Selektive Bestimmung von IPG F2α mittels LC-DMS-MS/MS in Urin von Tauchern vor und nach einer Sauerstoffbelastung durch Tauchgänge
A misregulation between oxidative processes and the antioxidative defense of the organism can lead to oxidative stress. Here Isoprostanes can be formed from arachidonic acid by non-enzymatic oxidative processes. From the multitude of isoprostanes the isomer IPG F2α is regarded as a stable marker substance and gold standard for the analysis of oxidative stress in vivo. The purpose of this study was to examine whether an increase of IPG F2α concentrations in urine after dives is measurable due to oxygen exposure in divers as a model for the generation of oxidative stress. For the selective determination of the target analyte IPG F2α a quantitative LC-DMS-MS/MS method was established and validated for the first time. The extraction types (Solid Phase Extraction and Supported Liquid Extraction) tested during the method development showed strong suppression effects for the matrix urine. It was therefore questionable whether the low concentrations of IPG F2α suspected in the diver samples could be reliably measured. In order to reduce matrix effects, chromatography was modified to achieve better separation of the suppressing matrix components. This made time-consuming and error-prone sample preparation unnecessary. By simply diluting the samples with a solution of the internal standard (dilute & shoot), a clear signal of the target analyte IPG F2α could be measured in each urine. The application of the validated method to urine samples of divers (n = 234) showed that the IPG F2α concentration per creatinine in the median immediately after a dive was significantly increased by +6 % (mean +10 %) compared to the initial value before physical exercise under oxygen exposure. For the first time this increase was correlated with the oxygen load responsible for it. A significant linear dependence between the increase of the measured values and the oxygen load during the dives was observed. In each sample an additional signal of the isomer 15(R)-IPG F2α, clearly separated from the target analyte, was measured. Therefore, this isomer was evaluated as a potential alternative marker of oxidative stress. A lower and not significant increase of the concentration per creatinine of +1 % in the median (mean +4 %) after a dive was observed. Thus, the isomer 15(R)-IPG F2α was a less sensitive marker of oxidative stress than the target analyte IPG F2α. The correlation of the increase in concentration with the oxygen load, which was also carried out here for the first time, showed a significant linear correlation between the increase in measured values and the oxygen load due to the dives. Using the diver urines (n = 234), measurements with and without differential mobility spectrometry (DMS) were compared. It was examined whether the measurement method used could also be used with LC-MS/MS systems without a DMS-module. The use of DMS resulted in +3 % higher IPG F2α readings. This statistically significant difference was not relevant for practical application, especially for before-and-after comparisons of samples of the same individual. The developed method could also be used without DMS add-on equipment. Nevertheless, the DMS-module showed a higher selectivity in the measurement series and the signal-to-noise ratio (S/N) improved by a factor of 3.5 compared to measurements without DMS.
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