Sample Preparation for Determination of Alpha-tocopherol in Erytrocyte Membrane by High Performance Liquid Chromatography

In this thesis, a new method for isolation of α-tocopherol from human erythrocyte membranes, using ultracentrifugation and solid phase extraction (SPE), for next HPLC analysis, was developed. Rightly SPE is nowadays one of the most effective and reliable modern methods permitting appropriate preparation of biological material sample before HPLC analysis. The developed SPE procedure is the following: 1) Washing of taken erythrocytes: 2,5 ml of blood is centrifugated 10 min at 1300×g, 4žC and the upper layer of plasma is separated. 0.01% saline BHT solution is added to the erythrocytes to a final volume of 2.5 ml and the centrifugation is repeated. The supernatant is removed and the whole operation is repeated three times. Washed erythrocytes are stored at a temperature of -86žC. 2) Proper α-tocopherol extraction from washed erythrocytes: 500 μl of 50 mmol/l D-mannitol solution and 500 μl of 5 mmol/l HEPES are introduced in the ultracentrifuge cuvette. Next, 100 μl of defrost washed erythrocytes are taken using a piston pipette. The pipette is carefully cleaned using cellulose and erythrocytes are introduced into the cuvette. 11 μl of 1 mol/l CaCl2 solution are then added and the sample is centrifugated for 3 min, at 90 000 rpm (288 000×g, rAV = 31,8 mm), at 4žC. Afterwards, 500 μl of supernatant is..