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Abstract

Chemical space coverage remains a major challenge in analytical chemistry. Even with modern instruments and data-processing tools, only a small percentage of all existing organic compounds is detected, let alone identified. Broad range of molecular polarity is one of the main factors influencing this: the most hydrophilic and hydrophobic compounds are often missed, especially in non-targeted workflows that seek broad coverage. This thesis focuses on highly polar compounds analysis and demonstrates the potential of hydrophilic interaction liquid chromatography (HILIC), an increasingly popular but underutilized chromatographic technique.
The work explores HILIC-based LC–MS across three fields: exposure science, metabolomics, and pharmacotherapy. Despite their differences, all three involve analytes that are often poorly retained by conventional reversed-phase (RP) chromatography. This thesis shows that incorporating HILIC into analytical workflows can meaningfully improve their detection and expand the accessible chemical space.
Two studies demonstrated the complementarity of HILIC and RP-based analyses of biologically relevant compounds in non-targeted profiling. An exposure-science-focused study was performed on an ovarian-cancer cohort, applying a fractionation-based sample-preparation approach and discovering 106 exposure biomarkers, six of which differed across groups. HILIC’s contribution was assessed as significant since it accounted for 23% of the annotations. The second study, also combining HILIC- and RP-based analysis, was focused both on metabolomics as well as exposure science, analyzing serum and urine samples of women with polycystic ovary syndrome (PCOS), with the aim to identify potential biomarkers of exposure and effect. The analysis revealed alterations in lipids, amino acids, and steroid-related metabolites, spanning a wide polarity range, again supporting the rationale behind the two-column analytical approach.
The pharmacotherapy section of the thesis focuses on bleomycin (BLM), an extremely polar cytostatic drug that has very limited number of published quantitative analytical methods. Two methodological papers are presented, introducing two high-performance analytical methods: one was developed and validated for the quantification of BLM in clinically relevant concentrations and matrices (blood-derived samples), and the other for the quantification of intracellular BLM to support in vitro experiments in the optimization of electrochemotherapy procedures. To the best of our knowledge, these studies represent the first validated analytical methods enabling reliable quantification of bleomycin in the respective experimental and clinical contexts. Beyond the analytical methods themselves, both studies also address practical considerations and common analytical challenges associated with the measurement of highly polar and structurally complex compounds.
The studies in this thesis demonstrate that incorporating HILIC into LC–MS workflows can substantially improve access to the polar end of chemical space across biomedical and environmental applications. The work provides validated methods, transferable workflows, and practical guidance that support more comprehensive analytical strategies in both targeted and non-targeted settings.