I've been trying to analyze CoQ10 levels in cell lines +/- treatment with oxidative stress-inducing agents. I've tried methanol/hexane, ethanol/hexane and isopropanol hexane extraction from 5x10^6 cells, followed by drying down in a speedyvac and resuspension in ethanol/methanol FeCL3 prior to reverse phase HPLC (using Ethanol:Methanol:Perchloric acid mobile phase). However, I don't see any peaks corresponding to that obtained with control preparations of CoQ10. What am I doing wrong? Any help would be much appreciated.
The method (HPLC) is the same as used for other tissues, after ubiquinone extraction. Colomé C, Artuch R, Vilaseca MA, Sierra C, Brandi N, Cambra FJ, Lambruschini N, Campistol J. Clin Biochem. 2002 Feb;35(1):81-4.
We normally use a single dilution step extracting 50 ul of a cellular suspension with 250 ul isopropanol. after centrifugation we inject directly and with the ECD method we are able to quantify as low as 1x10^5 cells
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