Sviluppo di tecniche di analisi ifenate per la quantificazione di biomarcatori di esposizione a tossici ambientali: gli acidi mercapturici

Human biomonitoring (HBM) is an ideal tool for evaluating toxicant exposure in health risk assessment. Chemical substances or their metabolites related to environmental pollutants can be detected as biomarkers of exposure using a wide variety of biological fluids. Individual exposure to aromatic hydrocarbon compounds (benzene, toluene, and o-xylene –“BTX”) were analysed with a liquid chromatography coupled to electrospray ionisation-mass spectrometry (μHPLC-ESI-MS/MS) method for the simultaneous quantitative detection of the BTX exposure biomarker SPMA, SBMA and o-MBMA in human urine. Urinary S-phenylmercapturic acid (SPMA) is a biomarker proposed by the American Conference of Governmental Industrial Hygienists (ACGIH) for assessing occupational exposure to benzene (Biological Exposure Index of 25 microg/g creatinine). Urinary S-benzylmercapturic (SBMA) and o-methyl S-benzyl mercapturic acid (o-MBMA) are specific toluene and o-xylene metabolites of glutathione detoxicant pathways, proposed as reliable biomarkers of exposure. To this aim a pre-treatment of the urine with solid phase extraction (SPE) and an evaporation step were necessary to concentrate the mercapturic acids before instrumental analysis. A liquid chromatography separation was carried out with a reversed phase capillary column (Synergi 4u Max-RP) using a binary gradient composed of an acquous solution of formic acid 0.07% v/v and methanol. The mercapturic acids were determinated by negative-ion-mass spectrometry and the data were corrected using isotope-labelled analogs as internal standards. The analytical method follows U.S. Food and Drug Administration guidance and was applied to assess exposure to BTX in a group of 396 traffic wardens. The association between biomarker results and individual factors, such as age, sex and tobacco smoke were also investigated. The present work also included improvements in the methods used by modifying various chromatographic parameters and experimental procedures. A partial validation was conducted to evaluate LOD, precision, accuracy, recovery as well as matrix effects. Higher sensitivity will be possible in future biological monitoring programmes, allowing evaluation of very low level of BTX human exposure. Keywords: Human biomonitoring, aromatic hydrocarbons, biomarker of exposure, HPLC-MS/MS.

Original analytical methods for the determination of psychoactive compounds in complex matrices

This thesis work aims to develop original analytical methods for the determination of drugs with a potential for abuse, for the analysis of substances used in the pharmacological treatment of drug addiction in biological samples and for the monitoring of potentially toxic compounds added to street drugs. In fact reliable analytical techniques can play an important role in this setting. They can be employed to reveal drug intake, allowing the identification of drug users and to assess drug blood levels, assisting physicians in the management of the treatment. Pharmacological therapy needs to be carefully monitored indeed in order to optimize the dose scheduling according to the specific needs of the patient and to discourage improper use of the medication. In particular, different methods have been developed for the detection of gamma-hydroxybutiric acid (GHB), prescribed for the treatment of alcohol addiction, of glucocorticoids, one of the most abused pharmaceutical class to enhance sport performance and of adulterants, pharmacologically active compounds added to illicit drugs for recreational purposes. All the presented methods are based on capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) coupled to various detectors (diode array detector, mass spectrometer). Biological samples pre-treatment was carried out using different extraction techniques, liquid-liquid extraction (LLE) and solid phase extraction (SPE). Different matrices have been considered: human plasma, dried blood spots, human urine, simulated street drugs. These developed analytical methods are individually described and discussed in this thesis work.