Dissertation Klaus Heitkamp 1999

0 Abstract 10 AbstractTo develop a number of photometric and fluorimetric procedures for sensitive determinationof lipid hydroperoxides (LHPs) in cuvettes and in HPLC systems, biologically relevantLHPs were synthesised either isomerically pure or as mixtures of isomers by oxidation offatty acids with lipoxidase or 1 O 2 . The methods of previous work were used as starting pointsfor the syntheses, but individual steps were substantially improved and the yield of byproductsgreatly reduced. The compounds synthesised were identified mainly by 1 H- and13 C-NMR-spectroscopy.The detection limits of the various photometric methods for lipid hydroperoxides werecompared with a newly developed procedure. This test is based on the Fe 2+ -catalysedconversion of N,N,N',N'-tetramethyl phenylene diamine to a blue radical cation by LHP. Thedetection limit of ca. 8-11 µmol/l was in the region of other well-known methods (e.g. FOX2test, measurement of the I 3 - -absorption). Literature claims that the TBA test does not permitany quantitative statement about the degree of oxidation of oil or fat samples wereconfirmed.Normal-phase HPLC proved to be an efficient method for separating the hydroperoxideproducts of the photosensitised oxidation of fatty acid methyl esters. However, since therestill were no selective detection methods for lipid peroxides in the NP-HPLC system, aprocedure was developed for a post-column reaction of hydroperoxides, followed byUV/VIS detection. This method is based on the oxidation of the colourless 1,5-diphenylcarbohydrazide to the red 1,5-diphenyl carbazone with succeeding photometric detection.The optimisation of the detection included a statistical procedure that made it possible tovary several parameters at the same time. The detection limit achieved with the system thusoptimised was ca. 17 µmol/l, somewhat higher than that of other detectors used in RP-HPLC.The use of immobilised microperoxidase-11 for the selective fluorescence detection ofhydroperoxides in RP-HPLC was described for the first time. To this end, MP-11 wasimmobilised on micro-porous glass (CPG BEADS) without the use of spacer molecules.Substantially higher immobilisation and peroxidase activity could be achieved in this way.Neither the pore dimensions nor the pore volume of the type of beads used had any influenceon the peroxidase activity. Fixed-bed reactors were prepared for use in an HPLC system withimmobilised MP-11. The detection limits achieved with this system, 1-3 µmol/l, were similarto those of the original system with microperoxidase dissolved in a reagent solution.The efficiency of the enzyme reactors developed was shown in the investigation of oxidisedoil samples. An attempt was made to confirm a dependency of the oxidation of fats in modelfoods on the water content of the sample, as described in the literature. As analyticalmethods there were available several photometric test methods and UV/VIS- andfluorescence-HPLC methods with non-enzymatic post-column derivatisation as well asenzymatic post-column derivatisation with the newly developed enzyme reactors. As itturned out, the effects described in the literature could not be reproduced, but the resultsfrom all the methods used were in agreement, so that the enzyme reactor developed in thiswork can be seen as a sensitive and economical means for the selective detection of lipidhydroperoxides in real samples.In the study of oxidised oils, the sample preparation was also considerably simplified by theuse of direct transesterification with sodium methylate, so that the complicated and errorproneremoval of non-oxidised sample constituents by solid-phase extraction could beavoided. The high proportion of non-oxidised fatty acid esters remaining in the samples hadno unfavourable effects on the HPLC analysis.