Oxidative stress: Determination of 4-hydroxy-2-nonenal by gas chromatography/mass spectrometry in human and rat plasma.

The lipid peroxidation product 4-hydroxynonenal (HNE) is a biomarker of oxidative stress which is essentially involved in the pathophysiology of many diseases. The analysis of HNE is challenging because this aldehyde is extremely reactive and thus unstable. Hence, we adopted a gas chromatography-mass spectrometry (GC-MS) method based on derivatization of HNE with pentafluorobenzyl-hydroxylamine-HCl followed by trimethylsilylation to trimethylsilyl ethers. Ions representative for a negative ion chemical ionization mode were recorded at m/z = 152 for HNE and at m/z = 162 for the deuterated analogon (HNE-d11) as internal standard. This excellent stable and precise GC-MS method was carefully validated for HNE, and showed good linearity (r(2) = 0.998), and high specificity and sensitivity. Within-day precisions were 4.4-6.1% and between-day precisions were 5.2-10.2%. Accuracies were between 99% and 104% for the whole calibration range (2.5-250 nmol/L) of HNE. To examine the versatility of this modified GC-MS method, we analyzed HNE in ethylenediaminetetraacetic acid (EDTA) plasma in a well-defined collective of migraine patients; recently published. The results underline our former observations that women with migraine are afflicted with increased levels of HNE. Patients with thyroidal dysfunction showed no significant HNE alterations. This was confirmed by normal HNE EDTA plasma levels in hyper- und hypothyroid Sprague-Dawley rats. Taken together, the GC-MS method presented herein is of excellent quality to record oxidative stress-related bioactive HNE levels. This is important for a reorientation of oxidative stress analytics in other human diseases first of atherosclerosis and cancer.

Measurement of total and free malondialdehyde by gas-chromatography mass spectrometry--comparison with high-performance liquid chromatography methology.

Malondialdehyde (MDA) is considered to be a biomarker for enzymatic degradation and lipid peroxidation of polyunsaturated fatty acids. Usually, MDA determination from different biological materials is performed by reaction with thiobarbituric acid (TBA) followed by high-performance liquid chromatography (HPLC) analysis and fluorometric detection. As this method lacks specificity and sensitivity, we developed a gas chromatography-mass spectrometry (GC-MS) method based on derivatization of MDA with 2,4-dinitrophenylhydrazine. Representative ions in negative ion chemical ionization (NICI) mode were recorded at m/z 204 for MDA and at m/z 206 for the deuterated analogon (MDA-d2) as internal standard. This stable and precise GC-MS method showed good linearity (r² = 0.999) and higher specificity and sensitivity than the HPLC method and was validated for both total MDA (t-MDA) and free MDA (f-MDA). Within-day precisions were 1.8-5.4%, between-day precisions were 4.8-9.2%; and accuracies were between 99% and 101% for the whole calibration range (0.156-5.0 µmol/L for t-MDA and 0.039-0.625 µmol/L for f-MDA). Although comparison of t-MDA levels from GC-MS and HPLC results using Passing-Bablok regression analysis as well as Bland-Altman plot showed a correlation of the data, a tendency to increased results for the HPLC values was detectable, due to possible formation of unspecific products of the TBA reaction.