Method performance characteristics for individual FAOOH and FAOH isomers.
To overcome this and to provide a more accurate estimation of total lipid peroxidation, alkaline hydrolysis of lipid esters to yield free fatty acid hydroperoxides (FAOOHs) and hydroxy fatty acids (FAOHs), followed by HPLC separation and direct UV detection of the conjugated dienes has been described (16).
The present report improves this methodology to achieve resolution of regioisomers of linoleic, linolenic, arachidonic, and docosahexaenoic acid FAOOHs and FAOHs. The method is applied to the analysis of FAOOHs and FAOHs generated by in vitro oxidation of human plasma by metal-catalyzed oxidation with the [Cu.sup.2+] ion and hydrogen peroxide.
Bulk FAOHs were synthesized by reduction of FAOOHs with sodium borohydride in ice-cold methanol followed by chloroform re-extraction.
Pure bulk preparations of FAOOHs and FAOHs were obtained by reaction with lipoxidase followed by reduction with sodium borohydride.
Thomas and Jackson (16) reported the conversion of small amounts of FAOOHs to FAOHs during alkaline hydrolysis of lipid esters, and the reaction of FAOOHs with strong alkali has been described in detail by Gardener et al.
For all linoleic acid- and linolenic acid-derived FAOOHs and FAOHs, which gave sharp peaks and were well resolved, the MDQ was 5-7 pmol on column.
The moderate reproducibility is also likely to be a function of the reduction of FAOOHs to FAOHs during alkaline hydrolysis, which was evident in the low recovery of the FAOOH compounds (87-93%).