Chromatographic methods of analysis applied to coumarins and cell organelles

The clinical use of any drug requires a complete understanding of its biotransformation properties. The drug coumarin has for years been used in clinical applications. High performance liquid chromatography (HPLC) and capillary electrophoresis (CE) were applied to the study of coumarin metabolism, in particular the phase I and phase II metabolites, 7- hydroxycoumarin and 7-hydroxycoumarin glucuronide. Glucuronidation is the principal pathway for detoxification of many drugs and is catalysed by the enzyme uridine diphosphate glucuronyl transferase (UDPGT). Both chromatographic methods were applied to the analysis of the UDPGT activity in rabbit tissue samples. CE was applied to the study of [3- glucuronidase activity for the same samples. Microchip-based capillary electrophoresis with laser induced fluorescence detection was used to investigate antibody based detection methods for the anticoagulant, warfarin, and the pesticide parathion. A novel approach for the assay used an easily labelled drug-protein conjugate as tracer, rather than the conventional free drug with fluorescent tag. However, problems associated with heterogeneity of the antibody preparation and also with the labelled drug protein, did not allow complete resolution of antibody-drug-protein from free drugprotein to be effected. Knowledge of what happens on a sub-cellular level is also required for a complete understanding of drug disposition. The use of capillary electrophoresis for the analysis of cell organelles was examined. Liposomes were chosen as a model as they could be tailored to mimic certain characteristics of different organelles (size, membrane composition), and are also manufactured industrially for use in drug delivery/targeting. The method developed provides critical information on size and membrane composition on a single liposome basis that can be used to monitor liposome preparations for use in clinical applications. Having successfully established this method, CE was then applied to the analysis of mitochondria, isolated from Chinese hamster ovary cells. Two specific organelle probes were used, i.e. nonyl acridine orange (NAO) for mitochondrial mass analysis, and rhodamine 123 (Rhl23) for membrane potential studies. NAO is a membrane specific probe and its uptake is independent on the energetic stale of the mitochondria, binding to the cardiolipin content in a 2:1 ratio, while rhodamine 123 requires a membrane potential for its uptake. Measurement of fluorophore content of each individual mitochondrion gives information on membrane size and activity. A preliminary analysis of the mitochondrial proteome was carricd out by capillary gel electrophoresis following the purification of protein from isolated mitochondria and labelling with a fluorescent tag