HPLC-chemiluminescence and thermospray LC/MS study of hydroperoxides generated from phosphatidylcholine

Lipid hydroperoxides generated from phosphatidylcholine (PC) by two commonly employed phosphatidylcholine hydroperoxide (PCOOH) generation methods were examined by HPLC-chemiluminescence (CL) and thermospray LC/MS assay. This HPLC-CL assay is specific for hydroperoxides. In the HPLC-CL chromatograms, a major peak eluted at 4.7 min for the samples generated by the photooxidation of PC in the presence of methylene blue. The direct LC/MS analysis of the hydroperoxides contained in this peak determined that the hydroperoxides are mono- and di-PCOOH. Quantitation showed that over 90% of the hydroperoxides generated by photooxidation are PCOOH. In contrast, a different major peak appeared at 3.7 min for the hydroperoxides generated by the incubation of PC with the azo compound AMVN. We determined by LC/MS analysis that the hydroperoxides contained in this peak were not equivalent to either mono- or di-PCOOH. Indeed, 70%-95% of the hydroperoxides generated by AMVN incubation were not PCOOH, but rather a large portion were AMVN-derived hydroperoxides. The hydroperoxides contained in the 4.7-min peak (i.e., PCOOH) were preferentially responsive to cytochrome c-luminol CL cocktail (about 100-fold more responsive than the hydroperoxides in the 3.7-min peak), whereas the hydroperoxides in the 3.7-min peak (including AMVN-derived hydroperoxide) were preferentially responsive to microperoxidase-isoluminol CL cocktail (about 20-fold more responsive than the PCOOH), suggesting a substrate specificity for the CL cocktail.