Margaret Knight (Presenter)
Newcastle University
Bio: Research Technician at Newcastle University Medical Toxicology Centre. Working on the application of mass spectrometric techniques to clinical and toxicological investigations
Authorship: Margaret Knight, Michael Dunn, Clair Roper, Simon Hill, Simon Thomas, Peter Blain
Medical Toxicology Centre, Newcastle University, United Kingdom
| Short Abstract Untargeted high resolution accurate mass spectral data acquisition, combined with untargeted or targeted data analysis, is used for the systematic investigation of Novel Psychoactive Substances – NPS- in clinical samples. We will demonstrate the utility of this analytical method by detecting emerging psychoactive substances such as novel synthetic cannabinoids and phenethylamines, in anonymised urine and plasma specimens. This work will enable the collation of information about NPS implicated in episodes of acute toxicity in the UK, assisting patient management. |
Long Abstract
Introduction: 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 con¬tained 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 emerging psychoactive substance 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 admitted to UK National Health Service hospitals. Psychoactive substances 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 600 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 625 Da, with a 20 Da SWATH isolation window. Data were acquired with AnalystTF 1.6 and processed with MasterView 1.0 operated with PeakView 2.0, LibraryView 1.0, with direct access to the Chemspider chemical structure database and an in-house mass spectral data base.
Results: The aim of this study was to prove the utility of the non-targeted SWATH LC-QqTOF methodology for the screening of NPS in clinical samples – serum, plasma and urine. This data independent MS/MS acquisition has successfully been used to identify novel synthetic cannabinoids 5F-PB-22, 5F-AKB-48, MMB-CHIMINACA, AB-CHIMINACA, FUB-PB-22 and BB-22, phenylethylamine 25-I-N-BOMe and methiopropamine, a thiophene ring-based structural analogue of methamphetamine, in clinically relevant samples. Limits of detection using solid supported liquid/liquid extraction of 0.2 to 1.4ng of identified NPS per mL of urine or plasma were achieved, with recoveries > 82%. The quadrupole filtered mass ranges employed by the SWATH method provides excellent specificity resulting in cleaner MS/MS spectra, enabling low LODs despite co-eluting components from a complex matrix, and high confidence when interrogating the data against a mass spectra data base. Identifications were verified against standard MRM based methods.
Conclusions: In this proof of concept study, we have shown that the SWATH data independent acquisition technique is suitable for the screening and of small molecules - in this case emerging novel psychoactive substances, in clinically relevant samples The quanlitative abilities of SWATH were proven by successful validation against comparable with MRM based methods. In authentic samples, different classes of NPS were identified. Since MS/MS spectra are acquired for all unknown compounds in the sample, newly emerging compounds can be identified retrospectively via data re-interrogation. Mass spectra of identified NPS can be incorporated into spectral libraries for detecting NPS in forensic laboratories.
References & Acknowledgements:
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