Microperoxidase hydroperoxide determination

The first FI-CL system for lipid hydroperoxide determination, published in 1993, was based on luminol as the CL reagent and microperoxidase as the catalyst. The optimized system was used for measuring lipid hydroperoxides in native low-density lipoprotein (LDL) (Cominacini et al. 1993). The FI-CL system consisted of two pumps, an autosampler, and a chemiluminescent detector with a T-mixing coil. Samples were injected by using an autosampler and mixed with luminescent reagent (3 (iM luminol and 1 xM microperoxidase in 0.1 M carbonate buffer, pH 10). The calibration curve was obtained using linoleic acid, and a detection limit of 3 pmol linoleic acid hydroperoxide was reached. 

The CLD methods for HPLC using isoluminol (190) with microperoxidase catalysis, for determination of lipid hydroperoxides in clinical fluids, have been reviewed. Determination of phospholipids hydroperoxides by luminol (124) CL has been reviewed . A fast RP-HPLC method (retention times 1 to 2 min) for determination of hydroperoxides and other peroxide compounds includes UVD, which is not always effective, and CLD, attained on injection of luminol (124), the CL reagent (Scheme 3), hemin (75a), a catalyst, and NaOH to raise the pH of the solution. A FLD cell may act as CLD cell if the excitation source is turned off. The selectivity of CLD is of advantage over UVD in industrial analysis thus, for example, UVD of a sample from a phenol production line based on cumene oxidation (equation 13) shows peaks for cumyl hydroperoxide (27), unreacted cumene, cumyl alcohol and acetophenone, whereas CLD shows only the 27 peak. The... 

A procedure for determination of lipid hydroperoxides in human plasma is based on kinetic measurement of the CL of luminol (124) with hemin (75a) catalysis . CLD of microperoxidase-catalyzed oxidation of luminol (124) or isoluminol (190) was applied to detection and determination of amino acid hydroperoxides after exposure to UV and y-irradiation A method for determination of hydroperoxides in the phospholipids of cultured cells uses isoluminol (190) and microperoxidase as catalyst " . Simultaneous determination of phosphatidylcholine hydroperoxides and cholesteryl ester hydroperoxides in human serum is carried out by quantitative extraction of the lipids, HPLC separation by column switching and CLD using isoluminol (190) with microperoxidase catalysis . ... 

The first step within this project was to improve the HPLC-fluorescence method [1] in order to determine not only hydrophilic but also lipophilic hydroperoxides of up to seven carbon atoms. Among the aspects investigated were colunm length, mixed solvent and gradient elution and the use of microperoxidase instead of horseradish peroxidase in the detection system [2, 3]. The use of microperoxidase let us add secondary, tertiary and other branched hydroperoxides to the list of peroxide species we can determine. For full analysis a number of hydroperoxides were synthesised as standards for retention time and quantitation methyl hydroperoxide (MHP), ethyl hydroperoxide (EHP), 1- and 2-propyl hydroperoxides (PHPs), 1-butyl hydro-peroxide (1-BHP), hydroxymethyl hydroperoxide (HMHP), 1-hydroxyethyl hydroperoxide (1-HEHP), 2-hydroxyethyl hydroperoxide (2-HEHP), 1-hydroxypropyl hydroperoxide (1-HPHP), 1-hydroxybutyl hydroperoxide (1-HBHP), 1- hydroxypentyl hydroperoxide (1-HPentHP),... 

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