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Capillary electrophoresis for blood doping analysis : modification of the electroosmotic flow for the separation and detection of blood doping agents in human whole blood


Blood doping is an illegal method, prohibited by World anti-doping agency to prevent the athletes increase their oxygen carrying capacity during the professional sports. The rapid detection of the dopants in the complex human whole blood sample is a difficult task and requires high throughput doping detection methods. The main focus of our work includes the separation and detection of artificial oxygen carriers such as hemoglobin based oxygen carriers (HBOCs) and perfluorocarbon emulsions; autologous blood transfusion markers present in the whole blood by using the capillary electrophoresis (CE). Chapter 2 describes the preparation of HBOCs, sample pretreatment and capillary zone electrophoresis (CZE) separation method development. The phospholipid bilayer capillary coating is important in this method to suppress the electroosmotic flow and prevent the protein adsorption. The molecular weight cut-off filter purified and concentrated the HBOCs which were detected with a sensitivity below the doping amount. This method is much more simplified and rapid than the methods reported before. The CE UV-Vis on-column detection is associated with the difficulty of poor sensitivity because of the small path length. To overcome this difficulty, an online sample stacking method has been developed. Chapter 3 describes the development and optimization of large volume sample stacking (LVSS) method with the same conditions as in HBOCs-CZE method. Simultaneous application of voltage and back pressure to remove the sample matrix; double step stacking are the crucial steps in this method. The detection sensitivity significantly improved compared to CZE (more than 100 fold) when a standard protein mixture was tested. Autologous blood transfusion is a potential blood doping method carried out by transfusing the athlete's own stored blood. Because there is no direct detection method, we investigated the applicability of CZE for the detection of the transfused blood marker glycated proteins present in the doped blood which is described in chapter 4. The standard glycated proteins were successfully separated and detected. A significant signal enhancement has been observed with the trypsin digests of RBCs membrane ghosts in the normal CZE-LVSS. However, because of the presence of intrinsic glycated proteins in normal individuals this method will not be a direct doping test

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