Development and application of instrumental LC-MS/MS analytical platforms and chemometric data treatment for the study of complex matrices in the environmental and nutritional fields.

This PhD project has been carried out to develop high throughput LC-MS/MS platforms exploiting both targeted and untargeted approaches capable to untangle the intricate data information from complex samples of environmental and nutritional interest. To this extent, different types of (analytically challenging) samples have been investigated, including fruit extracts, wastewaters, technical mixtures, and human biofluids (i.e., urine and serum). The development and/or application of (i) LC-MS/MS wet lab methods and (ii) dry lab chemometric workflows will generate informative and accurate data that could help to increase the knowledge on the composition of the investigated samples. In this regard, when applicable, LC-MS/MS data have been publicly shared in available repositories (e.g., MassIVE) to contribute to dissemination within the scientific community. A brief description of the activity presented in this PhD thesis is reported in the following lines. Activity 1: supporting the principles of environmental sustainability, the quantitative data (e.g., fruit yield, sugars, (poly)phenols etc.) on the quality of strawberry (Fragaria x ananassa, cv. Camarosa) fruits irrigated with four types of treated wastewaters were analysed. Statistical and multivariate analyses were performed to assess the impact of wastewater irrigation. Activity 2: the profiling of the antioxidant and bioactive compounds occurring in Rubus idaeus L. fruits compared to Rubus occidentalis L. black raspberry was provided by the adoption of a comprehensive untargeted metabolomics approach developed with UHPLC-MS/MS, using the SWATH® PhD Course in Chemical Sciences acquisition protocol. Chemometrics was used to highlight genotype effects. Activity 3: Analytical Quality by Design is used to optimize the chromatographic separation and mass spectrometric detection of a wide group of structurally heterogeneous model pharmaceutical compounds and their major transformation products. The optimized method allowed for analysing the target analytes in a single chromatographic run, adopting a mixed acquisition mode based on scheduled acquisition windows comprising both single polarity and continuous polarity switching. Activity 4: different chemometric selection workflows for potential exposure marker (PEM) discovery, using multivariate or univariate parametric or non-parametric data analyses, were comparatively tested and evaluated. The PEM selection protocols were applied to a small-sample-size untargeted LC-MS/MS study of a longitudinal set of serum samples from 20 volunteers after a single intake of (poly)phenolic-rich Vaccinium supplements. Activity 5: The investigation of the metabolite discovery capacity and the possible extension of the annotation coverage of the Feature-Based Molecular Networking approach was performed in combination with public and curated mass spectral libraries in comparison with the currently accepted non-commercial annotation protocols. The postprandial urinary metabolome analysis within a two-arm intervention on the intake of Vaccinium supplements was selected as a case study, analysing the postprandial behaviour of the annotated metabolites by PPMC correlation. Activity 6: an online comprehensive LC×LC-HRMS method was developed for the non-targeted identification of poly- and perfluorinated compounds (PFASs) in fire-fighting aqueous-film forming foams (AFFFs). The method exploited the combination of mixed-mode weak anion exchange-reversed phase with octadecyl stationary phase, separating PFASs according to ionic classes and chain length. Activity 7: thermal desorption was investigated for the first time in on-line SPE-RP-LC-MS/MS, using water as SPE eluent. This desorption strategy was tested in the on-line SPE-LC-MS/MS analysis of a model group of structurally heterogeneous analytes including 34 human gut metabolites characterized by a wide range of physicochemical and structural properties, including isobaric compounds, thus representing a challenging analytical issue.