| This thesis presents enhanced analytical methods developed for bioanalysis and drug discovery. These methods are based on solid phase microextraction (SPME) as a sample preparation method. SPME was developed specifically to address the need for fast sample preparation both in the laboratory and on-site. With this technique, a small amount of extracting phase dispersed on a solid support is exposed to a sample for a well-defined period of time. Target compounds are extracted without the need to remove the sample from its environment. The SPME process results in partial extraction of target compounds, and the amount removed from the sample is related to the concentration of macromolecules that bind the analyte. Accordingly, supplementary information such as binding constants and concentration of macromolecules can be obtained. The method is extremely useful for binding studies, in vivo investigations, and for very small samples.; The research is systematized in three successive stages. For the initial one, new SPME coatings based on restricted access materials and hollow membranes are developed. These new coatings for SPME offer the possibility to combine all initial sample preparation steps into a single one, even for complex biological samples such as whole blood or plasma. The technology is successfully applied for the fast determination of angiotensin 1 in whole blood and for the determination of paclitaxel free concentration in liposome formulations.; During the second stage, previously designed coatings, as well as existing ones, are used to develop analytical procedures for investigating the complex interactions between drugs and proteins. The theoretical background and practical approaches for studying ligand-receptor binding by SPME sampling are discussed, along with methods simultaneous calculation of receptor, free, and total ligand concentrations. The usefulness of this SPME approach for binding studies is further demonstrated by determining drug plasma protein binding, a parameter that is crucial in drug development.; In the third and final stage, the newly developed analytical methods are applied in pharmacokinetic studies, both in vivo and in vitro. In addition to the total drug concentration, methods based on SPME permit investigation of supplementary pharmacological parameters. The in vitro work consists of studying the pharmacokinetic profile of chlorhexidine in human saliva. (Abstract shortened by UMI.)... |
