Improved HPLC column-switching determination of Coenzyme Q and Vitamin E in plasma

A novel isocratic modified column-switching HPLC method for automated quantitative analysis of Vitamin E and Coenzyme Q, in the reduced and oxidized form, is described. Many column-switching HPLC methods are found in the literature, also for determining antioxidant substances, but we developed a different system of column-switching. An empty column, 5 cm long, was connected to the switching valve, before the sample loop and the extraction column. The sample loop was connected directly after the empty column. The inserted column, containing about 1.4 ml of the extraction eluent simulated a gradient elution, enhancing sensitivity and resolution. When switching the columns, the empty column is placed right before the extraction column and acts as a static mixer for the extraction phase and the incoming analytical phase. Samples were cleaned from interfering compounds by transfer onto a extraction-column, using a C-8 silica. Separation was performed onto an analytical column C18 3 m icrom, 150 mm x 4.6 mm at a flow rate of 1.0 ml/min with 20 mmol/l lithium perchlorate/perchloric acid, pH3.0 in Ethanol as analytical eluent. Detection was performed with a ESA Coulochem 5100 A model. The method was found to be suitable for automated analysis of Coenzyme Q, reduced and oxidized form, and Vitamin E in serum.