Abstract
Abstract BACKGROUND Oxidative stress is implicated in many pathological conditions. Herein, we report on our development and validation of a sensitive and rapid LC-MS/MS method for the determination of oxidative stress biomarkers glutathione (GSH), glutathione disulfide (GSSG), cysteine (Cys) and cystine (CySS) in human brain and glioblastoma tissue. METHODS Freshly-acquired human glioblastoma tissue was homogenized with N-ethylmaleimide solution to prevent thiol oxidation. Analytes were then extracted from homogenate samples by protein precipitation with 2% sulfosalicylic acid (SSA). Stable isotope-labeled analytes were used as internal standards. Independent calibration curves for thiols and disulfides were prepared in analytical solutions. Three levels of quality controls were prepared in human brain homogenate. The detection was performed on Sciex QTRAP 6500+ mass spectrometer in positive electrospray ionization mode. RESULTS Linear regression model was used to cover a concentration ranging 0.4-100 µmol/L for GSSG/CySS and 1-400 µmol/L for GSH/Cys. Chromatographic separation was optimized on Intrada Amino Acid column with total run time of 5 min using gradient elution. For all analytes the maximum coefficient of variation for intra- and inter-day precision was 11.4% and the accuracy was within 80.9-113.7% in analytical solution and matrix. The analytes were stable in brain homogenate for 1 hour and 3 hours at room temperature (RT) and 4 °C, respectively. Stability at -80°C was demonstrated for at least 35 days in human brain homogenate. Stability of stock and working solutions was demonstrated for at least 4 hours (RT) and 25 days (-20°C). CONCLUSIONS A bioanalytical method to quantify GSH, GSSG, Cys, and CySS is successfully developed and validated. The method is currently applied to measure thiols and related disulfides in human glioblastoma tissue undergoing 5-aminolevulinic acid sonodynamic therapy (NCT04559685).
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