Entwicklung von MEKC- und HPLC-Methoden zur Bestimmung von Fettsäuren, Fettsäurehydroperoxiden und Hydroxyfettsäuren
In this work, MEKC and HPLC methods were developed for determining unsaturated fatty acids and the hydroperoxy and hydroxy fatty acids derived from them. Triglycerides and phospholipids from biological samples were first enzymatically or chemically hydrolysed. The products were quantified by UV/VIS-diode-array or UV/VIS detection or by post-column derivatisation reactions, which increased the sensitivity and selectivity through fluorescence or chemiluminescence.
One focus of the work was the chromatographic and electrophoretic separation. MEKC allowed separation of the isomeric hydroxy and hydroperoxy derivatives of oleic, linoleic, linolenic and arachidonic acids under both reversed-flow and normal-flow conditions. The normal-flow system had significant advantages in the analysis time and the number of theoretical plates (ca. 106 m-1). Detection limits of 4 to 100 µM were achieved routinely by MEKC. The use of bubble-cell detectors led to a further improvement by a factor of 5-7. In addition, a previously developed post-column derivatisation technique for MEKC was applied successfully. In this technique, hydroperoxy fatty acids and p-hydroxyphenyl acetic acid (PES) yield selectively, in the presence of microperoxidase-11 (MP-11), a product for LIF detection (λ ex = 325 nm, λ em = 415 nm); the MP-11 for the reaction was immobilised on the wall of a reaction capillary spliced, without the introduction of dead volume, onto the end of the separation capillary (fused silica). Computer simulation of the processes in the reaction capillary made it clear that, to prevent loss of resolution, either the conversion must be very rapid or the fluorescent product must migrate at a rate similar to those of the analytes. It was shown in experiments that under optimal reversed-flow conditions the product from PES indeed migrates with the fatty acid hydroperoxides; PES was found to be the best reagent among a number of commercially available analogues.
RP-HPLC methods with UV detection at 195 or 234 nm were also used for routine analysis of fatty acids and hydroperoxy and hydroxy fatty acids liberated from triglycerides and phospholipids. The use of a fluoroalkyl phase (Fluofix) allowed the partial separation of individual isomers of the oxidised fatty acids. Fast HPLC reduced the analysis time to less than four minutes. The method proved to be linear over several orders of magnitude, from the detection limit of 0.2 to 0.9 µM up to 1500 µM. For the determination of hydroperoxy fatty acids in more complex matrices, the sensitivity and selectivity were improved further by the use of post-column derivatisation.
The methods developed were applied to the study of oxidised and non-oxidised lipids in biological samples such as oils and lecithin from soya and egg, and even to samples with more complex matrices such as blood serum, synovia and LDL (low-density lipoprotein). In the hydrolysis of the phosphatidylcholins, it was shown that the lipases degrade part of the hydroperoxides, thereby greatly reducing the recovery, especially when hydroperoxide concentrations are low; for such samples, hydrolysis with sodium hydroxide was more appropriate than enzymatic hydrolysis. MEKC had the advantage over HPLC of separating interfering matrix components from the analytes; thus, important hydroxy fatty acids in native LDL could be successfully analysed by MEKC with UV detection at 234 nm. The concentrations of the hydroxy fatty acids were found to be ca. 18 µg HODE and 30 µg HETE per gram LDL and thus some 600 times as great as those of the hydroperoxides. Since the hydroxy fatty acids in biological matrices are apparently only slowly degraded, they are better markers for lipid peroxidation than the hydroperoxides.