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 pentafl uorobenzylhydroxylamine-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-d(11)) 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 specifi city 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 modifi ed GC-MS method, we analyzed HNE in ethylenediaminetetraacetic acid (EDTA) plasma in a well-defi ned collective of migraine patients; recently published. The results underline our former observations that women with migraine are affl icted with increased levels of HNE. Patients with thyroidal dysfunction showed no signifi cant HNE alterations. This was confi rmed 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.