Automatic determination of hydroperoxides of phosphatidylcholine and phosphatidylethanolamine in human plasma.

An automatic method for the determination of hydroperoxides of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is reported. Sample plasma was deproteinized with a fourfold volume of methanol. After centrifugation, the supernatant was injected directly into an HPLC system without further treatment. The hydroperoxides of PC and PE were concentrated and washed on an ODS column followed by introduction into two analytical columns, a silica gel and an aminopropylsilica gel column, which were connected in series, by column switching. After the separation, they were detected by postcolumn detection with diphenyl-1-pyrenylphosphine. The compounds were determined at picomole levels within 30 min with good reproducibilities. By using only a silica gel column as an analytical column, PC hydroperoxides were determined within 20 min, and samples could be injected into it at 15-min intervals. Those methods made it possible to inject a sample of up to 2 ml at one time and up to 8 ml by repeated injections and to determine phospholipid hydroperoxides in human plasma at picomole levels.