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.

Application of different instrumental techniques to the selective evaluation of bio-sensible compounds in foodstuff of animal and vegetable origin

This PhD thesis describes the application of some instrumental analytical techniques suitable to the study of fundamental food products for the human diet, such as: extra virgin olive oil and dairy products. These products, widely spread in the market and with high nutritional values, are increasingly recognized healthy properties although their lipid fraction might contain some unfavorable components to the human health. The research activity has been structured in the following investigations: “Comparison of different techniques for trans fatty acids analysis” “Fatty acids analysis of outcrop milk cream samples, with particular emphasis on the content of Conjugated Linoleic Acid (CLA) and trans Fatty Acids (TFA), by using 100m high-polarity capillary column” “Evaluation of the oxidited fatty acids (OFA) content during the Parmigiano-Reggiano cheese seasoning” “Direct analysis of 4-desmethyl sterols and two dihydroxy triterpenes in saponified vegetal oils (olive oil and others) using liquid chromatography-mass spectrometry” “Quantitation of long chain poly-unsatured fatty acids (LC-PUFA) in base infant formulas by Gas Chromatography, and evaluation of the blending phases accuracy during their preparation” “Fatty acids composition of Parmigiano Reggiano cheese samples, with emphasis on trans isomers (TFA)”

Studies on the interaction of surfactants and neutral cyclodextrins by capillary electrophoresis. Application to chiral analysis

In the present study, mixed systems composed of SDS in the presence of neutral cyclodextrins were considered. Firstly, the effect of the CDs on the CMC of the surfactant was evaluated by CE experiments. Furthermore, a new CE approach based on electric current measurement was developed for the estimation of the stoichiometry as well as of the binding constants of SDS-CDs complexes. The results of these investigations were compared to those obtained with a different technique, electronic paramagnetic resonance (EPR). The obtained results suggested that methylated CDs, in particular (2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD), strongly affect the micellization of SDS in comparison to the other studied CDs. This effect also paralleled the chiral CD-MEKC performance, as indicated by the enantioresolution of (+/-)-Catechin, which was firstly selected as a model compound representative of important chiral phytomarkers. Then a CD-MEKC system, composed of sodium dodecyl sulfate as surfactant (90 mM) and hydroxypropyl-beta-cyclodextrin (25 mM) as chiral selector, under acidic conditions (25 mM borate – phosphate buffer, pH 2.5) was applied to study the thermal epimerisation of epi-structured catechins, (-)-Epicatechin and (-)-Epigallocatechin, to non epi-structured (-)-Catechin and (-)-Gallocatechin. The latter compounds, being non-native molecules, were for the first time regarded as useful phytomarkers of tea sample degradation. The proposed method was applied to the analysis of more than twenty tea samples of different geographical origins (China, Japan, Ceylon), having undergone different storage conditions and manufacturing processes.