Integrated Micro-Analytical Tools for Life Science

Advances in life science require knowledge of active molecules in complex biological systems. These molecules are often only present for a certain time and at limited concentrations. Integrated micro-analytical tools for sampling, separation and mass spectrometric (MS) detection would meet these requests and are therefore continuously gaining interest. An on-line coupling of analytical functions provides shorter analysis time and less manual sample handling. In this thesis, improved compatibility of microdialysis sampling and multidimensional separations coupled to MS detection are developed and discussed. Microdialysis was used in vitro for determination of the non-protein bound fraction of the drug ropivacaine. The sampling unit was coupled on-line to capillary column liquid chromatography (LC) followed by ultraviolet or MS detection. For MS detection, the system was extended with a desalting step and an addition of internal standard. A method for MS screening of microdialysates, collected in vivo, was also developed. The method involved sampling and measurements of the chemical pattern of molecules that generally are ignored in clinical investigations. Chemometric tools were used to extract the relevant information and to compare samples from stimulated and control tissues. Complex samples often require separation in more than one dimension. On-line interfaces for sample transfer between LC and capillary electrophoresis (CE) were developed in soft poly(dimethylsiloxane) (PDMS). MS detection in the LC-CE system was optimised on frequent sampling of the CE peak or on high resolution in mass spectra using time-of-flight (TOF)MS or Fourier transform ion cyclotron resonance (FTICR)MS, respectively. Aspects on electrode positioning in the LC-CE interface led to development of an on-column CE electrode. A successful method for deactivation of the PDMS surface using a polyamine polymer was also developed. The systems were evaluated using peptides and proteins, molecules that are gaining increased attention in bioscience, and consequently also in chemical analysis