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The development of analytical protocols for the determination of analytes at trace levels in complex matrices (e.g. biological fluids or contaminated water) is a crucial point for the environmental assessment and monitoring as well as for scientific research in the field of disease biomarkers. An essential part of analytical method development is represented by sample preparation due to its significant impact on most of the subsequent steps and the data quality. In recent years, the application of pro-ecological, automated, solvent-free sample preparation approaches or techniques employing a minimal amount of solvents or safe and non-toxic extractants has become one of the most popular research topics in analytical chemistry. In this context, microextraction techniques represent a suitable choice for the extraction of analytes from complex matrices because these techniques use less organic solvent and allow to perform in a single step extraction and concentration of analytes. Moreover, the use of microextraction techniques for sample preparation reduces the number of errors that commonly result from multi-stage procedures, and limits the negative impact on the environment and the health of analytical chemists performing laboratory work. The goal of this Ph.D project was the development and optimization of analytical methods based on the use of microextraction techniques for the assay of disease biomarkers and environmental contaminants in biological fluids and environmental matrices. The microextraction techniques employed in this thesis were solid phase microextraction (SPME) and microextraction by packed sorbent (MEPS). SPME was used to evaluate the applicability of a new fiber (PDMS/DVB/PDMS) as analytical sampling tool for investigation in raw human urine. The PDMS/DVB/PDMS fiber was exploited to develop a DI-SPME-GC-MS method for the assay of polycyclic aromatic hydrocarbons (PAHs) with 2-6 aromatic rings in untreated human urine samples. Moreover, in the light of the increasing demand of faster and easier protocols allowing the assessment of disease biomarkers, SPME was applied to develop a reliable and rapid GC-MS approach for the determination of polyamines in human urine. Indeed, polyamines are widely recognized as among the most important cancer biomarkers for early diagnosis and treatment. SPME was also applied for the extraction of nine phthalates monoesters in urine samples. These compounds are important metabolites of phthalates and their assay can reliably rank exposures to phthalates over a period. MEPS was used to extract organophosphate ester flame retardant in aqueous matrices and, again, monoesters phthalates in urine. In both methods, in order to improve method sensitivity, programmed temperature vaporization (PTV) was chosen as gas chromatographic injection technique. For polyamines and phthalates monoesters, a prior derivatization step with suitable reagents was carried out before gas chromatographic analysis so as to improve chromatographic elution and resolution by decreasing volatility and polarity of analytes. Derivatization reaction was performed directly in aqueous samples using alkyl chloroformates. The combined use of alkyl chloroformate as derivatizing reagent and SPME for analyte extraction was chosen to develop a simple protocol involving minimal sample handling and no consumption of toxic organic solvents. The variables affecting the different steps of the proposed protocols were optimized by the multivariate approach of experimental design which has allowed for the simultaneous investigation of the different factors in the entire experimental domain and the possible synergic effects between variables. In this thesis, experimental design was used to optimize the parameters influencing SPME extraction, MEPS extraction, PTV process and derivatization reaction. Gas chromatographic analyses were carried out using a GC-QqQ-MS instrument in selected reaction monitoring (SRM) acquisition which has allowed to obtain reconstructed chromatograms with well-defined chromatographic peaks and to achieve high specificity through the selection of appropriate precursor-product ion couples, improving the capability in analyte identification. Finally, during the period as visiting Ph.D student at University Duisburg-Essen, Faculty of Chemistry, Instrumental Analytical Chemistry, the object of research activity, coordinated by Professor Torsten C. Schmidt, concerned the extraction of fatty acid methyl esters (FAMEs) in wastewater by solid phase microextraction arrow (SPME arrow). Dottorato di Ricerca Medicina Traslazionale. Ciclo XXXI Università della Calabria

Il presente lavoro di tesi relativo all‘attività di ricerca svolta durante il triennio di dottorato ha riguardato la messa a punto di metodi analitici per la determinazione di analiti in due distinte aree di interesse. La prima di ambito clinico ed ha riguardato la quantificazione in urina di metaboliti riconosciuti come marker in diagnostica clinica. In particolare ci si occupati della sarcosina come biomarker del tumore alla prostata, e di tre acidi: acido omovanillico (HVA), acido vanilmandelico (VMA) ed acido 5-idrossindoloacetico (5-HIAA) come marker urinari del neuroblastoma. Il secondo ambito di lavoro ha riguardato la quantificazione di inquinanti in matrici acquose, vale a dire carbammati ed acidi perfluoroalchilici. In particolare gli analiti, previa derivatizzazione con alchilcloroformiati (eccetto i carbammati), sono stati estratti dalle matrici acquose (acqua e urina) tramite la tecnica della microestrazione in fase solida (SPME) e successivamente analizzati mediante un gascromatografo con analizzatore di massa a triplo quadrupolo (GC-QqQ-MS). Le variabili significative della microestrazione in fase solida in ciascun metodo sono state ottimizzate tramite l‘approccio multivariato dell‘ ― Experimental Design‖. L‘utilizzo della tecnica SPME ha consentito di poter estrarre gli analiti direttamente dal campione da analizzare minimizzando i tempi di preparazione dello stesso e riducendo l‘uso di solventi organici, ottenendo metodi poco costosi e basso impatto ambientale. L‘utilizzo dello spettrometro di massa triplo quadrupolo, ha consentito di raggiungere livelli di sensibilità molto elevati e, nel contempo, di identificare gli analiti con maggiore sicurezza. In tutti i metodi sviluppati, sono stati ottenuti ottimi risultati in termini di linearità accuratezza e precisione. Anche i valori dei limiti di rilevabilità (LOD) e dei limiti di quantificazione (LOQ) ottenuti in ciascun metodo possono essere considerati soddisfacenti. Dottorato di Ricerca in Organic Materials of Pharmacological Interest, Ciclo XXV, a.a. 2011-2012 Università degli Studi della Calabria

We describe a new method to detect trace levels of ptaquiloside (Pta), a major carcinogen of bracken fern in biological samples such as milk from farm animals. The method involves the absorption of analyte on carbograph followed by elution with solvents mixtures. The unstable analyte is then converted into Br-Pt (II), which is specific for Pta, as it is not a natural decay product of the glycoside in aqueous media. An internal standard, the Br-pterosine-d(2), prepared in our laboratories has been used. Detection and quantification are possible with gas chromatography/mass spectrometry (GC/MS) in single ion monitoring mode (SIM). The detectable amount is in the range of ppb. The method allowed us to detect Pta not only in the milk from bracken fern-poisoned cattle but also, for the first time, in the milk from healthy farm animals such as sheep, goat, horse, and donkey mares. (c) 2010 Elsevier Ltd. All rights reserved.