Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electroscopy MS

Measurement of lipid peroxidation is a commonly used method of detecting ox idative damage to biological tissues, but the most frequently used methods, including MS, measure breakdown products and are therefore indirect. We ha ve coupled reversed-phase HPLC with positive ionization electrospray MS (LC -MS) to provide a method for separating and detecting intact oxidized phosp holipids in oxidatively stressed mammalian cells without extensive sample p reparation. The elution profile of phospholipid hydroperoxides and chlorohy drins was first characterized using individual phospholipids or a defined p hospholipid mixture as a model system. The facility of detection of the oxi dized species in complex mixtures was greatly improved compared with direct -injection MS analysis, as they eluted earlier than the native lipids, owin g to the decrease in hydrophobicity. In U937 and HL60 cells treated in vitr o with t-butylhydroperoxide plus Fe2+, lipid oxidation could not be observe d by direct injection, but LC-MS allowed the detection of monohydroperoxide s of palmitoyl-linoleoyl and stearoyl-linoleoyl phosphatidylcholines. The l evels of hydroperoxides observed in U937 cells were found to depend on the duration and severity of the oxidative stress. In cells treated with HOCl, chlorohydrins of palmitoyloleoyl phosphatidylcholine were observed by LC-MS , The method was able to detect very small amounts of oxidized lipids compa red with the levels of native lipids present. The membrane-lipid profiles o f these cells were found to be quite resistant to damage until high concent rations of oxidants were used, This is the first report of direct detection by LC-MS of intact oxidized phospholipids induced in cultured cells subjec ted to oxidative stress.