Evaluation of the triphenylphosphine assay for quantitating hydroperoxides in oxidized lipids
Description
TitleEvaluation of the triphenylphosphine assay for quantitating hydroperoxides in oxidized lipids
Date Created2019
Other Date2019-05 (degree)
Extent1 online resource (xii, 89 pages) : illustrations
DescriptionThere is increasing need to measure low levels of hydroperoxides in oxidized lipids both to assess degradation extent and to track alternate pathways in mechanisms studies. Reaction of triphenylphosphine (TPP) with hydroperoxides to generate triphenylphosphine oxide (TPPO) offers promise to detect nanomoles of hydroperoxides selectively and recently has been applied to measuring lipid hydroperoxides. However, conditions of reactions have not been fully tested or validated for either qualitative or quantitative accuracy.
Accordingly, to develop a sensitive, accurate hydroperoxide assay that can be standardized for use in both research and quality control, this study reacted standardized cumene hydroperoxide (CuOOH) with TPP, then separated reactants and products by high pressure liquid chromatography to follow and quantitate progress of reaction, determine reaction stoichiometry, and identify conditions required for full and accurate reaction. TPP, TPPO, and CuOOH peaks were monitored at 260, 220, and 206 nm, respectively, using a photodiode array detector. Conditions giving rapid complete reaction and 1:1:1 stoichiometry (one mole TPP reacted with one mole CuOOH to generate one mole TPPO) were TPP (5 mM stock solutions ) with CuOOH in 5:1 molar ratio heated under argon for 20 minutes at 40 C. Side reactions and stoichiometry variability increased at higher reaction temperatures and TPP concentrations. After complete reaction, products were stable up to ~14 hours, long enough for analyzing many samples in autosampler queues. Limits of detection and quantification were 0.05 and 2 nmoles CuOOH per reaction, respectively.
These conditions were then applied to a model lipid, methyl linoleate (ML). TPP reaction was specific for hydroperoxides. It did not react with aldehydes, epoxides, or alcohols but did detect trace hydroperoxides generated during oxidation of unsaturated aldehydes. The greatest challenge was to know reactant concentrations to use when extent of lipid oxidation unknown. Even with low oxidation, ML required dilution before reacting with TPP. TPP detected higher peroxide levels than either iodometric titration or xylenol orange analyses, despite analyzing only 2 mg. With this sensitivity, TPP is particularly useful for detecting trace levels of oxidation in very early stages of lipid oxidation. HPLC adaptations will be required for analyzing triacylglycerols or phospholipids.
NoteM.S.
NoteIncludes bibliographical references
Genretheses, ETD graduate
LanguageEnglish
CollectionSchool of Graduate Studies Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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