Innovative materials and methods for HPLC and UHPLC: design of new bidentate urea-type stationary phases. Stereodynamic studies of artemisinin derivatives.

This Ph.D. thesis focuses on three main topics: 1) Design and evaluation of new stable bidentate urea-type stationary phases for HPLC and UHPLC. New totally synthetic bidentate urea-type phases were envisioned for HPLC and UHPLC, characterized by a high stability, a wide application range and an excellent suitability for LC-MS. Four conceptually new stationary phases have been developed for the separation of a wide variety of polar compounds by hydrophilic interaction chromatography (HILIC) through a facile one-pot two-step procedure with the aim of obtaining high hydrolytic stability in a variety of elution conditions. A full chemical characterization of the new materials has been obtained through solid-state NMR (both 29Si and 13C CPMAS) spectroscopy. A major application field of the bidentate urea-type stationary phase with free amino groups USP-HILIC-NH2-sil was sugars analysis, usually hampered by anomer peak splitting and instability of the stationary phases under conditions normally employed to suppress it. Complex mixtures of mono-, di- and oligosaccharides were successfully resolved under mild chromatographic conditions, which also allowed an easy interface with mass spectrometry. The potential of such materials was shown in the separation of other highly polar compounds, including polyols, hydroxybenzoic acids, nucleobases, and vitamins. A parallel project was developed using a very similar synthetic scheme where enantiopure diamines were treated with (3-isocyanatopropyl) triethoxysilane to yield reactive ureido selectors that were eventually attached to unmodified silica particles through a stable, bidentate tether. The two steps were part of an easy and time-saving one-pot procedure, as was the case for the HILIC phases. These new monomeric brush-type chiral stationary phases, hereafter called “Crab-like” CSPs, are characterized by an excellent stability and enantioselectivity, a high permeability and efficiency, as well as an extended compatibility with all solvents and no memory effect, which allowed us to operate under normal phase, reversed phase and polar organic mode conditions. Comparative studies were performed with commmercial polymeric brush-type CSPs bearing the same chiral selector (1,2-diaminocyclohexane and 1,2-diphenyl-1,2-ethylenediamine) in order to evaluate the resolution and performance skills of the new “Crab-like” CSPs in different elution modes. 2) Extending the use of “Inverted Chirality Columns Approach (ICCA)” for enantiomeric excess determination in absence of reference samples: application to a water-soluble camptothecin derivative. The aim of the present study was to extend the use of the “Inverted Chirality Columns Approach (ICCA)” previously developed for the identification and quantitation of the trace enantiomer in highly enriched samples of the camptothecin (CPT) family of drugs to a novel water-soluble CPT derivative, namely namitecan (ST1968), currently undergoing phase I clinical trials as anticancer agent. Namitecan, identified from a series of hydrophilic 7-oxyiminomethyl derivatives, contains a free terminal amino group, which traditionally hampers the analysis under normal-phase HPLC conditions. Nevertheless, commercially available Pirkle-type chiral stationary phases (CSPs) available in both the enantiomeric forms (i.e., having the same bound selector with opposite configuration) mainly operate under normal-phase HPLC conditions. For this reason, namitecan was pre-columnN-derivatized with isocyanates A–D and their sulfur analogues E–H to reduce its polarity by converting the amino group into a fragment compatible with the chiral recognition mechanism (i.e., ureido and thioureido groups). Once the optimal columns system and derivatizing agents were selected, an original enantioselective HPLC–MS/MS technique was developed on the Whelk-O1 CSPs. 3) Kinetic and thermodynamic studies of the epimerization processes in Artemisinin-derived antimalarial drugs by Dynamic High Performance Liquid Chromatography (D-HPLC). Stereodynamic investigations have been performed on two active artemisinin derivatives having labile stereogenic centres, namely dihydroartemisinin (DHA), and an in vivo degradation product of DHA containing an endoperoxide, an aldehyde and a ketonic moiety, namely MKA (monoketoaldehyde). We were able to obtain MKA through pyrolysis or basic degradation of DHA and have fully characterized its structure through a variety o analytical techinques. The stereodynamic studies performed have focused on a stereolabile hemiacetalic function present in both molecules. Linear Solvation Energy Relationships (LSER) analysis was used to study the thermodynamics of the epimerization process in DHA and MKA. Computational studies have provided further data on the differential stability and acidity of the two epimeric forms of MKA, while a kinetic investigation is introduced.