Development of new analytical methods for the identification of novel psychoactive substances (nps), related metabolites and biomarkers of intake in biological matrices

The market for new psychoactive substances (NPS) has increased significantly in recent years and there is now a steady stream of compounds appearing every year. NPS present challenges for detection due to the numerous generations and structural variations circulating in communities and the illicit market at any given time. Currently, no validated screening or quantification methods exist that cover a wide range of NPS. The aim of this work was to investigate and develop new approaches and methods to address some of the shortcomings of current analytical strategies in forensic chemistry; the use of liquid chromatography coupled with mass spectroscopy enabling the detection of a large number of NPS allowing this emerging problem to be tackled by looking at it from different point of view. The developed procedures were designed for use with biological samples, in particular urine and oral fluid, but they can be easily adapted to other specimens such as plasma, serum and whole blood. Each of these matrices is crucial for forensic purposes, as they provide different types of information regarding the timing of substance intake and possess unique characteristics; in addition, in forensic investigations, other matrices are interesting, such as seizures from law enforcement agencies. The development of methods for NPS monitoring and/or quantification, based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), both low and high-resolution MS, was the purpose of this PhD project. LC-MS is widely recognized as the benchmark for qualitative and quantitative analysis offering high specificity, sensitivity and speed. Various chromatographic conditions were assessed to achieve optimal separation of the target compounds and revelation of the unknown analytes. A first part of the project was devoted to the development of a suspect screening method for NPS detection. To enhance selectivity, a method based on a scheduled multi-reaction monitoring (sMRM) survey triggering enhanced product ion (EPI) acquisition has been created; this method could be used to investigate NPS in real samples without the need for certified reference standards. The method was successfully applied to oral fluid samples, yielding good results. In a second part of the project, the attention was paid to the study of indirect approaches to detect the consumption of psychoactive substances belonging to specific classes, such as synthetic opioids; the conspicuous difficulties in investigating and discovering unexpected drugs were circumvented by the creation of an indirect screening strategy, based on an untarget metabolomics LC-HRMS workflow, focused on the study of endogenous urinary metabolites alteration after the consumption of opioids. Last but not least, a pilot study was conducted involving principal component analysis-based prediction model for identifying NPS using LC-MS. The developed approach allows retrospective analysis of mass spectra, enabling the identification of class-specific fragments and neutral losses; in this context the use of multivariate statistical techniques expand the panorama of possibilities to potentially identifying new drugs of abuse.