised, mainly by O-demethylation, O,O-bis-demethylation, debenzylation, acetylation, glutathione adduction, hydroxylation with or without demethylation, as well as by glucuronidation and sulphation of the main phase I metabolites.
Conclusions: Identification of NPS metabolites is critically important to provide potential targets for analytical methods and candidates to test for pharmacological and toxicological effects. We have shown that the SWATH data independent acquisition technique and software assisted data mining, is suitable for the identification of the metabolic profile of both phase I and II metabolites of the N-methoxybenzyl-substituted phenethylamine 25I-NBOMe, in clinically relevant samples. The qualitative abilities of SWATH acquisition were proven by successful validation against comparable MRM based methods. The mass spectra of identified metabolites can be incorporated into spectral libraries for detecting NPS in forensic laboratories. Since SWATH MS/MS spectra are acquired for all unknown compounds in the sample, newly emerging psychoactive substances and there metabolites can be identified retrospectively via data re-interrogation.
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