In recent years, the spread of new psychoactive substances (NPS) has been one of the main challenges for forensic and clinical toxicology. These compounds, often synthesized with the aim of evading existing legislative controls, include a wide range of molecules, including fentanyl analogues and synthetic cathinones, characterized by high pharmacological potency, structural variability and often unpredictable toxicological profiles. Timely and accurate identification of NPS is essential for the clinical management of acute intoxications, forensic investigations, and epidemiological monitoring of substance abuse. In this context, it is necessary to develop highly sensitive and selective analytical methods, capable of adapting to the rapid evolution of the illicit drug market. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is now the reference tool for the identification and quantification of these substances in biological matrices. However, the choice of the biological matrix to be analyzed also plays a crucial role, especially where it is only necessary to search for recent consumption. Urine, in this analytical/identifying context, is characterized as the most suitable biological matrix available to the toxicologist. However, the lack of information on the stability of the compounds of interest, when stored in an aqueous solution, combined with the logistical difficulty of storing hundreds of urine samples at suitable temperatures, has prompted the laboratory to look for new substrates that can overcome these critical issues: among the emerging alternatives to conventional matrices, Dried Matrix Spots (DMS), and in particular the Dried Urine Spots (DUS), offer significant logistical and analytical advantages. In fact, DUS allow for non-invasive collection, greater analyte stability, simplified transport and storage management, and a reduction in biological risk, making them particularly suitable for large-scale clinical, forensic and screening settings. The present thesis project stems from the need to validate an innovative bioanalytical approach that combines the use of DUS with LC-MS/MS technology, for the selective and quantitative determination of a panel of NPS of particular toxicological relevance. In particular, the study focused on three fentanyl analogues (fentanyl, carfentanyl, furanyl fentanyl) and two synthetic cathinones (MDPV and 3,4-MD-α-PHP), selected for their diffusion in the illicit market, pharmacological potency and clinical hazard. The main objectives of the study were: To develop and optimize an LC-MS/MS analytical method for the detection of five NPS in urine samples deposited on a DUS support, with attention to the selection of the most suitable chromatographic and ionization parameters for each compound. Validate the method according to the international criteria of accuracy, precision, selectivity, linearity, limit of detection (LOD) and limit of quantification (LOQ). Monitor the stability of the investigated compounds on DUS substrates over a period of 3 weeks.
Negli ultimi anni, la diffusione di nuove sostanze psicoattive (NPS, New Psychoactive Substances) ha rappresentato una delle principali sfide per la tossicologia forense e clinica. Questi composti, spesso sintetizzati con l’obiettivo di eludere i controlli legislativi esistenti, comprendono una vasta gamma di molecole, tra cui analoghi del fentanyl e catinoni sintetici, caratterizzate da elevata potenza farmacologica, variabilità strutturale e profili tossicologici spesso imprevedibili. L’identificazione tempestiva e accurata delle NPS è essenziale per la gestione clinica delle intossicazioni acute, per le indagini medico-legali e per il monitoraggio epidemiologico dell’abuso di sostanze. In questo contesto, si rende necessario lo sviluppo di metodiche analitiche altamente sensibili e selettive, capaci di adattarsi alla rapida evoluzione del mercato illecito delle droghe. La cromatografia liquida accoppiata alla spettrometria di massa tandem (LC-MS/MS) rappresenta oggi lo strumento di riferimento per l’identificazione e la quantificazione di tali sostanze in matrici biologiche. Tuttavia, anche la scelta della matrice biologica da analizzare riveste un ruolo cruciale, soprattutto ove si renda esclusivamente necessario ricercare un consumo recente. L’urina, in questo contesto analitico/identificativo, si caratterizza come la più adatta matrice biologica a disposizione per il tossicologo. Tuttavia, la mancanza di informazioni in merito alla stabilità dei composti di interesse, quando conservati in una soluzione acquosa, unita alla difficoltà logistica di conservare centinaia di campioni di urina ad idonee temperature, ha spinto il laboratorio a cercare nuovi substrati che possano superare queste criticità: tra le alternative emergenti alle matrici convenzionali, i Dried Matrix Spots (DMS), e in particolare i Dried Urine Spots (DUS), offrono significativi vantaggi logistici e analitici. I DUS permettono infatti una raccolta non invasiva, una maggiore stabilità degli analiti, una gestione semplificata del trasporto e dello stoccaggio e una riduzione del rischio biologico, rendendoli particolarmente adatti per contesti clinici, forensi e di screening su larga scala. Il presente progetto di tesi nasce dalla necessità di validare un approccio bioanalitico innovativo che combini l’uso dei DUS con la tecnologia LC-MS/MS, per la determinazione selettiva e quantitativa di un panel di NPS di particolare rilevanza tossicologica. In particolare, lo studio si è focalizzato su tre analoghi del fentanyl (fentanyl, carfentanyl, furanyl fentanyl) e due catinoni sintetici (MDPV e 3,4-MD-α-PHP), selezionati per la loro diffusione nel mercato illecito, la potenza farmacologica e la pericolosità clinica. Gli obiettivi principali dello studio sono stati: 1. Sviluppare e ottimizzare una metodica analitica LC-MS/MS per la rilevazione di cinque NPS in campioni di urina depositati su supporto DUS, con attenzione alla selezione dei parametri cromatografici e di ionizzazione più idonei per ciascun composto. 2. Validare la metodica secondo i criteri internazionali di accuratezza, precisione, selettività, linearità, limite di rivelabilità (LOD) e limite di quantificazione (LOQ), 3. Monitorare la stabilità dei composti investigati sui substrati DUS in un periodo di 3 settimane.
Sviluppo e validazione di una metodica LC-MS/MS per la determinazione di Nuove Sostanze Psicoattive (NPS) su Dried Urine Spot (DUS)
POLLASTRINI, GABRIELE
2024/2025
Abstract
In recent years, the spread of new psychoactive substances (NPS) has been one of the main challenges for forensic and clinical toxicology. These compounds, often synthesized with the aim of evading existing legislative controls, include a wide range of molecules, including fentanyl analogues and synthetic cathinones, characterized by high pharmacological potency, structural variability and often unpredictable toxicological profiles. Timely and accurate identification of NPS is essential for the clinical management of acute intoxications, forensic investigations, and epidemiological monitoring of substance abuse. In this context, it is necessary to develop highly sensitive and selective analytical methods, capable of adapting to the rapid evolution of the illicit drug market. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is now the reference tool for the identification and quantification of these substances in biological matrices. However, the choice of the biological matrix to be analyzed also plays a crucial role, especially where it is only necessary to search for recent consumption. Urine, in this analytical/identifying context, is characterized as the most suitable biological matrix available to the toxicologist. However, the lack of information on the stability of the compounds of interest, when stored in an aqueous solution, combined with the logistical difficulty of storing hundreds of urine samples at suitable temperatures, has prompted the laboratory to look for new substrates that can overcome these critical issues: among the emerging alternatives to conventional matrices, Dried Matrix Spots (DMS), and in particular the Dried Urine Spots (DUS), offer significant logistical and analytical advantages. In fact, DUS allow for non-invasive collection, greater analyte stability, simplified transport and storage management, and a reduction in biological risk, making them particularly suitable for large-scale clinical, forensic and screening settings. The present thesis project stems from the need to validate an innovative bioanalytical approach that combines the use of DUS with LC-MS/MS technology, for the selective and quantitative determination of a panel of NPS of particular toxicological relevance. In particular, the study focused on three fentanyl analogues (fentanyl, carfentanyl, furanyl fentanyl) and two synthetic cathinones (MDPV and 3,4-MD-α-PHP), selected for their diffusion in the illicit market, pharmacological potency and clinical hazard. The main objectives of the study were: To develop and optimize an LC-MS/MS analytical method for the detection of five NPS in urine samples deposited on a DUS support, with attention to the selection of the most suitable chromatographic and ionization parameters for each compound. Validate the method according to the international criteria of accuracy, precision, selectivity, linearity, limit of detection (LOD) and limit of quantification (LOQ). Monitor the stability of the investigated compounds on DUS substrates over a period of 3 weeks.File | Dimensione | Formato | |
---|---|---|---|
Tesi Gabriele Pollastrini 454330.pdf
accesso aperto
Dimensione
1.46 MB
Formato
Adobe PDF
|
1.46 MB | Adobe PDF | Visualizza/Apri |
È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/29790