Development and validation of triphenylphosphine assay for nanomolar levels of lipid hydroperoxides

Abstract: Growing interest in alternate pathways of lipid oxidation and effects of different oxidation conditions on product distributions creates need for more sensitive, accurate methods to quantify all lipid oxidation products, especially hydroperoxides. Oxidation of triphenylphosphine (TPP) to triphenylphosphine oxide (TPPO) by lipid hydroperoxides (TPP + LOOH ® TPPO + LOH) has attracted attention for its nanomolar sensitivity, but the reaction needs full testing for development into a standardizable assay.

TPP reaction was validated with cumene and t-butyl hydroperoxides then extended to methyl ester hydroperoxides in argon-sparged acetonitrile. Reaction progress and completion were tracked by separating hydroperoxide, lipid alcohol, TPPO, and TPP by reversed phase HPLC (RP-HPLC) using a pentafluorophenyl column, acetonitrile:water solvent, and uv detection at 220, 233, and 260 nm. TPP reacted specifically with hydroperoxides to generate TPPO as the only product with 1:1 stoichiometry and linear detection range of 20.6 picomols to 12.4 nmols of LOOH injected. Optimized reaction and analysis times of 15 min each were achieved. Reagents and reaction products are stable for about 5 hours after reaction.

For triacylglycerol and oil hydroperoxides, the TPP assay was switched to normal phase HPLC to dissolve oils easily, avoid gradients and long elution times, and separate unreacted triacylglycerols early. Reactions were run in 1:1 toluene:acetonitrile with 20 mM BHT to inhibit continued oxidation. Eluting solvent was isocratic 5% ethanol in hexane. Methyl esters, trilinolein, triolein, and soybean, canola, high oleic sunflower, and fish oils were oxidized, analyzed for LOOH by iodometric titration, then volumes containing nanomolar LOOH were reacted with excess TPP. TPPO generation was linear from 7.5 pmols to 99.8 nmols LOOH injected, quantitative with 1 TPPO:1 LOOH stoichiometry, and independent of the LOOH source. Accuracy, sensitivity, and limited handling thus make TPP assay of LOOH a promising method for quality control, shelf-life analyses, and basic research.