Although reactive oxygen/nitrogen species (ROS/RNS) have a fundamental role in physiological processes, enhanced ROS/RNS production induced by exogenous sources, including drugs and other xenobiotics, may result in serious damage to biomolecules. Oxidative/nitrosative stress is being intensively investigated and might be responsible for a variety of health side effects. The present liquid chromatography-tandem mass spectrometry (LC-MS/MS) method provides reliable and accurate simultaneous measurement of malondialdehyde (MDA) and 3-nitrotyrosine (3-NT) in cultured human hepatoma (HepG2) cells. Sample preparation process involving ultrasonic homogenization, alkaline hydrolysis of protein-bound MDA and 3-NT, deproteination, derivatization of MDA by 2,4-dinitrophenylhydrazine and solid-phase extraction was optimized, ensuring the isolation and purification of desired analytes. Additionally, nonprotein thiols and nonprotein disulfides were measured using HPLC-UV. The established lower limit of quantification (0.025 nmol/mL for MDA; 0.0125 nmol/mL for 3-NT) allowed their LC-MS/MS determination in HepG2 cells exposed to model oxidizing agent, tert-butyl hydroperoxide (t-BOOH). The results show significant changes in MDA and 3-NT concentrations and alterations in thiol redox-state in dependence on the t-BOOH concentration and duration of its incubation in HepG2 cells. Concurrent evaluation of oxidative/nitrosative stress biomarkers in the in vitro model may significantly facilitate assessment of toxicity of newly developed drugs in preclinical trials and thus improve their safety profile.

Department of Chemistry Faculty of Science University of Hradec Kralove Hradec Kralove Czech Republic

Department of Toxicology and Military Pharmacy Faculty of Military Health Sciences University of Defence Hradec Kralove Czech Republic

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