Determination of phosphatidylcholine hydroperoxide in plasma and serum by HPLC/postcolumn method

Phosphatidylcholine hydroperoxide (PC-HPO) in human plasma and bovine serum was determined by high preformance liquid chromatography. PC-HPO was separated on a silicagel column eluted with chloroform/methanol/water followed by postcolumn reaction with a fluorescence reagent, diphenyl-1-pyrenylphosphine. By this system, PC-HPO was determined at the range of 1650 pmol and the detection limit was 1 pmol (S/N=3). The relative standard deviation of the peak area was 1.8% (108 pmol, n=7) and 3.4% (21.6 pmol, n=7). PC-HPO in biological fluids were extracted twice with methanol/dichloromethane and the solvent was evaporated under reduced pressure. After dissolving in chloroform/methanol, its aliquot was injected into the HPLC system. Phosphatidylcholine, β-NBD-aminohexanoyl-γ-palmitoyl, was added as an internal standard detected by UV at 475 nm. The relative standard deviation of 7 extracts from the same plasma was 6.5%. Recovery of PC-HPO from human plasma was 84.3%. By this method, PC-HPO was determined as 3075 nM (n=13) in human plasma, and 9100 nM (n=67) in bovine serum.