Multi-sample analytical workflow for the determination of F2-isoprostane in oral fluid by HPLC-MS/MS

An imbalance between antioxidant defense systems and reactive oxygen species (ROS) can occur due to the overproduction of ROS or their low degradation rate, leading to a condition of oxidative stress (OS) [1], which is involved in cardiovascular and neurodegenerative diseases, diabetes, and carcinogenesis. The F2 isoprostanes (F2-IsoPs) demonstrated greater chemical stability compared to other OS biomarkers of endogenous oxidative damage to DNA and proteins. IsoPs are formed in situ from the oxidation of arachidonic acid, esterified to phospholipids by the direct action of ROS in the cellular membrane. The aim of this work was the development of a sensitive, rapid, and reproducible method for the quantification of lipid oxidative stress biomarkers in OF through HPLC-MS/MS. The extraction step was developed using a novel approach based on parallel artificial liquid membrane extraction (PALME) [2]. Target analytes were extracted through a three-phase system, exploiting a pH gradient to facilitate mass transfer of uncharged analytes from the original matrix to an aqueous extracting phase, across an organic solvent, immobilised within the pores of a membrane, leading to very low consumption of organic solvents for extraction (only 3 μL). PALME also provided a high enrichment factor (40-fold) and minimized the matrix effect to ≤ 10%. The whole method has been validated according to the FDA guideline. This approach could provide useful diagnostic tools for the early detection of pathologies, enabling timely medical intervention with appropriate therapies.