A rapid liquid microextraction method for the determination of F2-Isoprostanes in oral fluid by means of PALME coupled with UHPLC-MS/MS analysis

A dangerous imbalance between antioxidant defense systems and ROS can occur due to the overproduction of reactive oxygen species (ROS) or their missing degradation; in this scenario ROS exert their toxic action, leading to a condition called oxidative stress (OS)1. OS has been implicated in a variety of pathological events such as cardiovascular and neurodegenerative diseases, diabetes, and carcinogenesis. The best-known biomarkers for endogenous oxidative damage to lipids, DNA, and proteins are 8-iso prostaglandin F2α (8-isoPGF2α), 8-hydroxy2'-deoxyguanosine (8-OHdG), and 3-nitro-l-tyrosine (3-NT)2, respectively. Since the lipid oxidative stress biomarkers, F2-isoprostanes (F2-IsoPs), are more chemically stable than other analytes, their study is more advantageous for analytical purposes. 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 determination and analysis of OS markers is usually based on rapid methods such as colorimetric tests, and immunochemical techniques (ELISA and enzyme immunoassays)3. Anyway, being IsoPs found at low concentrations in biological fluids, a need to develop highly sensitive methods of analysis, and the use of instrumental analysis such as liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) started to be mandatory in the last years. The elective biological matrices for this proposal are blood, plasma, and urine. However, an alternative matrix, like oral fluid (OF), was used in this work. The sampling of this matrix is favourable because it is a fast patient-friendly approach, moreover, it is characterized by a simpler composition than blood and urine. The aim of this work was the development of a new rapid, reproducible, and sensitive method for the quantification of lipid oxidative stress biomarkers in OF through UHPLC-MS/MS. The procedure involved an extraction step which was developed according with the aim of reducing the volume of organic solvent, using the novel approach based on parallel artificial liquid membrane extraction (PALME)4. In the PALME set up, target analytes were extracted through a three phases 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, led to the low consumption of organic solvents for extraction. Moreover, this technique allows an high enrichment factor (40 fold) without an increasing of matrix effect (below 20%). Noteworthy, for the first time, a microextraction technique in the 96-well format was developed for the extraction of IsoPs, to obtain satisfactory extraction levels in the PALME step, it was necessary to pay attention to different parameters: the nature of organic solvent, volumes of biological sample, composition and volume of donator and acceptor solution, pH, ionic strength of donator solution and agitation time.