Abstract :
[en] During the last decades, a great interest has been shown for miniaturised separation techniques. The use of microfluidic techniques fulfils the constant needs for increasing sample throughput and analysis sensitivity, while reducing costs and sample volume consumption.In this work, we studied the quantitation potentialities of an integrated miniaturised liquid chromatographic device coupled to mass spectrometric detection, the Agilent HPLC-chip-MS system.In the first part of this work, a method was developed to quantify fluoxetine and its N-desmethylated metabolite, norfluoxetine, in rat serum. Because samples were only available in limited volumes, special attention was paid to develop a method that required low sample volume while providing good sensitivity. Design of experiments was employed as a tool to find the most appropriate chromatographic parameters allowing fast and sensitive analysis. The combination of miniaturised sample preparation by solid-phase extraction to the HPLC-chip-MS system was successfully employed to achieve fluoxetine and norfluoxetine quantitation in the concentration range of interest. The developed method was finally fully validated.The second part of this work was dedicated to the assessment of the feasibility of peptide quantitation by the HPLC-chip system. Hepcidin, a peptide biomarker, was selected as model for method development. Design of experiments was used to optimise mobile phase and sample composition, in order to obtain the greatest sensitivity and the lowest carry-over. Method was prevalidated to estimate the limits of quantitation and find the most appropriate regression model for further hepcidin quantitation. A sample preparation method was then developed to allow the analysis of biological samples. Finally, a calibration curve was successfully built in human plasma in the physiopathological range of interest to use hepcidin as biomarker. Finally, the impact of sample and mobile phase composition on peptide retention and sensitivity was studied to improve the knowledge of peptide chromatographic behaviour. Design of experiments and principal component analysis were employed to highlight the relationships between peptide physicochemical properties, experimental conditions and peptide retention and sensitivity. A generic method for peptide analysis was proposed as starting point for rapid method development applied to peptide analysis. A fundamental study was undertaken to better understand the retention mechanisms of peptides in RP-LC.
