New Mass Spectrometric Approaches for the Evaluation of Drug Disposition

The aim of the thesis was to develop and evaluate novel analytical techniques for the generation of qualitative and quantitative ADME data on biological samples.

The quantitative aspect of the thesis focused on two approaches on how instrumentation improvement supports sensitivity enhancement of analytical methods developed on LC-MS systems.

In a first set of experiments, an innovative injection set-up composed of three valves that allows the injection of two distinct volumes with a 200-fold difference (2 µL and 10 nL) on a single LC-MS system was explored. By applying this novel approach of using two different injection volumes, it was possible to double the dynamic range of the liquid chromatography - triple quadrupole mass spectrometer used. The application of nL injections also allowed to analyze biological samples without sample preparation. More than 1,000 direct injections of untreated plasma were successfully carried out using the nanoliter valve with no measurable effect on LC performances.

In a second set of experiments focusing on mass spectrometry, a new atmospheric pressure ionization (API) source was evaluated. A selection of relevant pharmaceutical and biological small molecules was used to investigate the ionization mechanism of the new API source. All experiments were done in comparison with an electrospray ionization (ESI) source. Further investigations of the new API source in LC-MS mode were further conducted. LC-MS performances (matrix effects and dynamic range) were evaluated and compared to ESI for a selection of classic pharmaceutical compounds and matrices commonly encountered in bioanalysis. Also, post-column addition of water was evaluated and showed an improvement for the sensitivity of very late eluting compounds. Finally, a practical application for the quantitative analysis of endogenous prostaglandins (PGs) and thromboxanes (TXs) in complex biological matrices was performed on the new API source. Linearity, accuracy, precision, limit of detection and repeatability of the analytical method used for the analysis of PGs and TXs were evaluated and compared to ESI.

The qualitative aspect of the thesis was explored with the development of an analytical method for the derivatization of metabolite with DESI-MS as an alternative to classical derivatization methods. Metabolites were collected after liquid chromatography separation (e.g. by fraction collection), dried on a glass plate with printed Teflon spots and then a one-drop derivatization was performed. Once the spot was dried again, the derivatized compound was analyzed by DESI-MS.

The different analytical techniques and methods presented were evaluated and developed to improve the generation of qualitative and quantitative data for ADME profiling. The UniSpray source and the three valves set-up were used to enhance method sensitivity, therefore covering the quantitative aspect. The DESI-MS derivatization method offers a new tool for metabolite identification and cover the qualitative part of the thesis. The results presented in this thesis in addition to the results from the 12 other PhD students from the ARIADME initial training network will hopefully provide a significant improvement to ADME profiling and fulfill the objective of the project.