HPLC determination of oxidative stress

Reactive oxygen and nitrogen species have a physiological role in the organism, but their extensive production can damage cells and result in many diseases. By interaction of biomolecules with reactive oxygen and nitrogen species are biomolecules damaged. Damaged lipid, protein and DNA molecules then serve as biomarkers of oxidative stress and allow its evaluation. Oxidative stress can be caused by external factors such as drugs and then it may occur as adverse and toxic effects. It is important to monitor a drug's ability to induce oxidative stress to monitor drug safety. A high-performance liquid chromatography method coupled with tandem mass spectrometry has been developed to determine malondialdehyde from the cell pellet as a biomarker of lipoperoxidation. The greatest emphasis was given to the optimization of sample preparation by deproteination, derivatization and solid-phase extraction. The method was validated with acceptable specificity, accuracy, precision, recovery and matrix effect. The method complies with the requirements of the European Medicines Agency for the validation of bioanalytical methods. The method can determine intracellular malondialdehyde in the concentration range 0,1-2 μmol∙dm-3 . This method was successfully applied to the analysis of biological samples from in vitro..