Drug of abuse analysis: new methods in herbal and biological matrices for medicinal chemistry and forensic investigation

In this Ph.D. project, original and innovative approaches for the quali-quantitative analysis of abuse substances, as well as therapeutic agents with abuse potential and related compounds were designed, developed and validated for application to different fields such as forensics, clinical and pharmaceutical. All the parameters involved in the developed analytical workflows were properly and accurately optimised, from sample collection to sample pretreatment up to the instrumental analysis. Advanced dried blood microsampling technologies have been developed, able of bringing several advantages to the method as a whole, such as significant reduction of solvent use, feasible storage and transportation conditions and enhancement of analyte stability. At the same time, the use of capillary blood allows to increase subject compliance and overall method applicability by exploiting such innovative technologies. Both biological and non-biological samples involved in this project were subjected to optimised pretreatment techniques developed ad-hoc for each target analyte, making also use of advanced microextraction techniques. Finally, original and advanced instrumental analytical methods have been developed based on high and ultra-high performance liquid chromatography (HPLC,UHPLC) coupled to different detection means (mainly mass spectrometry, but also electrochemical, and spectrophotometric detection for screening purpose), and on attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) for solid-state analysis. Each method has been designed to obtain highly selective, sensitive yet sustainable systems and has been validated according to international guidelines. All the methods developed herein proved to be suitable for the analysis of the compounds under investigation and may be useful tools in medicinal chemistry, pharmaceutical analysis, within clinical studies and forensic investigations.

Dati preliminari dello studio multicentrico caso-controllo "Grave danno epatico acuto indotto da farmaci"

Il danno epatico indotto dall'assunzione di farmaci viene comunemente indicato con il termine inglese DILI (Drug-Induced Liver Injury). Il paracetamolo rappresenta la causa più comune di DILI, seguito da antibiotici, FANS e farmaci antitubercolari. In particolare, i FANS sono una delle classi di farmaci maggiormente impiegate in terapia. Numerosi case report descrivono pazienti che hanno sviluppato danno epatico fatale durante il trattamento con FANS; molti di questi farmaci sono stati ritirati dal commercio in seguito a gravi reazioni avverse a carico del fegato. L'ultimo segnale di epatotossicità indotto da FANS è associato alla nimesulide; in alcuni paesi europei come la Finlandia, la Spagna e l'Irlanda, la nimesulide è stata sospesa dalla commercializzazione perché associata ad un'alta frequenza di epatotossicità. Sulla base dei dati disponibili fino a questo momento, l'Agenzia Europea dei Medicinali (EMA) ha recentemente concluso che i benefici del farmaco superano i rischi; un possibile aumento del rischio di epatotossicità associato a nimesulide rimane tuttavia una discussione aperta di cui ancora molto si dibatte. Tra le altre classi di farmaci che possono causare danno epatico acuto la cui incidenza tuttavia non è sempre ben definita sono gli antibiotici, quali amoxicillina e macrolidi, le statine e gli antidepressivi.Obiettivo dello studio è stato quello di determinare il rischio relativo di danno epatico indotto da farmaci con una prevalenza d'uso nella popolazione italiana maggiore o uguale al 6%. E’ stato disegnato uno studio caso controllo sviluppato intervistando pazienti ricoverati in reparti di diversi ospedali d’Italia. Il nostro studio ha messo in evidenza che il danno epatico da farmaci riguarda numerose classi farmacologiche e che la segnalazione di tali reazioni risulta essere statisticamente significativa per numerosi principi attivi. I dati preliminari hanno mostrato un valore di odds ratio significativo statisticamente per la nimesulide, i FANS, alcuni antibiotici come i macrolidi e il paracetamolo.

Instrumental and sensory analyses for the characterization of food, phytotherapeutic or pharmaceutical hemp products

To address the request to develop rapid and easy methods for determining the cannabinoids, an HPLC-UV method (8 min) to separate and quantify the 10 main cannabinoids in hemp inflorescences was developed, and in-house validated. Moreover, the antioxidant activity of cannabidiol (CBD) in two oily matrices was investigated and compared to that of α-tocopherol, in relation to the growing market of oily solutions containing cannabidiol. Then, since no univocal legislation on the evaluation of quality and authenticity of hemp seed oil (HSO) exists, the composition and quality of cold-pressed HSOs were also explored, highlighting a great variability in terms of oxidative state minor compounds content. From the sensory point of view, a panel was trained, a specific sensory wheel and a profile sheet were developed. Due to the Covid-19 pandemic, the sensory evaluation was also performed at home. The panel showed a good performance both in the laboratory and remotely. Moreover, a focus group was used to investigate consumers’ attitudes, pointing out that a high-quality HSO has to be cold-pressed and green for them. Then, the evaluation of stability during the storage of HSOs was investigated. The results showed that photo-oxidation did not seem to significantly affect the quality of the oil during the first 3 months of storage. Finally, a study about the evolution of the volatile profile of 9 HSOs, under accelerated oxidation conditions, allowed identifying volatile markers of HSOs oxidation and freshness. This Ph.D. was developed in the context of the scholarship “Harmonized procedures of analysis of medical, herbal, food and industrial cannabis: development and validation of cannabinoids’ quality control methods, of extraction and preparation of derivatives from the plant raw material, according to the product destination” funded by Enecta S.r.l.